WO2019200411A1

WO2019200411A1 - Biomimetic tissue systems and methods of using the same

Info

Publication number: WO2019200411A1
Application number: PCT/US2019/027567
Authority: WO
Inventors: Walter Lee MURFEE; Jessica Margaret Mary MOTHERWELL
Original assignee: The Administrators Of The Tulaneeducational Fund
Priority date: 2018-04-13
Filing date: 2019-04-15
Publication date: 2019-10-17

Abstract

The present disclosure generally relates to a cell culturing system, and specifically to a three-dimensional cell culturing system for mesenteric mesometrial tissues with structural and functional characteristics that mimic those of in vivo peripheral tissues with the capability for perfusion across the system.

Description

BIO MIME TIC TISSUE SYSTEMS AND METHODS OF USING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/657,043 filed on April 13, 2018, which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This disclosure was made, in part, with support provided by the United States government under Grant No. R01AG049821, awarded by the National Institutes of Health. The Government may have certain rights in this disclosure.

FIELD

The present disclosure generally relates to a tissue culture system, and specifically to a three-dimensional cell culturing system comprising mesenteric or mesometrial tissue that mimics in vivo tissues. Methods of monitoring toxicities of agents, such as therapeutic agents, in the systems are also disclosed.

BACKGROUND

With advancements in microscale tissue engineering, vascular engineering has been harnessed for creating in vitro models of vascularized tissue for studying physiology and disease, toxicity screening, and drug discovery. The use of advanced in vitro testing is a powerful tool to develop predictive cellular assays suitable for simulating disease pathologies and improving the high attrition rates of novel pharmaceutical compounds. Inability of current models, at both the pre-clinical in vitro and in vivo levels, to correctly predict safety and efficacy in humans is largely responsible for clinical and post-market failure of pharmaceutical compounds. In order to better evaluate clinical relevancy using benchtop models, quantifiable outcomes must be representative of organ-specific physiological endpoints. It is critical that such model systems move beyond three-dimensional versions of conventional cell-based assays to models that truly replicate functional aspects of organ physiology that can be evaluated to screen for drug safety and efficacy.

Microvascular growth and remodeling is a common denominator for multiple pathologies such as diabetic retinopathy, myocardial ischemia, and tumor growth, among others, a need still exists to fully comprehend the multicellular dynamics during angiogenesis, which is defined as the growth of new blood vessels from existing ones. Ideal experimental models enable the observation or even tracking of specific cell types at different temporal stages of growth at certain locations across a microvascular network. However, live imaging of single cell dynamics is currently not possible with high temporal resolution in vivo, even with the use of dorsal skinfold chambers. Thus, the capability to induce microvascular growth into rat or mouse mesentery or mesometrium as is disclosed herein supports the use of the ex vivo model as a valuable tool for investigators in this field.

Mesentery and mesometrium tissue is a useful tool in understanding microcirculation. The thinness of these tissues has provided unique views of the network, vessel, and cellular levels that remain largely unobtainable with other tissues. From vasoreactivity, immunohistochemistry, and more recently, ex vivo tissue culture studies, the use of the rat mesentery has provided insights into cell-cell interactions, phenotypic dynamics, basic network architecture, and dysfunction associated with multiple disease scenarios.

Observations made in the rat mesentery in many ways have helped define the fundamental areas of microvascular research focused on hemodynamics, white blood cell mechanics, red blood cell flow, and microvascular network remodeling. Utilizing this tissue has shed light on different endothelial cell and pericyte phenotypes and network responses to various growth factors. Further, the tissues’ characteristics have allowed for the investigation and manipulation of vessel-specific hemodynamic stresses, and for the observation of leukocyte responses to altered environments. The tissue has also offered fundamental characterization of microvascular innervation and the structure of initial lymphatic networks.

In the context of tool development, ex vivo biomimetic models have emerged as powerful experimental platforms for basic science discovery and pre-clinical drug testing. A challenge for microvascular research, however, is recapitulating the complexity associated with intact networks in real tissue scenarios.

SUMMARY OF EMBODIMENTS

The present disclosure relates to a tissue culture system or a container comprising a three dimensional tissue comprising a plurality of cells within one or a plurality of vessels. In some embodiments, the cell types forming vascular rat and mouse mesentery and mesometrium, which have the following advantages: 1) investigation of multicellular interactions, i.e. endothelial cells, smooth muscle cells, pericytes, and macrophages, 2) similar to in vivo, angiogenesis most commonly occurs along venules and capillaries versus arterioles, 3) perivascular cells such as smooth muscle cells and pericytes remain functional during culture, and 4) the ability to capture time-lapse images during angiogenesis and lymphangiogenesis at the same time.

In the present disclosure, a novel tissue culture system is disclosed comprising tissue derived from mammalian mesentery or mesometrium. In some embodiments, the system comprises at least a first vessel, the first vessel comprising at least one contiguous surface that defines a volume within which the tissue is positioned. In some embodiments, the system comprises cell media at a volume sufficient to submerge the tissue in the medium tissue derived from mammalian mesentery or mammalian mesometrium tissue, In some embodiments, the mesentery tissue or mesometrial tissue comprises a plurality of endothelial cells in a number sufficient to create at least a first portion of vasculature within the tissue. In some embodiments, the mesentery tissue or mesometrial tissue comprises a plurality of endothelial cells in a number sufficient to create at least a first and a second portion of vasculature within the tissue. The disclosure also relates to a tissue culture system comprising a cell incubator. In some embodiments, the system relates to a pump operably connected to a first portion of tubing and a reservoir of cell medium, the tubing in fluid communication with the reservoir and at least the first vasculature of the tissue, such that cell medium is perfused through the reservoir, first portion of tubing and vasculature.

Disclosed are tissue culture systems comprising a solid substrate or vessel comprising at least one contiguous surface defining a volume; a three dimensional tissue from a mammalian mesentery or mammalian mesomentrium, the tissue comprising a plurality of endothelial cells and the tissue free of intestinal wall; a volume of cell medium sufficient to submerge the three dimensional tissue in the vessel; reservoir of cell medium; and at least a first length of tubing in fluid connection with the reservoir and the three-dimensional tissue. In some aspects, the tissue culture system can further comprise a pump in operable connection to the tubing or the reservoir.

Disclosed are methods of measuring angiogenesis or capillary sprouting comprising exposing any of the tissues disclosed herein in culture to an agent; and measuring growth of vasculature or capillary sprouting in the tissue before and after exposure to the agent. Disclosed are methods of evaluating the toxicity of an agent comprising culturing one or plurality of a mesenteric and/or a mesometrial tissue in any of the compositions described herein; exposing at least one agent to the one or more cells; measuring and/or observing one or more morphometric changes of the one or more mesenteric and/or a mesometrial tissue; and correlating one or more morphometric parameters of the one or more mesenteric and/or mesometrial tissues with the toxicity of the agent, such that, if the morphometric parameters are indicative of decreased cell viability, the agent is characterized as toxic and, if the morphometric parameters are indicative of unchanged or positive cell viability, the agent is characterized as non-toxic.

Also disclosed are methods of inducing growth of one or a plurality of cells in a three dimensional tissue comprising contacting one or a plurality of any of the isolated mesenteric or mesometrial tissues described herein with the solid substrate, said solid substrate comprising at least one exterior surface, at least one interior surface and at least one interior volume defined by the at least one interior surface and accessible from a point exterior to the solid substrate through at least one opening; positioning one or a plurality of any of the isolated mesenteric or mesometrial tissues described herein to the at least one interior volume; applying a cell medium into the culture vessel with a volume of cell medium sufficient to cover the at least one interior volume; affixing at least a one or a plurality of fluid linkage elements to at least the first vasculature.

Also disclosed are methods of detecting and/or quantifying cell mobility in vitro comprising culturing one or a plurality of any of the mesenteric or mesomentrial tissues disclosed herein in any of the systems disclosed herein; and exposing the tissue or tissues to a known number of cells in the composition after culturing for a time period sufficient to allow cell migration of the one or plurality of cells.

Also disclosed are methods of measuring vasculature growth within a tissue comprising positioning one or a plurality of tissues in any of the compositions disclosed herein; quantifying the one amount or density of vasculature in the one or plurality of tissues; contacting the one or plurality of tissues to one or a plurality of agents; quantifying the number or density of vasculature after contacting the one or plurality of tissues to one or a plurality of agents; and calculating the difference in the number or density of vasculature prior to and after the step of contacting the one or plurality of tissues to one or a plurality of agents. Also disclosed are methods of measuring intracellular or extracellular recordings comprising culturing one or a plurality of tissues in any of the composition disclosed herein; and measuring a recording across the one or a plurality of tissues.

Also disclosed are methods of real-time imaging of tissue comprising culturing tissue within any of the tissue culture systems disclosed herein; and exposing the tissue culture system to digital imaging.

Also disclosed are methods of making any of the tissue culture systems disclosed herein comprising forming an interior chamber within a solid substrate; affixing mesenteric or mesomentiral tissue from a subject to the solid substrate; positioning at least one fluid linkage element into the tissue in fluid communication with a feeding arteriole; culturing the tissue in cell culture medium at about 37 degrees Celsius; attaching at least a first length of tubing to the at least first fluid linkage element; placing a reservoir of cell medium in fluid connection with at least the first length of tubing; placing a pump in operable connection to the at first length of tubing; and, optionally sealing the tissue within the solid substrate, such that the tissue is positioned within an internal cavity of the solid substrate in fluid communication with the reservoir.

A method for testing the efficacy of a test substance comprising: exposing a three dimensional tissue comprising mesentery or mesomentrial cells to the test substance, in which the three dimensional cell culture comprises tissue secured to a solid substrate and in a culture chamber; and determining the effect of the test substance by measuring or observing a change in the three dimensional cell culture.

Also disclosed are methods of producing a tissue culture, in-vitro model of rat mesentery tissue wherein the tissue comprises blood and lymphatic microvascular networks, endothelial cells, smooth muscle cells, immune cells, neural cells, and pericytes, the method comprising harvesting rat mesentery tissue from a rat and securing it to a solid substrate.

Also disclosed are methods of printing cells on any of the tissue culture systems described herein; and evaluating the cells wherein the evaluating comprises tracking growth and/or interactions of the cells within the tissue culture system.

Also disclosed are methods of producing a tissue culture, in-vitro model of mouse mesentery tissue comprising blood and lymphatic microvascular networks, endothelial cells, smooth muscle cells, immune cells, neural cells, and pericytes, the method comprising inducing vascularizaton of mouse mesentery by injecting said mouse with tamoxifen, sunflower oil, of VEGF for 5 consecutive days before harvesting the mouse mesentery.

Also disclosed are methods of manufacturing a tissue culture, in-vitro model of mouse mesometrium tissue comprising blood and lymphatic microvascular networks, endothelial cells, smooth muscle cells, immune cells, neural cells, and pericytes, the method comprising harvesting the mouse mesometrium and securing the mouse mesometrium to a solid substrate.

In some embodiments, the system comprises a closed system for fluid flow in a fluid circuit. In some closed system embodiments, the tissue culture system comprises a cell medium reservoir a first portion of tubing, a second portion of tubing and a three-dimensional mesentery or mesometrium tissue comprising a first vasculature of endothelial cells, the first portion of tubing in fluid connection with at least the first portion or feeding blood vessel of the vasculature and the second portion of tubing in fluid communication with an exiting blood vessel. In such embodiments, the tissue culture system comprises a pump (i.e. a peristaltic pump), in operable connection with a first portion of tubing, said pump capable of generating fluid flow through the first portion of tubing into the first portion of vasculature, from the feeding portion of the vasculature through the exiting portion of the vasculature and into the second portion of tubing. The second portion of tubing is in fluid communication with an inlet of the pump such that cell media can be circulated through a fluid circuit. The tissue culture system can also comprise a gas exchanger for introduction of gas such as carbon dioxide into the system and/or a heating element. In some embodiments, the tissue culture system is maintained at about 37 degrees Celsius an about 5% carbon dioxide.

In some embodiments, the tissue is free of intestinal endothelial cells or free of intestinal wall. In some embodiments, the tissue comprises a thin sheet of a plurality of cells, such cells comprising the microvasculature is accessible on one portion of the model contains blood vessels, lymphatic vessels, endothelial cells, smooth muscle cells, and pericytes, providing a physiologically relevant in-vitro tissue model for research and drug development, evaluation, and design. The tissue culture model can be cannulated and perfused, providing even more relevant physiological data. Shear stress caused by blood flow is known to play a role in endothelial cell morphology and proliferation as well as angiogenesis, thus it is novel to incorporate flow through the disclosed model system. The perfusion system disclosed can be closed or open loop allowing flow to perfuse continuously through the tissue or exit as waste and not be re-introduced into the system. The system disclosed can also be single or double cannulated, to allow flow to enter through an arteriole or vein and either drain out of the tissue in a single cannulated system or to exit from a second cannula in another corresponding arteriole or vein in the double cannulated system.

The mesentery and mesometrium models disclosed provide cellular interactions and growth within an intact, shear stress induced, and real microvascular network.

The tissue culture model disclosed provides an ability to observe and understand angiogenesis, lymphangiogenesis, pericyte-endothelial cell interactions, and anti-angiogenic drug testing. The model disclosed also provides the ability to time-lapse image the culture model and track cellular changes in the model at various timepoints.

Interestingly, the analogous transparent connective tissue in the mouse is avascular, preventing the impact of using the mesentery in combination with transgenic mouse strains for cell lineage or targeted inhibition studies has not been realized. The model disclosed herein provides a method of inducing vascularization in the normally avascular mouse mesentery. This vascularization can be induced by injecting the mice to be used for the culture model disclosed with organic sunflower seed oil (Spectrum), 10 mg/mL of tamoxifen T5649 (Sigma-Aldrich), or VEGF.

The tissue culture model also disclosed can utilize mouse mesometrium tissue to be a source of tissue to be harvested for the model. A potential analog to the rat mesentery is the mouse mesometrium, which is the thin connective tissue of the uterine horns. The disclosed disclosure demonstrates the ability to visualize and study angiogenesis and other cell interactions in the mesometrium tissue, providing another tissue type for real-time and biomimetic analysis.

The tissue culture model disclosed herein also provides the capability to be seeded with various cell types for investigation of growth and tracking. Through cell printing methods, such as laser direct writing cell printing, various cells, including but not limited to stem cells and cancer calls can be pre-positioned and cultured to evaluate cellular interactions with the new culture model.

The disclosure relates to a method of measuring angiogenesis or capillary sprouting comprising exposing a tissue in culture to a therapeutic and monitoring growth of vasculature or capillary sprouting in the tissue. The disclosure also relates to a method of measuring the toxicity of a substance or therapeutic, the method comprising exposing a tissue disclosed herein with the substance or therapeutic and monitoring the viability of the cells in the tissue after the step of exposing.

BRIEF DESCRIPTION OF DRAWINGS

Fig.l depicts how imaging during perfusion culture can be achieved by either positioning a microscope objective optionally operably connected to a camera, above the bioreactor or below using simple air objectives or water immersion.

Fig.2 depicts a closed-loop double cannula perfusion system where the main feeding arteriole and venule of the tissue is cannulated to allow flow to enter through the arteriole side and exit through the venule side of the vasculature. The tissue is secured in the bioreactor and placed into a culture chamber. The flow is generated by a delivery pump from the perfusate reservoir monitored by a pressure sensor positioned on the arteriole inlet side. Flow will pass through the vasculature and exit into the perfusate reservoir to be recycled through the tissue again, hence the closed-loop system. The data acquisition will collect recordings from the pressure sensor for monitoring. The filter in the perfusate reservoir is to help maintain sterility of the fluid

Fig.3 depicts an open-loop single cannula perfusion system where the main feeding arteriole of the tissue is cannulated and fluid is allowed to flow through the vasculature and drain out of the tissue. The tissue is secured in the bioreactor and placed into a culture chamber. The flow is generated by a delivery pump from the perfusate reservoir monitored by a pressure sensor positioned on the arteriole inlet side. Flow will pass through the vasculature and exit into the waste reservoir to be discarded, hence the open-loop system. The data acquisition will collect recordings from the pressure sensor for monitoring. The filter in the perfusate reservoir is to help maintain sterility of the fluid.

Fig.4 depicts an open-loop double cannula perfusion system where the main feeding arteriole and venule of the tissue is cannulated to allow flow to enter through the arteriole side and exit through the venule side of the vasculature. The tissue is secured in the bioreactor and placed into a culture chamber. The flow is generated by a delivery pump from the perfusate reservoir monitored by a pressure sensor positioned on the arteriole inlet side. Flow will pass through the vasculature and exit into the waste reservoir to be discarded, hence the open-loop system. The data acquisition will collect recordings from the pressure sensor for monitoring. The filter in the perfusate reservoir is to help maintain sterility of the fluid.

Fig.5 depicts a bioreactor or solid substrate created by laser cutting the top and base pieces (#4 & #5). Threaded rods (#6) are screwed through the base piece. PDMS O-rings are made using a circular punch (#2). The vasculature of the tissue will be connected via the glass cannula embedded in PDMS (#7). The bioreactor is assembled in a“sandwich” fashion where the different pieces stack on top of each other and the tissue will lay parallel between two filter membranes (#3). The threaded rods and knurled nuts (#1) are used to apply pressure

perpendicular to the assembly to hold in place.

Fig. 6 depicts the top view of an assembled solid substrate.

Fig.7 depicts an expanded side view of a bioreactor or solid substrate.

Fig.8 depicts an assembled side view of a bioreactor or solid substrate.

Fig.9 depicts bioreactor within the culture chamber will be placed inside an incubator to maintain temperature and pH levels. The pump will deliver flow through the system and can be either external or internal to the incubator.

Fig.10 depicts a single tissue cannulated on the arteriole side and within a fully assembled bioreactor in a culture chamber with media. Below that image is a magnified view of the cannulated tissue in the chamber.

Fig.ll depicts a triple window tissue cannulated on the arteriole side and within a fully assembled bioreactor in a culture chamber with media. Below that image is a magnified view of the cannulated tissue windows in the chamber.

Fig.l2A- 12 F depict quantification of capillary sprouting per vessel type after (12A- 12C) bFGF and (12D-12F) VEGF stimulation. For each growth factor stimulation experiment the numbers of sprouts per vessel type length after 3 days in culture were compared across experimental groups: control (MEM alone), MEM + growth factor (GF), MEM + GF + NG2 antibody, and MEM + GF + Rabbit IgG. The rabbit IgG groups controlled for potential non specific antibody binding effects. * indicates significance against the control group (p < 0.05). + indicates significance against the growth factor group (p < 0.05). Values are mean ± s.e.m.

Fig.13 depicts the mesenteric tissue, harvested from the small intestine of an adult Wistar rat, is transferred into a culture dish, quickly spread out on the bottom of a well, secured in place with a membrane insert, and covered with media. Tissues are cultured in standard conditions (37°C, 5% C02). This process can be applied to harvesting mouse mesentery and mesometrium tissue.

Fig.l4A - 14D depict live/dead assay performed after culture showed a high ratio of live cells (green) to dead cells (red) specifically along the blood vessels (14A). Mesentery tissues were labeled with lectin and anti-NG2, to identify pericytes (dark grey) alongside vessels (grey) and to confirm that different types of cells are present in the post-culture tissues (14B). Tissues were also labeled against PECAM/LYVE-l to identify blood (dark grey) vessels from lymphatic (grey) vessels (14C). To investigate if microvascular cells undergo proliferation in the culture, mesentery tissues were labeled with lectin/anti-BrdET. On capillary segments labeled with lectin (grey), multiple cells were confirmed to be proliferative (dark grey), another indicator that cells in the rat mesentery culture model undergo normal cell life cycles (14D). Scale bars = 100 pm.

Fig.l5A - 151 depicts the evaluation of smooth muscle cell morphology in the rat mesentery culture model. A-F) Comparison of Day 0 and Day 3 + 10% FBS SMC morphology. PEC AM and aSMA labeling identified increased EC sprouting and decreased SMC bands after three days in culture with 10% FBS compared to bands in Day 0 tissues. Scale bars = 50 pm. A = arteriole, V = venule. G-I) Evaluation of SMC bands, where (G, H) are higher magnifications of the tissue regions indicated by the above squares. Comparison of Day 0 and Day 3 + 10% FBS tissues reveals a decrease in SMC bands after three days in culture with serum. Plus signs identify SMC bands. Scale bars = 10 pm. Black and white bars represent Day 0 and Day 3 +

10% FBS groups respectively. **** indicates a significant difference of p < 0.0001 by two-tailed Student’s t-test.

Fig.l6A - 16F depicts comparison of arteriole vasoconstriction responses to 50 mM KC1 and 20 nM ET-l between Day 0 (pre-culture) and Day 3 (cultured) ex vivo tissues. 16A-16D) Comparison between Day 0 (16 A, 16B) and Day 3 + 10% FBS (16C, 16D) groups before and after drug exposure demonstrates arteriole constriction in rat mesenteric tissues. Microvascular networks were visualized with BSI-lectin labeling. Arrows indicate points of constriction along the vessel. Scale bars = 100 pm. A = arteriole, V = venule. 16E, 16F) Percent vasoconstriction was quantified after five-minute exposure to 50 mM KC1 and 20 nM ET-l, respectively. Black, white, and striped bars represent Day 0, Day 3 + 10% FBS, and Day 3 + No FBS groups respectively. *, **, and *** indicates a significant difference of p < 0.05, p < 0.01, and p < 0.001 respectively by One-Way ANOVA and Student-Newman-Keuls post hoc method“ns” indicates no significant difference (p > 0.05).

Fig.l7A - 17B depicts time-lapse images demonstrate the ability to observe lymphatic and blood vessel patterning. Lymphatic (1) vessels can be distinguished from arterioles (a) and venules (v) based on labeling morphology on day 0 (A). On day 5 post-stimulation with 10% serum, lymphatic morphology is lost and vessels appear to have integrated with the nearby angiogenic blood vessels. Scale bars = 100 pm.

Fig.l8A - 18D depicts maintenance of nerves in culture. 18A-18B) Quantification of the NG2 nerve alignment as a percentage of network feeding arterioles and length along capillaries per vascular area in unstimulated tissues on day 0 and tissues cultured in minimum essential media or nerve media on day 3. 18C- 18D) Representative images of NG2 nerve alignment along network feeding arterioles and capillary regions. Comparison of unstimulated tissues on day 0 to tissues cultured in NBM, NGF, with 20% FBS for 3 days. Arrows indicate nerve presence.

Values are shown as mean ± SEM. * indicates significant difference (p < 0.05; Student’s t-test). Scale bars = 100 pm.

Fig.l9A - 19C depicts time-lapse imaging of the rat mesentery enables observing microvascular remodeling over the course of the culture. A robust angiogenic response was observed after 3 (B) and 5 days (C) of culture with 10% serum stimulation. Scale bars = 100 pm.

Fig.20A - 20F depicts microvascular networks in the rat mesentery culture model that were imaged before and after angiogenesis. Comparison of the same network labeled with lectin on day 0 and day 3 (20A, 20B) post-stimulation with 10 % serum identifies new vessels. Lectin also labels a population of unidentified interstitial cells. Quantification of vessel density (20C, 20D) and the number of capillary sprouts per vascular area (20E, 20F) confirmed an increase in both metrics for each tissue. 20C, 20E) Before (day 0) and after (day 3) comparisons per tissue. 20D, 20F) Comparison between day 0 and day 3 averages using a paired Student’s t-test confirmed a significant difference in both the average number of vessel segments (p < 0.0001) and the average number of sprouts (p < 0.00001) per vascular area. White bars represent day 0, and black bars represent day 3. Values are averages +/- SEM. For this representative analysis, 13 tissues were harvested from 2 rats. Scale bars = 100 pm.

Fig.21A and 21B depicts RFP-transfected MDA-MB-231 (dark grey) metastatic breast cancer cells interacting with PEC AM positive (grey) blood vessels (BV) and lymphatics (L) in the rat mesentery culture model. Post transplantation, cancer cells remain viable, migrate, and proliferation. 21 A) Example of high density vessel region apparently growing around a cluster of cancer cells. 21B) Example of individual cancer cells nearby lymphatic to blood vessel connections (arrows) in a high density vessel region. These cells were printed onto the tissue culture model by laser direct write method.

Fig.22A - 22D depicts quantification of angiogenesis inhibition following sunitinib treatment. The effect of 3 -day exposure to sunitinib on 10% serum growth was evaluated based on two angiogenic metrics: Vessel density (22A, 22B), and number of capillary sprouts (22C, 22D) per vascular area. Control tissues were stimulated with 10% serum only. 22A, 22C) Each pair of bars represents a tissue. The average increase in vessel segments (22B) and capillary sprouts (22D) per area in control group is plotted against the sunitinib-treated group. * represents a significant difference between control and sunitinib groups (p < 0.05 for vessel segments and p < 0.01 for capillary sprouts). White bars represent day 0 (before) and black bars represent day 3 (after).

Fig.23 depicts an example image of Dil-labeled aged human bone marrow derived stem cells after 5 days in cultured adult rat mesentery networks. Arrows identify stem cells in a pericyte location wrapped along capillaries. This image demonstrates the novel view provided by the rat mesentery culture model and establishes the feasibility of our ongoing analysis. Scale bar = 20 pm. These studies have also been done in Adult human bone marrow-derived stem cells, Aged human adipose-derived stem cells and Adult human adipose-derived stem cells.

Fig.24 depicts quantification of the percentage of adult and aged BMSCs in pericyte location along capillaries in adult mesenteric microvascular networks 5 days after culture. The data represents 32 (adult) and 31 (aged) tissues and 3 cell donors per group. Experiments have been conducted for a total of 8 adult and aged donors and the additional analysis of images is ongoing.

Fig.25A - 25C depict application of the rat mesentery culture model for aging research. 25A) In the Rat Mesentery Culture Model mesenteric tissues can be harvested from adult and aged rats and cultured for comparison of microvascular growth dynamics. Advantages of this tissue culture model include time-lapse observation of angiogenesis across intact microvascular networks, the ability to probe cell-cell interactions (2, 54). 25B) Representative image of an aged mesenteric network from an aged 24 month-old Fisher-344 rat immediately after harvesting and after culturing for 3 days in minimum essential media supplemented with 10% fetal bovine serum (FBS). PEC AM labeling identifies the hierarchy of intact networks including arterioles, “A,” venules,“V,” and capillaries. Angiogenesis in the cultured tissues is supported by the observation of regions with high vascular density“*” and capillary sprouting (arrows). 25C)

Data demonstrating the feasibility of comparing angiogenic responses in mesenteric tissues from adult (9 month) and aged (24 month) Fisher-344 rats in unstimulated (UN) and stimulated (ST) conditions (n = 8 tissues from 5 rats per group) n.s. indicates that numbers of vascular segments per area and capillary sprouts per vascular area were not significantly different between adult and aged networks (p > 0.05). * indicates significant difference between unstimulated and stimulated conditions (p > 0.05). These results suggest that angiogenesis in aged scenarios can be rescued by exogenous delivery of growth cues. Values are shown as mean ± SEM. Scale bars = 100 pm.

Fig. 26A and 26B depict a mouse mesometrium culture model. (26 A) Image showing a female mouse with the mesometrium tissue exposed (dashed oval) and (26B) a schematic demonstrating the harvested tissue for time-lapse imaging during culture.

Fig. 27A - 27C depict how angiogenesis is stimulated during culture in intact microvascular networks from mouse mesometrium tissues. Tissues were cultured with MEM supplemented with 20% FBS for 3, and 5 (27B) days and compared to day 0 tissues (27 A), Angiogenic response was quantified by counting the total number of sprouts (arrows) per total vascular length (27C). A = arteriole and V = venule. Data is shown as the mean + SEM and * represents p < 0.05. Scale bars = 100 pm.

Fig. 28A - 28D depict growth of microvascular networks in mouse mesentery. PECAM labeling identified endothelial cells along microvascular networks. Mouse mesentery tissue from 21 days post-injection with saline (28 A) and sunflower seed oil (28B). The lines denote border between the connective tissue and the adipose tissue (+). The quantification of vascularized tissue area (28C) and tissue density (28D) from each group are shown. A = arteriole, V = venule, and C = capillary. Data is shown as the mean + SEM, n=8. The * and *** represents p < 0.05 and p < 0.0001, respectively. Scale bars = 200 pm.

Fig. 29 depicts an evaluation of angiogenesis in microvascular networks cultured with flow and without flow. Microvascular networks from both experimental With Flow and Without Flow groups became angiogenic after 48 hours in culture, defined by an increase in vascular density and capillary sprouts. Angiogenic microvascular networks cultured without flow exhibited significantly (p=0.0027) denser vascular structures compared to networks cultured with flow. The *** indicates a significant difference of p<0.0l by two-tailed Student’s t-test.“ns” indicates no significant difference (p>0.05).

Fig. 30 depicts bioreactor system for perfused microvascular studies. (30A) Side-by-side comparison of illustrated and photographed cannulated mesentery tissue secured in biochamber. (30B) Diagram of the open-loop bioreactor system used for ex vivo mesentery perfusion studies. Flow is generated by the peristaltic pump and circulates from the media reservoir into the biochamber, entering the mesentery microvascular network, and exits into the waste reservoir. (30C) Expanded view of the assembled biochamber with cannulated mesentery tissue. The chamber is assembled in a“sandwich” fashion to hold the tissue flat and enable real-time imaging of microvascular networks. MR = media reservoir, WR = waste reservoir, P = pump, BC = biochamber, SC = secondary culture dish.

Fig. 31 depicts the evaluation of tissue viability with and without the intestinal loop in perfused bioreactor culture. Cell viability/cytotoxicity labeling with Calcein AM (Live - grey) and Ethd-l (dark grey- Dead) confirms tissues cultured without the intestinal loop remain alive and viable in the perfused bioreactor system, as indicated by the increased presence of positive Live labeling compared to the tissues with intestinal loop.

Fig. 32A - 32C depicts perfused microvascular networks from freshly harvested mesentery tissue. (A) Representative epifluorescence images of microvascular networks perfused with FITC- conjugated albumin (B). (C) Lectin labeling identified microvascular networks where blood and lymphatic vessels were distinguished based on their morphology and network structure. L = lymphatic vessel, V = venule, A = arteriole, C = capillary.

Fig. 33A and 33B depicts velocity measurements in perfused microvascular networks ex vivo. Velocities were calculated by tracking fluorescent microbeads (1 um diameter) flowing through capillary networks. Example images of the start (A) and end (B) of tracking a microbead along the path of a perfused capillary vessel over the time-course of 0.3 seconds. (C) Velocities and wall shear stresses calculated from capillary vessels across four microvascular networks are shown in the table.

Fig. 34A - 34F depicts microvascular networks maintain perfusion during culture. (A,B) Representative epifluorescence images of capillary networks perfused with FITC-conjugated albumin (green) after 48 hours in culture. The * symbol indicates newly formed capillary loops with perfusion culture. (C-F) Examples of newly formed capillary sprouts perfused with FITC- conjugated albumin (green). Microvascular networks were identified by lectin (red) labeling, where blood and lymphatic vessels were distinguished based on their morphology and network structure.

Fig. 35A - 35D depicts the evaluation of angiogenesis in microvascular networks cultured with flow (Perfused) and without flow (Static). Microvascular networks from both experimental Perfused (A) and Static (B) groups became angiogenic after 48 hours in culture, defined by an increase in vascular density (C) and capillary sprouts (D). Angiogenic microvascular networks cultured without flow exhibited significantly (p=0.0027) denser vascular structures compared to networks cultured with flow. Interestingly, no significant difference (p=0.9269) was observed in capillary sprouting between Perfused and Static groups. White and grey bars represent Perfused and Static groups respectively. The ** indicates a significant difference of p<0.0l by two-tailed Student’s t-test.“ns” indicates no significant difference (p>0.05).

DETAILED DESCRIPTION OF EMBODIMENTS

Various terms relating to the methods and other aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.

As used in this specification and the appended claims, the singular forms“a,”“an,” and “the” include plural referents unless the content clearly dictates otherwise.

The term“more than 2” as used herein is defined as any whole integer greater than the number two, e.g. 3, 4, or 5.

The term“plurality” as used herein is defined as any amount or number greater or more than 1.

The term "vessel" as used herein is defined as any vessel suitable for growing, culturing, cultivating, proliferating, propagating, or otherwise similarly manipulating cells. A culture vessel may also be referred to herein as a "culture insert". In some embodiments, the culture vessel is designed to comprise an interior chamber into which the disclosed tissue is positioned and various culture mediums. Wherever any of the phrases“for example,”“such as,”“including” and the like are used herein, the phrase“and without limitation” is understood to follow unless explicitly stated otherwise. Similarly“an example,”“exemplary” and the like are understood to be non-limiting.

The term“substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word“substantially” is not explicitly recited. Therefore, for example, the phrase“wherein the lever extends vertically” means“wherein the lever extends substantially vertically” so long as a precise vertical arrangement is not necessary for the lever to perform its function.

The terms“comprising” and“including” and“having” and“involving” (and similarly “comprises”,“includes,”“has,” and“involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning“at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example,“a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms“a” or“an” are used,“one or more” is understood, unless such interpretation is nonsensical in context.

The terms “physiologically effective rate” as used herein is defined as the rate of microfluidic flow in the three-dimensional tissue that is sufficient to maintain the viability of constitutive cells in the tissue at a rate comparable to the rate of microfluidic flow in the tissue in an animal from which it is derived. In some embodiments, the tissue culture system comprises a microfluidic flow that is free of oscillatory or general shear stress of above an amount of force that would damage the interior cells of the vasculature when exposed pressure. In some embodiments, the tissue culture system is free of shear stress of a magnitude higher than about 1 dynes per square centimeter of surface area exposed to the microfluidic flow. In some embodiments, the tissue culture system is free of shear stress of a magnitude higher than about 2 dynes per square centimeter of surface area exposed to the microfluidic flow. In some embodiments, the tissue culture system is free of shear stress of a magnitude higher than about 3 dynes per square centimeter of surface area exposed to the microfluidic flow. In some embodiments, the tissue culture system is free of shear stress of a magnitude higher than about 4 dynes per square centimeter of surface area exposed to the microfluidic flow. In some embodiments, the tissue culture system is free of shear stress of a magnitude higher than about 5 dynes per square centimeter of surface area exposed to the microfluidic flow. In some embodiments, the tissue culture system is free of shear stress of a magnitude higher than about 6 dynes per square centimeter of surface area exposed to the microfluidic flow. In some embodiments, the tissue culture system is free of shear stress of a magnitude higher than about 7 dynes per square centimeter of surface area exposed to the microfluidic flow. In some embodiments, the tissue culture system is free of shear stress of a magnitude higher than about 8 dynes per square centimeter of surface area exposed to the microfluidic flow. In some embodiments, the tissue culture system is free of shear stress of a magnitude higher than about 9 dynes per square centimeter of surface area exposed to the microfluidic flow. In some embodiments, the tissue culture system is free of shear stress of a magnitude higher than about 10 dynes per square centimeter of surface area exposed to the microfluidic flow. In some embodiments, the tissue culture system is free of shear stress of a magnitude higher than about 12 dynes per square centimeter of surface area exposed to the microfluidic flow.

The term "seeding" as used herein is defined as transferring an amount of cells or tissue into a culture vessel. The amount may be defined and may use volume or number of cells as the basis of the defined amount. The cells may be part of a suspension.

The term "recording" as used herein is defined as measuring the responses of one or more cells within the tissues disclosed herein. Such responses may be morphological changes to the cells within the tissue or may be immunohistological measurements or collected by visually inspecting the tissue for staining or quantifying the amount of fluorescence emitted from a fluorophore covalently bound to an antibody or antibody fragment specific for an epitope on one or a plurality of cells within the tissue. In some embodiments, the“recording” is (i) a velocity measurements in physiologically relevant microvascular networks, (ii) an observation of angiogenesis at specific locations within a vascular tree, and/or (iii) an evaluation of wall shear stress on capillary sprouting. In some embodiments, the methods disclosed herein comprise measuring one or a plurality of recordings.

Systems and Compositions

In some embodiments, the solid substrate or vessel can comprise at least one plastic material forming a flat or substantially flat surface relative and parallel to the ground and at least one or a plurality of sidewalls extending upward from the flat surface, the flat surface and the at least one or plurality of sidewalls defining a volume into which the three-dimensional tissue is positioned parallel to the surface such that the three-dimensional tissue has a single upper surface and a single bottom surface, wherein the bottom surface is in physical contact with the flat surface of the solid substrate. In some embodiments, once cell medium is added to the solid substrate/vessel, the three-dimensional tissue can lose contact with the flat surface of the solid substrate due to suspension in the cell medium.

In some embodiments, the tissue culture system can comprise an insert comprising at least one flat surface positioned adjacent to or substantially adjacent to a top surface of the three- dimensional tissue, such that the three-dimensional tissue is positioned between the flat surface of the solid substrate and the flat surface of the insert, and wherein the single upper surface is in physical contact with at least a portion of the flat surface of the insert and the bottom surface is in physical contact with the flat surface of the solid substrate.

The present disclosure relates to a solid substrate for holding any of the disclosed tissues. The solid substrate can be a dish, vessel, plastic stand with sidewalls, hollow fiber tube, conical, cylindrical, or rectangular in shape. In some embodiments, the solid substrate has a lid second component or top that can enclose a volume and define an interior chamber within which the tissue is positioned. In some embodiments, the solid substrate comprises plastic or other inert material on which biological material such as cells or tissue can grow or culture for hours or days at a time. The solid substrate can, in some embodiments, be sealed by one or a plurality of seals, in the form of a rubber stopper that, for instance, in a cylindrical dish rests circumferentially around the edge of a dish between the lid and the dish creating an air-tight or nearly air-tight edge. In some embodiments, the solid substrate may have one or a plurality of inlets or outlets (typically circular or semicircular in shape) through which tubing cannula and/or other fluid circuit components may be placed to feed the internal chamber with fluid or remove fluid from the internal chamber while the culture system is in operation.

In some embodiments, the solid substrate can have various thicknesses. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 100 mm. In some embodiments, the thickness of the solid substrate is from about 150 pm to about 800 pm. In some embodiments, the thickness of the solid substrate is from about 200 pm to about 800 pm. In some embodiments, the thickness of the solid substrate is from about 250 pm to about 800 pm. In some embodiments, the thickness of the solid substrate is from about 300 pm to about 800 pm. In some embodiments, the thickness of the solid substrate is from about 350 pm to about 800 pm. In some embodiments, the thickness of the solid substrate is from about 400 pm to about 800 pm. In some embodiments, the thickness of the solid substrate is from about 450 pm to about 800 pm. In some embodiments, the thickness of the solid substrate is from about 500 pm to about 800 pm. In some embodiments, the thickness of the solid substrate is from about 550 pm to about 800 pm. In some embodiments, the thickness of the solid substrate is from about 600 pm to about 800 pm. In some embodiments, the thickness of the solid substrate is from about 650 pm to about 800 pm. In some embodiments, the thickness of the solid substrate is from about 700 pm to about 800 pm. In some embodiments, the thickness of the solid substrate is from about 750 pm to about 800 pm. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 750 pm. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 700 pm. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 650 pm. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 600 pm. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 550 pm. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 500 pm. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 450 pm. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 400 pm. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 350 pm. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 300 pm. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 250 pm. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 200 pm. In some embodiments, the thickness of the solid substrate is from about 100 pm to about 150 pm. In some embodiments, the thickness of the solid substrate is from about 300 mih to about 600 mih. In some embodiments, the thickness of the solid substrate is from about 400 mih to about 500 mih.

In some embodiments, the solid substrate has a height from about 0.01 centimeters to about 100 centimeters. In some embodiments, the solid substrate has a width or diameter from about 1 centimeter to about 15 centimeters.

In some embodiments, the solid substrate can comprise a lid defining an interior chamber comprising the volume and into which the three-dimensional tissue is positioned. In some embodiments, the tissue culture system can further comprise at least one gasket forming an airtight or semi-air tight seal between the interior chamber and the space outside the solid substrate.

In some embodiments, the solid substrate can comprise at least one inlet and at least one outlet, the inlet defining a space through which the first length of tubing and/or a first fluid linkage element connect the interior chamber to the reservoir in fluid communication and the outlet defining a space through which fluid exits the interior chamber. In some embodiments, the outlet defines a space through which a second length of tubing and/ or a second fluid linkage element connects the interior chamber and a point exterior to the solid substrate in fluid communication. In some embodiments, the outlet defines a space through which a second length of tubing and/ or a second fluid linkage element connects the interior chamber and the reservoir in a fluid circuit. Fluid linkage elements comprise an inert material that connect the three-dimensional tissue to the tubing. In some embodiments, the fluid linkage element is a cannula, catherter, Luer lock valve (e.g. with a screw cap), plastic valve or other similar device having a fluid channel through a plane and a lip protrusion or tapered end designed to receive tissue or tubing. In some embodiments, one end of the element can be positioned within the feeding arteriole or the exiting venule of the vasculature within the three-dimensional tissue and the opposing end can be positioned in fluid communication with a length of tubing such that the fluid channel of the linkage is aligned with and opening of the tubing and an opening of the vasculature.

In some embodiments, a tissue culture system can further comprise an incubator enclosing the solid substrate. The incubator can comprise a heating element that maintains temperature at or about 37 degrees Celsius. Some embodiments of the tissue culture system comprise

In one embodiment, the present disclosure provides a composition or three-dimensional tissue culture system made from harvested rat mesentery tissue containing blood vessels, lymphatic vessels, endothelial cells, smooth muscle cells, and pericytes, providing a physiologically relevant in-vitro tissue model for research and drug development, evaluation, and design. The tissue culture model can be cannulated and perfused, providing flow through the tissue model thus providing even more relevant physiological data. Shear stress caused by blood flow is known to play an important role in endothelial cell morphology and proliferation as well as angiogenesis.

The disclosure also relates to a composition or tissue culture system comprising:

(i) a solid substrate or vessel comprising an internal surface and an outer surface, at least one contiguous region of the internal surface defining an interior volume;

(ii) a three-dimensional tissue from a mammalian mesentery or mammalian

mesometrium, the tissue comprising a plurality of endothelial cells;

(iii) a volume of cell medium sufficient to submerge the three-dimensional tissue in the vessel;

(iv) a reservoir of cell medium.

(v) at least a first length of tubing in fluid communication with the reservoir and the three-dimensional tissue. In some embodiments the tubing connects the reservoir of cell medium to the three-dimensional tissue. In some embodiments, the tissue culture system or composition comprises a pump operably linked to a fluid circuit connecting the reservoir with the three- dimensional tissue by the tubing, such that the pump supplies force for microfluidic flow within the fluid circuit. In some embodiments, the composition or tissue culture system comprises a waste collection unit in fluid communication with a second length of tubing connecting an exiting venule of the three-dimensional tissue. In such embodiments, microfluidic flow of fluid, such as cell medium can be pumped through a first length of tubing from the reservoir into a feeding arteriole of the three-dimensional tissue, through the micrvasculature of the three- dimensional tissue and out into the second length of tubing into the waste collection unit. In some embodiments, this is a close system. In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 25 dynes per square centimeter. In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 25 dynes per square centimeter.

In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 24 dynes per square centimeter. In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 23 dynes per square centimeter. In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 22 dynes per square centimeter. In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 21 dynes per square centimeter. In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 20 dynes per square centimeter. In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 19 dynes per square centimeter. In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 18 dynes per square centimeter. In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 17 dynes per square centimeter. In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 16 dynes per square centimeter. In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 15 dynes per square centimeter. In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 12 dynes per square centimeter. In some embodiments, the compositions or tissue culture systems disclosed herein generate shear stress force within the fluid system such that stress force does not exceed 10 dynes per square centimeter of fluid channel.

The disclosure also relates to to a composition or tissue culture system comprising:

(ii) a three-dimensional tissue from a mammalian mesentery or mammalian

mesometrium, the tissue comprising a plurality of endothelial cells;

(iii) a volume of cell medium sufficient to submerge the three-dimensional tissue in the vessel; (iv) a reservoir of cell medium.

(v) at least a first length of tubing in fluid communication with the reservoir and the three- dimensional tissue, wherein the density of capillaries within the vasculature of the three- dimensional tissue does not exceed 500 capillaries per square millimeter of tissue. In some embodiments, the density of capillaries within the vasculature of the three-dimensional tissue does not exceed 475 capillaries per square millimeter of tissue. In some embodiments, the density of capillaries within the vasculature of the three-dimensional tissue does not exceed 450 capillaries per square millimeter of tissue. In some embodiments, the density of capillaries within the vasculature of the three-dimensional tissue does not exceed 425 capillaries per square millimeter of tissue. In some embodiments, the density of capillaries within the vasculature of the three- dimensional tissue does not exceed 400 capillaries per square millimeter of tissue. In some embodiments, the density of capillaries within the vasculature of the three-dimensional tissue does not exceed about 375 capillaries per square millimeter of tissue. In some embodiments, the density of capillaries within the vasculature of the three-dimensional tissue does not exceed about 350 capillaries per square millimeter of tissue. In some embodiments, the density of capillaries within the vasculature of the three-dimensional tissue does not exceed about 325 capillaries per square millimeter of tissue the density of capillaries within the vasculature of the three-dimensional tissue does not exceed about 300 capillaries per square millimeter of tissue. In some embodiments, the density of capillaries within the vasculature of the three-dimensional tissue does not exceed 310 capillaries per square millimeter of tissue. In some embodiments, the density of capillaries within the vasculature of the three-dimensional tissue does not exceed about 320 capillaries per square millimeter of tissue. In some embodiments, the density of capillaries within the vasculature of the three-dimensional tissue does not exceed about 340 capillaries per square millimeter of tissue the density of capillaries within the vasculature of the three-dimensional tissue does not exceed about 360 capillaries per square millimeter of tissue.

In another set of embodiments, the three-dimensional tissue can be from a rodent. In some embodiments, the three-dimensional tissue can be derived from rat or mouse mesentery or mesometrium. In some embodiments, the present disclosure provides a three dimensional tissue culture system made from harvested, induced vascular, mouse mesentery tissue containing one or a combination of blood vessels, lymphatic vessels, endothelial cells, smooth muscle cells, neural cells and pericytes, providing a physiologically relevant in-vitro tissue model for research and drug development, evaluation, and design. For example, the three-dimensional tissue can comprise one or a combination of live cells chosen from: pericytes, immune cells, elongated endothelial cells, and blood cells. In some instances, the three-dimensional tissue comprises a plurality of pericytes and/or immune cells and/or blood cells. In some instances, the three-dimensional tissue is free of intestinal endothelial or epithelial cells. The tissue culture model can be cannulated and perfused, providing flow through the tissue model thus providing even more relevant physiological data. Shear stress caused by blood flow is known to play an important role in endothelial cell morphology and proliferation as well as angiogenesis.

In another embodiment, the present disclosure provides a three dimensional tissue culture system made from harvested mouse mesometrium tissue containing blood vessels, lymphatic vessels, endothelial cells, smooth muscle cells, and pericytes, providing a physiologically relevant in-vitro tissue model for research and drug development, evaluation, and design. For example, the three-dimensional tissue can comprise one or a combination of live cells chosen from: pericytes, immune cells, elongated endothelial cells, and blood cells. In some instances, the three- dimensional tissue comprises a plurality of pericytes and/or immune cells and/or blood cells. In some instances, the three-dimensional tissue is free of intestinal endothelial or epithelial cells. The tissue culture model can be cannulated and perfused, providing flow through the tissue model thus providing even more relevant physiological data. Shear stress caused by blood flow is known to play an important role in endothelial cell morphology and proliferation as well as angiogenesis.

In some embodiments, the three-dimensional tissue comprises an upper and bottom surface with the least one vasculature positioned in between the upper and bottom surfaces; and a portion of the upper and bottom surfaces comprising a translucent connective tissue through which the at least one vasculature is positioned; wherein the vasculature comprises at least one arteriole and at least one veinule pair positioned across at least a portion of the translucent connective tissue. In some embodiments, the three-dimensional tissue is maintained at about 37 degrees Celsius. In some embodiments, the three-dimensional tissue can be exposed to no more than about 5% carbon dioxide.

In some embodiments, the three-dimensional tissue can be exposed to fluid flow through its interior at a physiologically effective rate. In some embodiments, the three-dimensional tissue can be free of steady or oscillatory shear stress magnitudes greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 dynes per centimeter squared of tissue from fluid flow through the tissue. In some embodiments, a tissue culture system can further comprise a pump in operable connection to the tubing or the reservoir. In some instances, the pump can create fluid flow of cell medium across a surface of the three-dimensional tissue in a rate and volume sufficient to maintain the viability of the endothelial cells in the vasculature. In some instances, the pump can create fluid flow of cell medium across a surface of the three-dimensional tissue in a rate and volume sufficient to maintain the viability of the endothelial cells in the vasculature for no less than about 5, 10, 60, 120, 240, 480 minutes. In some instances, the pump can create fluid flow of cell medium across a surface of the three-dimensional tissue in a rate and volume sufficient to maintain the viability of the endothelial cells in the vasculature for no less than about 12 hours. In some instances, the pump can create fluid flow of cell medium across a surface of the three-dimensional tissue in a rate and volume sufficient to maintain the viability of the endothelial cells in the vasculature for no less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 14, 15 days. In some instances, the pump creates fluid flow of cell medium across a surface of the three-dimensional tissue at a rate and in a volume sufficient to maintain the viability of the endothelial cells in the vasculature, immune cells in the tissue, and pericytes in the tissue for no less than about 5, 10, 60, 120, 240, 480 minutes. In some instances, the pump creates fluid flow of cell medium across a surface of the three- dimensional tissue in at a rate and in a volume sufficient to maintain the viability of the endothelial cells in the vasculature, immune cells in the tissue, and pericytes in the tissue for no less than about 12 hours. In some instances, the pump creates fluid flow of cell medium across a surface of the three-dimensional tissue at a rate and in a volume sufficient to maintain the viability of the endothelial cells in the vasculature, immune cells in the tissue, and pericytes in the tissue for no less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 14, 15 days. In some embodiment, the cell medium can be pumped across the three-dimensional tissue at no less than about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 microliters per second

In some embodiments, a tissue culture system can further comprise a fluid linkage element operably linking the first length of tubing and a feeding arteriole within the three-dimensional tissue. In some embodiments, the fluid linkage element comprises or consists essentially of a cannula.

In some embodiments, a tissue culture system can further comprise a first and second fluid linkage element, the first fluid linkage element connecting the first portion of tubing to the three- dimensional tissue at one end of the vasculature and the second fluid linkage element connecting the three-dimensional tissue to a second length of tubing, such that the fluid linkage elements create a closed fluid system among the three-dimensional tissue, the reservoir and the first and second length of tubing. In some embodiments, the first and second fluid linkage elements can be cannulas.

In some embodiments, a tissue culture system can further comprise a valve and/or adapter in fluid communication with the first length of tubing, wherein the valve comprises at least a first and second operable condition; wherein, in a first operable condition the valve is closed preventing introduction of fluid into the three-dimensional tissue; and wherein, in the second operable condition, the valve is at least partially open allowing introduction of fluid into the three dimensional tissue. In some embodiments, a tissue culture system can further comprise a valve and/or adapter in fluid communication with the volume and/or interior chamber, wherein the valve comprises at least a first and second operable condition; wherein, in a first operable condition the valve is closed preventing introduction of fluid into the volume and/or interior chamber of the solid substrate; and wherein, in the second operable condition, the valve is at least partially open allowing introduction of fluid into the volume and/or the interior chamber.

In another embodiment of the invention, vascularization of the mouse from which mesentery is to be harvested will be induced by injecting the mice with organic sunflower seed oil (Spectrum), 10 mg/mL of tamoxifen T5649 (Sigma-Aldrich), and/or VEGF.

In one embodiment of the invention, the perfusion system can be a closed loop and single cannulated system. In this embodiment, the main feeding arteriole of the tissue is cannulated and fluid is allowed to flow through the vasculature and drain out of the tissue. The tissue is secured in the bioreactor and placed into a culture chamber. The flow is generated by a delivery pump from the perfusate reservoir monitored by a pressure sensor positioned on the arteriole inlet side. Flow will pass through the vasculature and exit into the culture chamber. Excess fluid will flow into the perfusate reservoir to be recycled through the tissue again, hence the closed-loop system. The data acquisition will collect recordings from the pressure sensor for monitoring. The filter in the perfusate reservoir is to help maintain sterility of the fluid.

In another embodiment of the invention, the perfusion system can be a closed-loop double cannula perfusion system where the main feeding arteriole and venule of the tissue is cannulated to allow flow to enter through the arteriole side and exit through the venule side of the vasculature. The tissue is secured in the bioreactor and placed into a culture chamber. The flow is generated by a delivery pump from the perfusate reservoir monitored by a pressure sensor positioned on the arteriole inlet side. Flow will pass through the vasculature and exit into the perfusate reservoir to be recycled through the tissue again, hence the closed-loop system. The data acquisition will collect recordings from the pressure sensor for monitoring. The filter in the perfusate reservoir is to help maintain sterility of the fluid. In another embodiment of the invention, the perfusion system can be an open-loop single cannula perfusion system where the main feeding arteriole of the tissue is cannulated and fluid is allowed to flow through the vasculature and drain out of the tissue. The tissue is secured in the bioreactor and placed into a culture chamber. The flow is generated by a delivery pump from the perfusate reservoir monitored by a pressure sensor positioned on the arteriole inlet side. Flow will pass through the vasculature and exit into the waste reservoir to be discarded, hence the open-loop system. The data acquisition will collect recordings from the pressure sensor for monitoring. The filter in the perfusate reservoir is to help maintain sterility of the fluid.

In another embodiment of the invention, the perfusion system can be an open-loop double cannula perfusion system where the main feeding arteriole and venule of the tissue is cannulated to allow flow to enter through the arteriole side and exit through the venule side of the vasculature. The tissue is secured in the bioreactor and placed into a culture chamber. The flow is generated by a delivery pump from the perfusate reservoir monitored by a pressure sensor positioned on the arteriole inlet side. Flow will pass through the vasculature and exit into the waste reservoir to be discarded, hence the open-loop system. The data acquisition will collect recordings from the pressure sensor for monitoring. The filter in the perfusate reservoir is to help maintain sterility of the fluid.

In another embodiment, the culture system maintains pH and temperature, eliminating the need for an incubator.

In another embodiment, a gravity driven flow is utilized in the system, eliminating the need for a pump to drive flow through the tissue

In one embodiment, the bioreactor (i.e. the solid substrate) disclosed is created by laser cutting the top and base piece of plastic. Threaded rods are screwed through the base piece. PDMS O-rings are made using a circular punch. The bioreactor is assembled in a“sandwich” fashion where the different pieces stack on top of each other. The threaded rods and knurled nuts are used to apply pressure perpendicular to the assembly to hold in place. An expanded side view of the assembly is also disclosed.

In one embodiment, the perfusion reactor disclosed includes the bioreactor within the culture chamber placed inside an incubator to maintain temperature and pH levels. The pump will deliver flow through the system and can be either external or internal to the incubator. In some embodiments, the pH levels are from about 6.9 to about 7.6. In some embodiments, the pH of the fluid in the system is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or above.

Methods

Disclosed are methods of measuring angiogenesis or capillary sprouting comprising exposing any tissue disclosed herein in culture to an agent; and measuring growth of vasculature or capillary sprouting in the tissue before and after exposure to the agent. In some embodiments, the method can further comprise a step of harvesting any of the disclosed three-dimensional tissues prior to performing a step of exposing the three-dimensional tissue to a therapeutic. In some embodiments, the method can further comprise a step of correlating the presence absence or quantity of growth of the vasculature or capillary to effect of the agent; and characterizing the agent as promoting angiogenesis if the agent stimulates vasoreactivity, growth and/or density of vasculature or if the agent stimulates sprouting of capillaries.

Also disclosed are methods of evaluating the toxicity of an agent comprising culturing one or plurality of a mesenteric and/or a mesometrial tissue in any of the compositions or systems described herein; exposing at least one agent to the one or more cells; measuring and/or observing one or more morphometric changes of the one or more mesenteric and/or a mesometrial tissue; and correlating one or more morphometric parameters of the one or more mesenteric and/or a mesometrial tissues with the toxicity of the agent, such that, if the morphometric parameters are indicative of decreased cell viability, the agent is characterized as toxic and, if the morphometric parameters are indicative of unchanged or positive cell viability, the agent is characterized as non toxic. In some instances, at least one agent can comprise a small chemical compound. For example, the at least one agent comprises one or a combination of small chemical compounds chosen from: chemotherapeutics, analgesics, cardiovascular modulators, cholesterol, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatories, and anti-microbial drugs. In some instances, the at least one agent comprises at least one environmental pollutant.

The present disclosure also relates to a method of evaluating the relative degree of toxicity of a first agent as compared to a second agent comprising: (a) culturing one or more three- dimensional tissues in any of the compositions or tissue culture systems disclosed herein; (b) exposing a first agent and a second agent to the one or more three-dimensional tissues in sequence or in parallel time periods (in sequence if on the same set of tissues or in parallel if on a second set of tissues— for instance, in a multiplexed system); (c) measuring and/or observing one or more morphometric changes of the one or more three-dimensional tissues; and (d) correlating one or more morphometric changes of the one or more three-dimensional tissues with the toxicity of the first agent; and (e) correlating one or more morphometric changes of the one or more three- dimensional tissues with the toxicity of the second agent; and (f) comparing the toxicities of the first and second agent; and (g) characterizing the first or second agent as more toxic or less toxic than the second agent. In some embodiments, when characterizing the first or second agent as more toxic or less toxic than the second agent, if the morphometric changes induced by the first agent are more severe and indicative of decreased cell viability to a greater extent than the second compound, the first agent is more toxic than the second agent; and, if the morphometric changes induced by the first agent are less severe and/or indicative of increased cell viability as compared to the second compound, then the second agent is more toxic than the first agent. The same characterization can be applied in embodiments in which electrophysiological metrics are observed and/or measured. Rather than observe cell viability, one can also observe tissue vascularization as a function of the presence or absence of one or a plurality of agents.

In some embodiments, the degree of toxicity is determined by repeating any one or more of the steps provided herein with one or a series of doses or amounts of an agent. Rather than comparing or contrasting the relative toxicities among two different agents, one of skill in the art can this way add varying doses of the same agent to characterize when and at what dose the agent may become toxic to the one or plurality of three-dimensional tissues.

The present disclosure also relates to a method of evaluating the toxicity of an agent comprising: (a) culturing one or more three-dimensional tissues in any of the compositions or systems disclosed herein; (b) exposing at least one agent to the one or more three-dimensional tissues; (c) measuring and/or observing one or more recordings of the one or more three- dimensional tissues; and (d) correlating one or more recordings of the one or more three- dimensional tissues with the toxicity of the agent, such that, if the recordings are indicative of decreased cell viability, the agent is characterized as toxic and, if the recordings are indicative of unchanged or increased cell viability, the agent is characterized as non-toxic; wherein step (c) optionally comprises and/or observing one or more morphometric changes of the one or more three-dimensional tissues; and wherein step (d) optionally comprises correlating one or more morphometric changes of the one or more three-dimensional tissues with the toxicity of the agent, such that, if the morphometric changes are indicative of decreased cell viability, the agent is characterized as toxic and, if the morphometric changes are indicative of unchanged or increased cell viability, the agent is characterized as non-toxic.

Also disclosed are methods of inducing growth of one or a plurality of cells in a three- dimensional tissue comprising contacting one or a plurality of isolated mesenteric or mesometrial tissues of any of claims 1 through 34 with the solid substrate, said solid substrate comprising at least one exterior surface, at least one interior surface and at least one interior volume defined by the at least one interior surface and accessible from a point exterior to the solid substrate through at least one opening; positioning one or a plurality of any of the isolated mesenteric or mesometrial tissues described herein to the at least one interior volume; applying a cell medium into the culture vessel with a volume of cell medium sufficient to cover the at least one interior volume; affixing at least a one or a plurality of fluid linkage elements to at least the first vasculature. In some embodiments, the methods can further comprise exposing one or plurality of isolated mesenteric or mesometrial tissues with at least one agent. In some instances, the at least one agent can comprise one or a combination of small molecules chosen from: chemotherapeutics, analgesics, cardiovascular modulators, cholesterol, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatories, and anti-microbial drugs. In some instances, the at least one agent can be one or a plurality of stem cells or modified T cells. In some embodiments, the methods can further comprise monitoring growth of cells after exposure of the tissue to one or a plurality of agents.

Also disclosed are methods of detecting and/or quantifying cell mobility in vitro comprising culturing one or a plurality of mesenteric or mesomentrial tissues of any of claims 1 through 34 in any of the systems disclosed herein; and exposing the tissue or tissues to a known number of cells in the composition after a culturing for a time period sufficient to allow cell migration of the one or plurality of cells. In some embodiments, the methods can further comprise measuring a recording, distance of migration or cell-to-cell interaction between the cell or plurality of cells and the tissue after the step of exposing the tissue or tissues to a known number of cells in the composition. In some embodiments, the methods can further comprise the step of detecting an internal and/or external recording of such one or more cells after culturing one or more tissues and correlating the recording with a measurement of the same recording corresponding to a known or control number of cells. In some embodiments, the methods can further comprise contacting the one or more tissues to one or more agents. In some embodiments, the methods further comprise a step comprising measuring an internal and/or external recording before and after the step of contacting the one or more tissues to one or more agents; and correlating the difference in the recording before contacting the one or more tissues to the one or more agents to the recording after contacting the one or more tissues to one or more agents. In some instances, the recording can be a distance between where the cell or cells were introduced or exposed into the system and where the cell or cells were positioned after allowing a time period sufficient to for the cell or cells to migrate through the tissue.

In some embodiments, the methods relate to allowing the cells to migrate for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more hours. In some embodiments, the methods comprise allowing for a time period of no more than 48 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than 48 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 36 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 24 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 20 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 18 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 16 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 14 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 12 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 10 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 8 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 6 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 5 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 4 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 3 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 2 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 1 hours before measuring a recording. In some embodiments, the methods comprise allowing for a time period of no more than about 8 hours before measuring a recording.

Also disclosed are methods of measuring vasculature growth within a tissue comprising positioning one or a plurality of tissues in any of the compositions or tissue culture systems disclosed herein; quantifying the one amount or density of vasculature in the one or plurality of tissues; contacting the one or plurality of tissues to one or a plurality of agents; and quantifying the number or density of vasculaure after contacting the one or plurality of tissues to one or a plurality of agents; and calculating the difference in the number or density of vasculature prior to and after the step of contacting the one or plurality of three-dimensional tissues to one or a plurality of agents. In some embodiments, the step of quantifying comprises staining the one or plurality of tissues. The staining can be performed using fluorescence or chemiluminesence or any known technique in the art. In some embodiments, the steps of quantifying the one amount or density of vasculature in the one or plurality of tissues, quantifying the number or density of vasculature after contacting the one or plurality of tissues to one or a plurality of agents, and/or calculating are performed via microscopy or digital imaging.

Also disclosed are methods of measuring intracellular or extracellular recordings comprising culturing one or a plurality of tissues in any of the composition or tissue culture system disclosed herein; measuring a recording across the one or a plurality of three-dimensional tissues. In some embodiments, the step of measuring comprises staining the one or plurality of tissues. The staining can be performed using fluorescence or chemiluminesence or any known technique in the art. In some embodiments, the step of measuring can be performed via microscopy or digital imaging. Also disclosed are methods of real-time imaging of tissue comprising culturing tissue within the tissue culture system described herein; and exposing the tissue culture system to digital imaging. In some embodiments, the method can further comprise exposing one or a plurality of isolated mesenteric or mesometrial tissues with at least one agent. In some instances, the at least one agent comprises one or a combination of small molecules chosen from: chemotherapeutics, analgesics, cardiovascular modulators, cholesterol, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatories, and anti-microbial drugs. In some instances, the at least one agent is one or a plurality of stem cells or modified T cells. In some embodiments, the methods can further comprise monitoring growth of cells in the tissue after exposure of the tissue to one or a plurality of agents. Monitoring the growth of cells can be done using any known technique in the art.

Also disclosed are methods of making any of the tissue culture systems disclosed herein comprising forming an interior chamber within a solid substrate; affixing mesenteric or mesomentiral tissue from a subject to the solid substrate; positioning at least one fluid linkage element into the tissue in fluid communication with a feeding arteriole; culturing the tissue in cell culture medium at about 37 degrees Celsius; attaching at least a first length of tubing to the at least first fluid linkage element; placing a reservoir of cell medium in fluid connection with at least the first length of tubing; placing a pump in operable connection to the at first length of tubing; and, optionally sealing the tissue within the solid substrate, such that the tissue is positioned within an internal cavity of the solid substrate in fluid communication with the reservoir. In some embodiments, the method can further comprise harvesting mesenteric or mesomentrial tissue from a subject prior to the step of affixing. In some embodiments, the methods can further comprise positioning at least a second fluid linkage element into the tissue in fluid communication with an exitng venule in the tissue; attaching at least a second length of tubing to the at least second fluid linkage element; and placing the second length of tubing in fluid communication with the reservoir and the tissue. In some instances, the tissue culture system can be an open or closed system. For example, it can be any of the open or closed systems described herein. In some embodiments, the methods can further comprise introducing fluid flow through the tissue. In some embodiments, the methods can further comprise attaching one or more valve and/or adapters in fluid communication with the first length of tubing, wherein the valve comprises at least a first and second operable condition; wherein, in a first operable condition the valve is closed preventing introduction of fluid into the three-dimensional tissue; and wherein, in the second operable condition, the valve is at least partially open allowing introduction of fluid into the three dimensional tissue. In some embodiments, the methods can further comprise attaching a syringe to the adaptor or valve such that the syringe becomes part of a fluid circuit within the system.

Also disclosed are methods for testing the efficacy of a test substance comprising: exposing a three dimensional tissue comprising mesentery or mesomentrial cells to the test substance, in which the three dimensional cell culture comprises tissue secured to a solid substrate and in a culture chamber; and determining the effect of the test substance by measuring or observing a change in the three dimensional cell culture. In some embodiments, the tissue comprises rat or mouse mesentery or rat or mouse mesometrium.

Also disclosed are methods of producing a tissue culture, in-vitro model of rat mesentery tissue wherein the tissue comprises blood and lymphatic microvascular networks, endothelial cells, smooth muscle cells, immune cells, neural cells, and pericytes, the method comprising harvesting rat mesentery tissue from a rat and securing it to a solid substrate. In some embodiments, the step of harvesting comprises exteriorizing the mesentery from the gut of the animal and harvesting the mesenteric window. In some embodiments, the rat can be chosen from one or a combination of: a transgenic rat, an aged rat and an immunocompromised rat. In some embodiments, the methods can further comprise cannulating the tissue to allow perfusion through the tissue into an arteriole or vein while the mesentery is exteriorized and secured with sutures on the cannulated blood vessel. In some embodiments, the methods can further comprise securing the tissue in a bioreactor or incubator and attaching tubing to one or a plurality of blood vessels within the tissue. In some embodiments, the methods can further comprise placing the bioreactor in a culture chamber, operably connecting the bioreactor and tubing to a pump; and operating the pump for a time sufficient to create physiologically effective perfusion of the tissue.

Also disclosed are methods of printing cells on any one of the tissue culture systems described herein; and evaluating the cells wherein the evaluating comprises tracking growth and/or interactions of the cells within the tissue culture system.

Also disclosed are methods of producing a tissue culture, in-vitro model of mouse mesentery tissue comprising blood and lymphatic microvascular networks, endothelial cells, smooth muscle cells, immune cells, neural cells, and pericytes, the method comprising inducing vascularization of mouse mesentery by injecting said mouse with tamoxifen, sunflower oil, of VEGF for 5 consecutive days before harvesting the mouse mesentery. In some embodiments, the mouse mesentery can be free of endothelial cells from intestinal walls of the mouse. In some embodiments, the methods can further comprise harvesting the mesentery tissue by exteriorizing the mesentery from the gut of the animal and harvesting the mesenteric window. In some embodiments, the mouse can be one or a combination of a transgenic mouse, an aged mouse, and an immunocompromised mouse. In some embodiments, the methods can further comprise cannulating the tissue to allow perfusion through the tissue and securing a first and/or second cannula with sutures. In some embodiments, the methods can further comprise placing the tissue into a bioreactor and attaching tubing to the first and or second cannula.

Also disclosed are methods of manufacturing a tissue culture, in-vitro model of mouse mesometrium tissue comprising blood and lymphatic microvascular networks, endothelial cells, smooth muscle cells, immune cells, neural cells, and pericytes, the method comprising harvesting the mouse mesometrium and securing the mouse mesometrium to a solid substrate. The solid substrate can be any of the solid substrates described herein. In some embodiments, the tissue is secured to the solid substrate using a staple, suture, clamp or tie. In some embodiments, the tissue is secure or affixed to the slide by physical contact of one or a plurality of inserts that fits within or snaps into the solid substrate by contact by the insert with one or a portion of the sidewalls. In some embodiments, the harvesting comprises exteriorizing the mesentery from the gut of the animal and harvesting the mesenteric window. In some embodiments, the mesometrium is free of uteran or ovarian cells. In some embodiments, the mouse can be one or a combination of mice chosen from: a transgenic mouse, an aged mouse, and an immunocompromised mouse. In some embodiments, the methods can further comprise cannulating the tissue by introducing a cannula into an arteriole or vein while the mesometrium is exteriorized and securing the cannula to the tissue with sutures. In some embodiments, the methods can further comprise placing the tissue in a bioreactor and attaching at least a first length of tubing to the cannula. In some embodiments, the methods can further comprise operably attaching a pump to the tubing and perfusing cell culture medium through the tissue.

Any and all journal articles, patent applications, issued patents, or other cited references disclosed herein are incorporated by reference in their respective entireties. EXAMPLE 1 - Inducing Microvascular Growth in Mouse Mesentery Tissue

All animal experiments were approved by Tulane University’s Institutional Animal and Care Use Committee. The procedure followed is a common protocol for tamoxifen induction in genetically engineered mice. Briefly, adult, female C57BL/6 mice were given a single intraperitoneal (IP) injection daily for 5 consecutive days. All injections were 0.1 mL in volume and warmed to 37°C. The three experimental groups receiving injections were as follows: Saline, Oil, and Tamoxifen (n = 4 mice per group). Sterile saline (Baxter), organic sunflower seed oil (Spectrum), and 10 mg/mL of tamoxifen T5649 (Sigma-Aldrich) were utilized. In a 1 mL centrifuge tube, 5 mg of tamoxifen and 0.5 mL of organic sunflower seed oil were added. Tamoxifen was dissolved by placing the tube on a shaker inside an oven set to 37°C for 2 hours. Mesentery tissues were harvested 21 days after the last injection. The mice were euthanized in a C02 chamber by asphyxiation followed by cervical dislocation. Similar to a previously established protocol by Stapor et al., 2013, the mesentery tissues were harvested. Briefly, the abdominal fur was shaved before sterilizing the abdomen with 70% isopropyl alcohol followed by 3 wipes with iodine. The abdominal skin and muscles were cut off with sterile scissors to expose the abdominal cavity. Using sterile techniques, the ileum was located as a reference point, and the mesentery was carefully spread on a sterile drape to expose the individual windows. Sterile saline, warmed to 37°C was dripped on the tissue to keep it from drying out. Utilizing microscissors and forceps, mesenteric windows were aseptically harvested. Once removed, most tissues were rinsed in phosphate buffered saline (PBS) and immediately fixed in 100% methanol. The tissues to be cultured were rinsed in sterile Dulbecco’s phosphate buffered saline (DPBS), and transferred to sterile minimum essential media containing Earle’s Salts (MEM; Gibco) and 1% Penicillin- Streptomycin (PenStrep; Gibco) warmed to 37°C. In a biosafety cabinet, the tissues were transferred to a 6-well culture plate with 4 mL of culture media in each well. Culture media contained sterile MEM, 1% PenStrep, and 20% fetal bovine serum (FBS). Tissues were then placed inside an incubator set to standard cell culture conditions (5% C02, 37°C) for 3 days, where the media was changed every 24 hours.

Immunohistochemistry: Before labeling, tissues were spread on a microscope slide and fixed in 100% methanol at - 20°C for 30 minutes. Briefly, all antibodies were diluted in an antibody buffer solution containing PBS, 0.1% saponin, and 2% bovine serum albumin. Tissues were incubated with all primary and secondary antibodies for 1 hour at room temperature. Tissues were then rinsed for 10 minutes three times with PBS and 0.1% saponin cooled to 4°C. The following antibodies were used to label the tissues: 1 :200 mouse platelet endothelial cell adhesion molecule (PECAM; BD Biosciences) with 1 :500 streptavidin secondary (Strep-CY2), 1 :200 CY3- conjugated a-smooth muscle actin (aSMA, Sigma-Aldrich), 1 : 100 rabbit neuron-glial antigen 2 (NG2, Millipore) with 1 : 100 goat anti -rabbit secondary (GAR-CY3), and 1 : 100 rabbit lymphatic vessel endothelial hyaluronan receptor- 1 (LYVE-l, AngioBio) with 1 : 100 GAR-CY3.

Quantification of Microvascular Growth. All quantifications were performed via blind analysis. Using the Java-based NIH ImageJ v. l .O image processing software, the percentage of vascularized tissue area and tissue density of the mesentery tissues were determined. The vascular area of each tissue was measured by outlining the perimeter of the vasculature within the window. The outline included open-ended vascular segments, and excluded vessels in the adipose border. The tissue area was measured 7 by outlining the entire window excluding the adipose border. The areas were calculated in mm2 based on the pixel to mm ratio of the image. The vascular area was then divided by the tissue area and converted to a percentage to calculate the vascularized tissue area. For tissue density, the number of segments per vascular area of a distinct network in the tissue was calculated. A segment was defined as the length of blood vessel between two nodes, defined as the branching points in the vascular network, where the segments do not have blind ends. A distinct network constituted of an arteriolar and venular pair with its corresponding capillaries. To differentiate between arterioles and venules, the diameter and morphology of endothelial cells were examined as arterioles have smaller diameters and more elongated endothelial cells due to their higher shear stress compared to venules. Statistical Analysis Data are presented as mean ± standard error of mean (SEM) and analyzed with one-way Analysis of Variance (ANOVA) test followed by Tukey’s multiple comparisons test to identify differences between the three groups. GraphPad Prism version 7 software was used for all statistical analyses. Statistical significance was achieved when p-value < 0.05.

After injecting mice with saline, oil or tamoxifen for 5 consecutive days and tissue harvesting 21 days post-injection, the mesenteries from the saline group were still avascular, while the mesenteries from the oil and tamoxifen groups contained complete microvascular networks. Here, we define the‘mesenteric windows’ as the thin, translucent connective tissue located between artery/vein pairs that feed the small intestine. The PEC AM-positive endothelial cells lined the microvascular networks which contained a distinct hierarchy with branched arterioles, venules and capillaries. Vessel types could be identified based on their endothelial cell morphologies and vessel diameters (Murfee et al. 2005, Yang et al. 2011). Every single mesenteric window from the oil (8/8) and tamoxifen (8/8) groups contained microvascular networks with arterioles, venules, and capillaries. In contrast, zero mesenteric windows (0/8) from the saline group contained a branching microvascular network. The percentages of vascularized tissue area or the oil and tamoxifen group were dramatically increased compared to avascular saline group (Saline: 0.0462 ± 0.0462%; Oil: 28.3 ± 8.61%, p = 0.0338; Tamoxifen: 35.6 ± 9.46%, p = 0.0073). A similar trend was observed when the tissue density was compared. The number of segments per vascular area were significantly higher for the oil and tamoxifen compared to saline (Saline: 4.19 ± 4.19 #/mm2 ; Oil: 290 ± 32.0 #/mm2 ; Tamoxifen: 275 ± 31.8 #/mm2 , p < 0.0001). Importantly, the heterogeneous size for the networks across the oil and tamoxifen groups were connected to vessels that originated from the periphery of the tissue suggesting ingrowth from the surrounding adipose border. The microvasculature that grew into the mesentery tissues also contained perivascular cells such as aSMA-positive smooth muscle cells and NG2 -positive pericytes. These two supporting cells displayed common wrapping morphologies and were present in their expected locations. Moreover, along with the vascularization of the mesentery, tissues also displayed the 9 growth of LYVE-l -positive lymphatics. Lymphatics were distinguished by LYVE-l- positive labeling, intensity of PEC AM, and endothelial cell morphology. Recently, the culturing of rat mesentery was introduced as a novel ex vivo model for investigating real-time cell dynamics involved in angiogenesis across the hierarchy of an intact network (Stapor et al. 2013, Yang et al. 2011). This motivated us to culture mouse mesentery tissues to observe whether angiogenesis can also be stimulated ex vivo, which would enable further experimental approaches using knockout and transgenic mice. Out results demonstrated that when mouse mesentery tissues from the oil or tamoxifen groups were cultured for 3 days in MEM supplemented with 20% FBS, angiogenesis, defined as capillary sprouts, was indeed observed. When compared to Day 0 uncultured tissues, Day 3 cultured tissues displayed more capillary sprouts which are blind-ended PECAM-positive endothelial cell segments protruding from existing microvessels.

Mesentery Tissue Culture Model Materials and Methods:

Surgical Procedure Setup 1. Autoclave instruments, surgical supplies, and culture supplies prior to surgery. Surgical supplies for each rat and/or mouse include: 1 drape, 1 drape with pre-cut hole (0.5 in x 1.5 in) in the center, gauze pads, and 1 absorbent underpad. Surgical instruments include: 1 scalpel with a number 10 blade, 2 pairs of tweezers, and a pair of fine scissors. Culture supplies include: 1 drape, 1 pair of tweezers, and prepared 6-well plate inserts with polycarbonate filters.

2. Sterilize a plexiglass platform, a surgical stage and a surgical benchtop space with 70% ethanol. Keep the surgical stage in a sterile bowl until use.

1. Create a surgical stage by drilling an approximately 2 in by 1 in hole in the center of a 100 mm culture dish. Next, use sandpaper to smooth any sharp edges and add a layer of silicone glue to the hole's edges to create a raised surface for the tissues.

2. Alternatively, design the surgical stage using CAD software and make by 3-D printing.

3. Place a sterile absorbent underpad down and lay a plexiglass platform on top of it. Place the drape, without a pre-cut hole, over a heated pad next to the absorbent underpad.

4. Pre-warm sterile phosphate-buffered saline (PBS), media and saline to 37 °C. Place media and PBS in separate culture dishes atop the heating pad and place saline in a 50 mL conical tube next to the surgical setup.

5. Make sure all packages are opened prior to the beginning of the surgery to ensure sterile handling of all materials.

Mesentery Tissue Harvesting

1. Use adult male Wistar rats (350 ± 25 g; 6 - 8 weeks of age). Other strains and ages of rats and mice can be substituted.

2. Anesthetize the rat via an intramuscular injection of ketamine (80 mg/kg body weight) and xylazine (8 mg/kg body weight). Confirm the rat is under anesthesia by pinching between the toes to check for a reflex response; there should be none. Pre-emptive analgesia for this terminal procedure is not necessary. If using mouse, euthanize by asphyxiation in a C02 chamber followed by cervical dislocation. 3. Shave the abdominal region and remove remaining hair using hair removal cream. Wipe abdominal skin twice with 70% isopropyl alcohol followed by povidone-iodine. For the wipes the surgeon should start at the center of the surgical site and move to the outside of the prepared area in a circular manner as to not overlap areas that have been previously scrubbed with the same piece of sterile gauze or sterile cotton swab. Then transfer animal to the sterile surgical setup and place atop the plexiglass platform.

4. Using a scalpel blade, make a 0.75 in - 1.25 in incision in the gut starting 1 in below the sternum. Be careful not to puncture the bowel or mesentery (1 layer of skin, 1 layer of connective tissue, and 1 layer of muscle).

5. Place a drape with a pre-cut hole over the incision and place a sterile surgical stage atop the drape. Ensure the opening aligns with the incision. Use sterile cotton-tipped applicators to locate and pull out the ileum through the surgical stage opening.

6. Pull 6 - 8 mesenteric windows through the stage using cotton-tipped applicators, and be careful not to touch the windows. Tissues are typically harvested from the ileum region of the small intestine starting near the cecum. Keep exposed tissues moist with warmed sterile saline as needed using a sterile syringe to drip the solution.

7. Euthanize the rat via intracardiac injection of pentobarbital sodium (0.2 mL per rat). Before removing mesenteric windows, ensure the rat is euthanized by palpating the heart; there should be no pulse.

8. Remove desired mesentery tissues by using tweezers to grab the fat pad and fine scissors to cut the window. Leave a border of fat (2 mm) around the window. Wash tissues once in warmed sterile PBS and once in media.

9. Return exteriorized ileum to the abdominal cavity and dispose of animal according to institutional guidelines.

Mesentery Tissue Culture for Time-Lapse Studies

1. Transfer autoclaved culture supplies (see section 1.1) and tissues to a sterile laminar flow hood.

2. Use tweezers to transfer each tissue atop a polycarbonate filter membrane. Grab tissues by the fat pad to avoid damaging the vasculature. 3. Quickly spread the tissue using the fat pad, being careful not to touch the window. Invert the insert with the tissue into the bottom of a 6-well plate and cover with 3 mL of media. Typical media used for this procedure includes Minimum Essential Media (MEM) with 1% Penicillin Streptomycin (PenStrep) and 10% Fetal Bovine Serum (FBS). Media can be supplemented with other serums and/or growth factors to stimulate angiogenesis and lymphangiogenesis.

4. Repeat steps 3.2 - 3.3 for each tissue and culture in standard incubator conditions (5% C02, 37 °C) for up to 5 days.

Time-Lapse Imaging of Mesentery Tissue

1. On the day of imaging, supplement the media in each well with conjugated BSI-Lectin and incubate under standard culture conditions for 30 min. Wash tissues twice with lectin-free media. BSI-Lectin stain will remain visible on the mesentery tissue for up to 3 days in culture.

2. Transfer the plate to a microscope stage. Identify blood and lymphatic vessels based on their morphology and network structure.

3. Locate a desired network region on each tissue and take images. Take note of the imaging location to ensure the same region will be captured for subsequent images. If using a motorized stage, document the coordinates.

4. Return tissues to the incubator and continue to culture until desired end point. Repeat steps 4.1 - 4.3 as needed depending on desired experimental time points.

5. Tissue Immunolabeling:

1. BSI-Lectin Labeling

1. Incubate tissues for 30 min at 37 °C with 1 :40 FITC-conjugated lectin in media (2.5 mL antibody solution per well in 6-well plate) followed by two rinses with media. For rinses, add media and then immediately replace.

2. Live/Dead Labeling

1. Incubate tissues for 10 min at 37 °C with 1 :500 2 mM ethidium homodimer-l and 1 :500 1 mM calcein AM in media (2.5 mL antibody solution per well in 6-well plate) followed by two rinses with media.

3. BSI-Lectin/NG2 Labeling

1. Spread tissues on a microscope slide (1 - 2 tissues/slide) and allow to dry. Remove excess fat with a scalpel by pressing down firmly to excise the fat. 2. Fix tissues in cold methanol for 30 min at -20 °C. Wash tissues with PBS (3 x 10 min).

3. For primary antibody labeling incubate tissues for 1 h at room temperature with 1 : 100 rabbit polyclonal NG2 antibody and 5% normal goat serum (NGS). Wash tissues with PBS (3 x 10 min).

4. For secondary antibody labeling incubate tissues for 1 h at room temperature with 1 : 100 goat anti-rabbit CY2-conjugated antibody (GAR-CY2) and 5% NGS. Wash tissues with PBS (3 x 10 min).

5. Incubate tissues for 30 min at room temperature with 1 :40 FITC- conjugated lectin in PBS followed by two rinses with PBS. For rinses, add PBS and then immediately replace.

6. To mount the slides, cover tissues with 50:50 PBS and glycerol solution and place coverslip on top. Seal the slide edges using nail polish.

4. L YVE- 1 /PEC AM Labeling

1. Spread tissues on a microscope slide (1 - 2 tissues/slide) and allow to dry. Remove excess fat with a scalpel by pressing down firmly to excise the fat.

2. Fix tissues in cold methanol for 30 min at -20 °C. Wash tissues with PBS + 0.1% saponin (3 x 10 min).

3. For primary antibody labeling incubate tissues for 1 h at room temperature with 1 :200 mouse monoclonal biotinylated CD31 antibody and 1 : 100 rabbit polyclonal LYVE-l antibody in PBS + 0.1% saponin + 2% bovine serum albumin (BSA) + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).

4. For secondary antibody labeling, incubate tissues for 1 h at room temperature with 1 :500 CY3 -conjugated streptavidin antibody and 1 : 100 GAR- CY2 in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).

5. To mount slides, cover tissues with 50:50 PBS and glycerol solution and place a coverslip on top. Seal the slide edges using nail polish.

5. BrdU/BSI-Lectin Labeling

1. Add 1 mg/mL BrdLT to media and replace tissue media with BrdLT solution. Incubate for 2 h at 37 °C. 2. Spread tissues on a microscope slide (1 - 2 tissues/slide) and allow to dry. Remove excess fat with a scalpel by pressing down firmly to excise the fat.

3. Fix tissues in cold methanol for 30 min at -20 °C. Wash tissues with PBS (3 x 10 min).

4. Denature tissue DNA in 2 M HC1 for 1 h at 37 °C. Wash tissues in PBS + 0.1% saponin (3 x 10 min).

5. For primary antibody labeling, incubate tissues for 1 h at room temperature with 1 : 100 monoclonal mouse anti-BrdU in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).

6. For secondary antibody labeling, incubate tissues for 1 h at room temperature with 1 : 100 goat anti-mouse Cy3 -conjugated antibody (GAM-Cy3) in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).

7. Incubate tissues for 30 min at room temperature with 1 :40 FITC- conjugated lectin in PBS followed by two rinses with PBS.

8. To mount slides, cover tissues with 50:50 PBS and glycerol solution and place coverslip on top. Seal the slide edges using nail polish.

6. BSI-Lectin/CDl lb labeling

3. For primary antibody labeling incubate tissues for 1 h at room temperature with 1 : 100 mouse anti-rat CDl lb in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).

4. For secondary antibody labeling incubate tissues for 1 h at room temperature with 1 : 100 GAM-Cy3 in PBS + 0.1% saponin + 2% BSA + 5% NGS. Wash tissues with PBS + 0.1% saponin (3 x 10 min).

5. Incubate tissues for 30 min at room temperature with 1 :40 FITC- conjugated lectin in PBS followed by two rinses with PBS. 6. To mount slides, cover tissues with 50:50 PBS and glycerol solution and place coverslip on top. Seal the slide edges using nail polish.

After 3 days in culture, tissues were labeled with a live/dead viability/cytotoxicity kit to demonstrate the viability of the microvasculature in the rat mesentery culture modelThe majority of cells present in the mesentery remained viable in the culture where endothelial cells were identified based on their location in microvascular segments. Endothelial cell proliferation was also confirmed by lectin/BrdU labeling. Smooth muscle cell and pericyte presence along vessels was confirmed with NG2 labeling. Labeling for LYVE1 and PECAM identified branching lymphatic and blood microvascular networks and confirmed the maintained lymphatic versus blood endothelial cell phenotype

The time-lapse feature of this model was utilized by labeling the microvascular networks with BSI-lectin at different time points and imaging the same region within the network over time; this capability is particularly valuable for investigating tissue specific angiogenic responses. The supplementation of media with 10% serum caused a robust angiogenic response after 3 days of stimulation. Additionally, new vessel segments and capillary sprouts were identified by day 5 of stimulation. The time-lapse imaging method allowed for the quantitative comparison of network regions before and after stimulation. For this representative study, which corroborates our previous results9, the number of vessels per vascular area and the number of capillary sprouts per vascular area were quantified from one 4X image per tissue. Blood vessel segments were defined as lectin positive blood endothelial cell segments present between two branch points and capillary sprouts were defined as blind ended segments originating from a host vessel. Time-lapse comparison of network regions also enabled tracking of endothelial cell segments and identification of blood/lymphatic vessel mis-patteming. Labeling of cultured tissues for lectin and CDl lb additionally confirmed the presence of interstitial resident macrophages in remodeling networks.

Mouse Mesometrium Tissue Harvesting and Culture Model

All animal experiments were approved by Tulane University’ s Institutional Animal and Care Use Committee. Adult, female C57BL/6 mice were euthanized by asphyxiation in a C02 chamber followed by cervical dislocation. The abdominal fur was shaved before sterilizing the abdomen with 70% isopropyl alcohol followed by 3 wipes with iodine. The abdominal skin and muscles were cut off with sterile scissors to expose the organs. Two forceps were utilized to move away all organs until the uterine horns were exposed. Each uterine horn was stretched to more clearly expose the mesometrium, the connective tissue attaching the uterine horns to the back wall, and then pinned down to facilitate harvesting. Sterile saline warmed to 37°C was dripped on tissue to prevent dehydration as needed. Using aseptic techniques, the mesometrium tissues were harvested with forceps and microscissors by pinching a side of the mesometrium and cutting around the uterine horn. Once removed, the tissues were immediately rinsed in sterile Dulbecco’s phosphate buffered saline (DPBS), and then transferred to sterile minimum essential media containing Earle’s Salts (MEM; Gibco) and 1% Penicillin-Streptomycin (PenStrep; Gibco) warmed to 37°C. The mesometrium tissues were transferred to a biosafety cabinet after harvesting. Using a 6-well culture plate, 4 mL of culture media was transferred into each well where a single tissue was placed. Cultured media was comprised of sterile MEM and 1% PenStrep. Tissues were then immediately placed inside an incubator set at standard cell culture conditions (5% C02, 37°C) for up to 7 days, where media was changed every 24 hours.

Immunohistochemistry

Tissues were first spread on a microscope slide and then fixed in methanol at -20°C for 30 min before labeling. The following antibodies were used to label the tissues: 1 :200 mouse platelet 5 endothelial cell adhesion molecule (PECAM; CD31) with 1 :500 streptavidin secondary (Strep- CY2), 1 :200 CY3 -conjugated a-smooth muscle actin (aSMA), 1 : 100 rabbit neuron-glial antigen 2 (NG2) with 1 : 100 goat anti -rabbit secondary (GAR-CY3), and 1 : 100 rabbit lymphatic vessel endothelial hyaluronan receptor-l (LYVE-l) with 1 : 100 GAR-CY3. Staining protocols were replicated as previously stated by Stapor et al., 2013. Briefly, all antibodies were diluted in antibody buffer solution which contained phosphate buffered saline (PBS) + 0.1% saponin + 2% bovine serum albumin. All primary and secondary antibodies were incubated for 1 hour at room temperature. After every antibody incubation, tissues were rinsed three times with cold PBS + 0.1% saponin for 10 min. Stimulation of Angiogenesis

To induce angiogenesis, tissues were cultured in sterile MEM supplemented with 20% fetal bovine serum (FBS; Gibco) or 400 ng/mL of recombinant mouse vascular endothelial growth factor-l64 (VEGF164; R&D Systems). Tissues were placed in the following groups: 1) Day 0 (n = 8 tissues from 4 mice), 2) Day 3 with 20% FBS (n = 8 tissues from 4 mice), 3) Day 3 without 20% FBS (n = 8 tissues from 4 mice), 4) Day 5 with 20% FBS (n = 8 tissues from 4 mice), 5) Day 5 without 20% FBS (n = 8 tissues from 4 mice), and 6) Day 5 with VEGF (n = 6 tissues from 3 mice). Tissues from day 0 group were fixed and labeled immediately after being excised.

Quantification of Angiogenesis

Using the Java-based NIH ImageJ v. l .O image processing software, capillary sprouts were quantified for the entire tissue. Capillary sprouts were defined as blind-ended PECAM-positive endothelial cell segments protruding from existing microvessels. The data was normalized per total vessel length. To differentiate between arterioles and venules, the diameter of the blood vessel and the morphology of endothelial cells were examined since arterioles are known to have smaller diameters and more elongated endothelial cells due to their higher shear stress compared to paired venules. All arterioles and venules that measured larger than 7 pm in diameter were analyzed.

Microscopy

Images were taken with a 4x (dry, NA = 0.1), lOx (dry, NA = 0.3), and 20x (oil, NA = 0.8; or air, NA = 0.75) objectives from an inverted microscope (Olympus 1X71) paired with a Photometries CoolSNAP EZ camera. Whole tissue images with PECAM labeling were acquired with a 4x or lOx objective to extrapolate vascular data. Time-lapse images were captured using the lOx (dry, NA = 0.3) objective.

Time-Lapse Imaging of Endothelial Cell Dynamics

Additional mesometrium tissues from adult, female FlkEGFP mice with a CD1 background were harvested using the same aseptic techniques. These transgenic mice were graciously provided by Dr. Meadows’ lab from the Department of Cell and Molecular Biology at Tulane University. In the biosafety cabinet, tissues were spread on a 1 pm polycarbonate filter fitted to a cell-crown insert (Sigma- Aldrich) and then inverted into a well from a 6-well culture plate with 4 mL of media supplemented with 20% FBS. The culture plate was immediately placed in the incubator, and media was changed every 24 hours. Images of the same venule were acquired with the lOx objective at least once every 24 hours for 7 days. For day 0 and 1, an image was captured every 24 hours; for day 2 and 3, images were captured every 4 hours; and for days 4-7, images were captured every 6 hours.

Statistical Analysis

Data are presented as mean ± standard error of mean (SEM). Angiogenesis data with serum was compared across experimental groups with two-way Analysis of Variance (ANOVA) 7 followed by Tukey’s multiple comparisons tests to notify differences between media and serum and between time periods. Angiogenesis data with VEGF was compared using an unpaired two tailed Student t-test. A p-value < 0.05 was considered statistically significant. Statistical analysis was executed using GraphPad Prism version 7 software.

Mouse Mesometrium Tissue Contains Intact Microvascular Networks

PEC AM-positive labeling of endothelial cells along the hierarchy of branched microvascular networks were present in the thin, connective mesometrium tissue of the uterine horns in female mice. In contrast, tissues harvested from the mouse mesentery were avascular as expected and displayed no evidence of PEC AM-positive labelled networks. Intact networks in the mesometrium were commonly characterized by branching arterioles, venules and capillaries. Vessel types were identified based on endothelial cell morphology and relative vessel diameters. Importantly, aSMA-positive smooth muscle cells and NG2-positive pericytes were present in their expected locations and displayed typical wrapping morphologies. Mesometrium tissues also demonstrated the presence of LYVE-l -positive lymphatics. Lymphatics were distinguished by the endothelial cell morphology, intensity of PEC AM, and LYVE-l -positive labeling. Angiogenesis Can be Stimulated in Cultured Mouse Mesometrium Tissue

Angiogenesis, defined as capillary sprouts, was observed in the mouse mesometrium tissues cultured in MEM supplemented with 20% FBS for 3 and 5 days. The number of PECAM- positive endothelial cell sprouts per total vascular length significantly increased for the groups supplemented with FBS for 3 (FBS: 3.19 ± 0.78 sprouts/mm, MEM: 0.60 ± 0.18 sprouts/mm, p < 0.0001) and 5 (FBS: 2.49 ± 0.05 sprouts/mm, MEM: 0.80 ± 0.13 sprouts/mm, p = 0.0033) days in culture when compared to the MEM control group. During culture, tissue viability was confirmed via Live/Dead assay. MEM supplementation with VEGF was also sufficient to induce an increase in the number of PEC AM-positive endothelial cell sprouts per total vascular length after 5 days in culture, (MEM:0.80 ± 0.13 sprouts/mm, MEM + VEGF: 2.35 ± 0.57 sprouts/mm, p = 0.0031). Capillary sprouts preferentially originated off venules compared to arterioles in all the angiogenic groups which is consistent with in vivo observations. While capillary sprout density was comparable at day 3 and 5, the length of capillary sprouts qualitatively appeared to be longer in day 5 tissues.

An important aspect for establishing the mouse mesometrium culture model is to investigate pericyte-endothelial cell interactions, which was supported by the angiogenic capillary sprouts covered by NG2-positive pericytes. Moreover, the identified NG2 and aSMA labeling was able to maintain phenotypic dynamics comparable to in vivo. In previous work, NG2 was characterized as an arteriole versus venule smooth muscle cell marker in quiescent tissues and was observed to be upregulated along venules during angiogenesis. Similar NG2 labeling patterns were observed in non-cultured mouse mesometrium tissues. During angiogenesis in culture, NG2- positive cells were present along venules. Along capillary sprouts, pericytes were typically NG2- positive and aSMA-negative

Time-Lapse Imaging Reveals Novel Endothelial Cell Dynamics During Capillary Sprouting

Time-lapse imaging of cultured mesometrium tissues harvested from FlkEGFP mice enabled the tracking of endothelial cell dynamics during capillary sprouting. Images recorded at least every 24 hours, revealed ‘endothelial cell jumping,’ defined by an endothelial cell disconnecting from a capillary sprout and subsequently connecting to a neighboring capillary sprout. EXAMPLE 2 - Perfusion Materials and Methods

Harvesting Method for Perfusion

The animal will be anesthetized and given an anti-coagulating agent to prevent blood from clotting. The abdominal cavity will be exposed and the mesentery exteriorized. The large mesenteric artery will be cannulated, the animal will be euthanized, and immediately after the mesenteric vein will be severed to allow blood to be flushed out of the mesentery vasculature. After flushing, a vascularized mesentery window will be harvested along with its feeding arteriole and venule.

Cannulation Method

The harvested mesentery window will be laid parallel and flat onto a heated stage and a cannula will be introduced into the arteriole by hand using tweezers. The cannula will be secured in place by tying suture knots along the cannulated vessel. The cannulated mesentery window is then transferred to the bioreactor and secured in place as shown in the“Expanded Side-View” schematic on slide 9. The bioreactor is then attached to tubing that connects to the perfusion system to enable perfused culture in an incubator. The various perfusion methods are described in slides 2-5. An example setup of perfusion culture is also shown on slide 10.

EXAMPLE 3 - Alternative Tissue Harvest and Perfusion

Mesentery tissue harvest

All animal experiments were approved by Institutional Animal and Care Use Committee. Adult male Wistar rats (350-400 g) were anesthetized via an intramuscular injection\n with ketamine (80 mg kg ¹ body weight) and xylazine (8 mg kg ¹ body weight). After confirming the effect of anesthesia, an intraperitoneal injection of heparinized saline (28 mg kg ¹ body weight) was administered and allowed to circulate for 10 minutes followed by euthanization via intracardiac injection of 0.2 mL beuthanasia. Mesentery windows, defined as the translucent connective tissue between artery/vein pairs feeding the small intestine, were aseptically exteriorized onto a custom PDMS stage. Two vascularized mesentery windows were identified and harvested, keeping with the feeding artery/vein pair intact; marginal vessels at the base of the tissue were ligated with 7-0 silk sutures. Tissues were immediately rinsed in sterile phosphate- buffered saline (PBS; Gibco, Grand Island, NY) with CaCl₂ and MgCh at 37 °C and immersed in sterile minimum essential media (MEM; Gibco, Grand Island, NY) containing 1% Penicillin- Streptomycin (PS; Gibco, Grand Island, NY).

Bioreactor assembly with mesentery tissue

The bioreactor assembly was designed as an open-loop system where media flows from the inlet reservoir into the arterial side of mesentery tissue and exits through the venous side into a collecting reservoir. Bioreactor tubing was connected to a peristaltic pump and three-way stopcock. Biochambers were assembled by first placing the PDMS base between the threaded rods of the acrylic biochamber base. Harvested mesentery tissues were then transferred to the biochamber and the feeding artery was cannulated with a 30G needle connected to bioreactor tubing. Residual blood was removed from microvascular networks in the mesentery tissue by perfusing approximately 3 mL of heparinized PBS (3 mg mL ¹). Once the microvascular effluence was clear, the mesentery tissue was covered with a filter membrane followed by adding the PDMS O-ring and acrylic top. The biochamber was sealed with knurled nuts and 5 mL of media was added to the well of the chamber to submerge the tissue. Approximately 250 mL of media was added to the inlet reservoir and the biochamber was connected to the bioreactor tubing and transferred to an incubator for perfusion culture.

Mesentery Cannulation

1. Surgical Procedure Setup

1.1) Sterilize surgical supplies, bioreactor assembly, and surgical tools prior to surgery according to section“Sterilization Methods and Procedures”.

1.2) Sterilize a surgical benchtop space and plexy glass platform with 70% ethanol.

1.3) Place the absorbent underpad down and lay the plexy glass platform on top of it. Place one blue drape over a heated pad next to the absorbent underpad.

1.4) Lay the other blue drape on the benchtop next to the heated pad and place all sterilized surgical tools on top. 1.5) Assemble the tubing with connectors and three-way stopcock to the peristaltic pump above the heating pad, keeping the tubing atop the blue drape to maintain sterility.

1.6) Place the sterile PDMS stage atop the plexy glass platform in the top left corner.

1.7) Pre-warm media and reagents to 37 °C. Place 10 mL MEM+l%PS and 10 mL PBS in separate 60 mm petri dishes atop the heating pad and place 100 mL saline in two 50 mL conical tubes next to the surgical setup.

1.8) Make sure all packages are open prior to the beginning of the surgery to ensure sterile handling of all materials. A complete list of the common tools used in this procedure are listed in the Table of Specific Surgical Materials and Tools.

2. Mesentery Tissue Harvesting

2.1) Use adult male Wistar rats (350 g). Other strains and ages of rats can be substituted.

2.2) Anesthetize the rat via an intramuscular injection of ketamine (80 mg/kg body weight) and xylazine (8 mg/kg body weight). Confirm the rat is under anesthesia by pinching between the toes to check for a reflex response; there should be none.

2.3) Shave the abdominal region and remove remaining hair using hair removal cream. Wipe abdominal skin three times with 70% isopropyl alcohol followed by povidone-iodine and transfer rat to the sterile surgical setup and place atop the plexy-glass platform.

2.4) Administer an intrap eritoneal injection of heparin (28 mg/kg) dissolved in 1 mL sterile 0.9% saline and allow to circulate for 10 minutes throughout the body of the anesthetized rat.

2.5) Euthanize the rat via intracardiac injection of Beuthanasia-D (0.2 mL per rat). Ensure the rat is euthanized by palpating the heart; there should be no pulse.

2.6) Using a scalpel blade, make a 2” incision along the abdominal midline starting 1” below the sternum. Be careful not to puncture the bowel or mesentery (1 layer of skin, 1 layer of connective tissue, and 1 layer of muscle).

2.7) Using dissecting scissors, make a 2” incision perpendicular to the midline on the left. First separate the layer of skin using lateral blunt dissection followed by sharp dissection of the muscle layer.

2.8) Rotate the rat 90° and roll over on its side to the left lateral position (the abdominal incision should be facing towards you). Place the PDMS stage against the abdomen of the rat with the rounded edge facing outward. Place gauze on each side of the stage between the rats’ legs to prevent saline spillage during harvest.

2.9) Use sterile cotton-tipped applicators to locate and pull out the ileum onto the PDMS stage. Continue to pull out mesenteric windows, careful not to touch them directly, until two windows of interest have been identified. Keep exposed tissues moist with warmed sterile saline as needed using a sterile syringe to drip the solution.

2.10) Secure the target mesentery tissue using 26G x ½” needles to pin the intestinal loop into the PDMS stage, ensure not to create tears that will leak contaminants onto the setup.

2.11) Remove desired mesentery tissues by first isolating the feeding arteriole and venule from their surrounding fat with the aid of a dissecting microscope. Use blunt dissection technique with the micro-scissors and Dumont forceps to remove any fat or connective tissue, being sure to clean the arteriole completely and separate from the venule.

2.12) Identify any branching vessels along the surrounding sides of the mesentery window and tie them off using 7-0 suture with the aid of a dissecting microscope. Ensure the vessels at the bottom corners of the tissue have been sutured closed as well.

2.13) Using the micro-scissors and Dumont forceps, begin separating the mesentery tissue from the intestinal loop, being careful not to snip the feeding arteriole/venule pair running along the base.

2.14) Continue separating the mesentery tissue up the sides of the window, leaving some of the adjacent tissue intact with the fat border.

2.15) Finally snip the feeding arteriole, leaving as much length as possible, followed by the venule and rinse the tissue in the warm sterile saline and place in MEM+l%PS.

2.16) Remove the 26G needles and put aside for further use. Clean any blood with saline and gauze from the PDMS stage.

2.17) Repeat steps 2.10 - 2.15 for the next mesentery tissue.

2.18) Return exteriorized tissue to the abdominal cavity and dispose of animal according to institutional guidelines.

2.19) Wipe the plexy glass platform with 70% ethanol and sterile gauze pad.

3. Mesentery Tissue Cannulation 3.1) Begin to partially assemble the sterile Biochamber assembly atop the plexy glass. Take the Acrylic Base and place PDMS Base between the threaded posts atop the acrylic inner lip.

3.2) Attach the 30G needle embedded in PDMS Needle Holder to the inlet tubing connected to the peristaltic pump. Position the Needle Holder along the outer lip of the Acrylic Base so the 30G needle is facing towards the center of the Biochamber.

3.3) Set the peristaltic pump setting to coarse #10; other flow rates can also be used. Make sure the side-port is closed in the three-way stopcock.

3.4) Begin filling the tubing with pre-warmed heparinized PBS until the solution exits the needle tip and then stop the flow.

3.5) Take one of the single looped 5-0 sutures and place onto 30G needle, keep the tie loose.

Drip a few drops of sterile saline onto the center of the PDMS Base.

3.6) Use forceps (straight or slight curve) to transfer one tissue atop the PDMS Base; grab tissues by the fat pad to avoid damaging the vasculature.

3.7) Carefully spread tissue using the fat pad, careful not to touch the window, and elongate the feeding arteriole so the vessel opening is near the 30G needle tip.

3.8) Use a dissecting microscope and Dumont forceps to gently push the opening of the feeding arteriole onto the 30G needle. Once cannulated, continue to push the vessel onto the needle to surpass the angled tip.

3.9) Use the Castroviejo needle holders to secure the cannulated arteriole onto the needle with the 5-0 suture loop.

3.10) Start the peristaltic pump and allow about 3 mL heparinized PBS to flow into the vasculature (the final volume will depend on how long it takes to clear blood from vessels). Check blood is draining from the vasculature with the dissecting microscope and use gauze and sterile saline to remove blood from the PDMS Base to prevent staining the mesentery window.

3.11) Once all blood has been flushed, stop the pump and change the three-way stopcock to close the outlet port.

3.12) Change the solution to MEM+l%PS+l0%FBS and start the pump. Allow media to exit through the stopcock onto a gauze pad until the lead bubble is removed from the line. Stop the pump and change the stopcock to open the outlet port. **Changing the tubing fluid sometimes causes bubbles in the line and this method ensures the removal before it enters the cannulated tissue** 3.13) Start the pump and allow media to flow through the line until it reaches the cannulated tissue and begins to flow through and exit the vasculature. Continue to use gauze pads to remove any RBCs from the PDMS Base.

3.14) Once media begins to exit the vasculature, stop the pump and change the stopcock to close the outlet port.

3.15) Use a 1 mL syringe to drip a few drops of MEM+l%PS+l0%FBS onto the tissue and surrounding edges.

3.16) Use tweezers (straight or slight curve) to place the sterile Filter Membrane onto the tissue, careful to keep the hole in the membrane away from the tissue. Ensure no bubbles form between the media and membrane.

3.17) Use tweezers (straight or slight curve) to place the PDMS O-ring atop the membrane.

3.18) Use sterile gloved hands to place the Acrylic Top through the threaded rods and onto the PDMS O-Ring.

3.19) Use sterile gloved hands to position the knurled nuts onto the threaded rods.

3.20) After sealing the biochamber by tightening the knurled nuts, add 5 mL of media into the reservoir of the biochamber.

3.21) Transfer the perfusion setup to a culture incubator. Place the biochamber inside the secondary culture chamber and position the peristaltic pump on the outside of the incubator.

3.22) Start the pump and be sure there are no bubbles in the line.

EXAMPLE 4 - Alternative Perfusion and Angiogenesis Study.

The objective of this study was to evaluate the effects of flow during angiogenesis in cultured ex vivo microvascular networks. Herein, we report a novel open-loop bioreactor system that enables perfusion of blood microvascular networks and live tissue imaging. The approach uses a “sandwich” method for assembling the bioreactor to keep mesentery tissue flat and facilitate live imaging. Tissues were stimulated to undergo angiogenesis during culture with and without perfusion to evaluate the influences of flow during angiogenesis. Our results suggest 1) that microvascular networks in rat mesentery tissue can be perfused ex vivo and maintain perfusion up to 48 hours in culture, and 2) the presence of flow during serum stimulation influences the spatial patterning of angiogenic microvascular networks. The findings from this study confirm that ex vivo microvascular networks can be cultured with flow, undergo angiogenesis by serum stimulation, and suggests the presence of flow influences the patterning of angiogenic networks. The bioreactor system developed for this study establishes a new mesentery tissue culture model to enable the evaluation of flow on microvascular networks in an ex vivo setting and supports the use as an experimental tool for microvascular research.

Materials and methods

Design and fabrication of mesentery bioreactor

The mesentery biochamber was fabricated using clear cast acrylic (McMaster Carr, Elmhurst, IL) with laser cutting techniques (Epilog Helix 24, 50-watt C02 laser system) to generate designed acrylic structures. The acrylic top and base were designed using Solidworks 3D CAD software (Solidworks, Waltham, MA). Threading was etched into the laser cut holes of the biochamber base and 316 stainless steel threaded rods (McMaster Carr, Elmhurst, IL) were inserted.

Polydimethylsiloxane (PDMS) structures were generated by mixing silicon elastomer base and curing agent (Ellsworth Adhesives, Germantown, WI) at a ratio of 10: 1, degassing in a vacuum chamber, and curing for 72 hours at room temperature in molds to form patterns. L/S13 tubing (Cole-Parmer, Vernon Hills, IL), which has an inner diameter of 0.8 mm, was used throughout the bioreactor with complimenting luer connectors (Cole-Parmer, Vernon Hills, IL) of the same dimensions. 316 stainless steel knurled nuts (McMaster Carr, Elmhurst, IL) were used to seal the biochamber after assembly and tissue cannulation. The biochamber and all bioreactor parts were autoclaved prior to experimental usage.

Mesentery tissue harvest

All animal experiments were approved by University of Florida’s Institutional Animal and Care Use Committee (protocol 201710060). Adult male Wistar rats (350-400 g) were anesthetized via an intramuscular injection with ketamine (80 mg kg-l body weight) and xylazine (8 mg kg-l body weight). After confirming the effect of anesthesia, an intraperitoneal injection of heparinized saline (28 mg kg-l body weight) was administered and allowed to circulate for 10 minutes followed by euthanization via intracardiac injection of 0.2 mL beuthanasia. Mesentery windows, defined as the translucent connective tissue between artery/vein pairs feeding the small intestine, were aseptically exteriorized onto a custom PDMS stage. Two vascularized mesentery windows were identified and harvested, keeping with the feeding artery/vein pair intact; marginal vessels at the base of the tissue were ligated with 7-0 silk sutures (Ethicon, Somerville, NJ). Tissues were immediately rinsed in sterile phosphate-buffered saline (PBS; Gibco, Grand Island, NY) with CaCl2 and MgCl2 at 37 °C and immersed in sterile minimum essential media (MEM; Gibco, Grand Island, NY) containing 1% Penicillin-Streptomycin (PS; Gibco, Grand Island, NY).

Bioreactor assembly with mesentery tissue

The bioreactor assembly was designed as an open-loop system where media flows from the inlet reservoir into the arterial side of mesentery tissue and exits through the venous side into a collecting reservoir. Bioreactor tubing was connected to a peristaltic pump (Living Systems, St. Albans City, VT) and three-way stopcock. Biochambers were assembled by first placing the PDMS base between the threaded rods of the acrylic biochamber base. Harvested mesentery tissues were then transferred to the biochamber and the feeding artery was cannulated with a 30G needle connected to bioreactor tubing. Residual blood was removed from microvascular networks in the mesentery tissue by perfusing approximately 3 mL of heparinized PBS (3 mg mL-l). Once the microvascular effluence was clear, the mesentery tissue was covered with a filter membrane (Millipore, Burlington, MA) followed by adding the PDMS O-ring and acrylic top. The biochamber was sealed with knurled nuts and 5 mL of media was added to the well of the chamber to submerge the tissue. Approximately 250 mL of media was added to the inlet reservoir and the biochamber was connected to the bioreactor tubing and transferred to an incubator for perfusion culture.

Albumin perfusion of microvascular networks

Perfusion in cannulated microvascular networks was assessed in freshly harvested tissues (t = 0 hours) and perfusion cultured tissues (t = 48 hours). Mesentery tissues were topically labeled with Alexa-Fluor™ 594-conjugated lectin (1 : 100; Invitrogen, Carlsbad, CA) to visualize microvascular networks. A solution of FITC-conjugated albumin (Sigma- Aldrich, St. Louis,

MO) was prepared in PBS (1 mg mL-l) and perfused through the cannulated artery. Images were taken before and during perfusion with a 4X and 10X objective from an inverted microscope Olympus 1X71 paired with a Photometries Cool SNAP EZ camera. Flow experiments

Flow was produced in the microvascular networks using a peristaltic pump set to a pre-defmed rate. Velocity measurements were obtained by adding 1 pm polystyrene FITC-fluorescent microspheres (Invitrogen, Carlsbad, CA) to the perfusate and measuring the distance they traveled through capillary vessels. Videos were captured at a rate of 1 frame sec-l with a 10X objective from an inverted microscope Olympus 1X71 paired with a Photometries Cool SNAP EZ camera. ImageJ was used to measure the distance microspheres traveled through capillary vessels using the following equation: v = L / 1 where L is the length the microsphere traveled and t is the elapsed time for the sphere to travel the measured distance. The mean wall shear stress for perfused capillaries was calculated from measured velocities assuming Hagen-Poiseuille flow in a cylindrical pipe: t = 8mn / D where t is the wall shear stress, m is viscosity of the perfusate, v is the measured velocity, and D is the average diameter of the vessel.

Angiogenesis perfusion culture

To examine the effects of flow during angiogenesis, culture media was supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY). Serum was chosen for this study because it produces a robust angiogenic response in mesentery tissues, as demonstrated in our previous publications. Tissues were harvested from adult male Wistar rats and divided into two experimental groups. For the Static group, tissues were harvested, cannulated, secured in the biochamber, and cultured without perfusion. For the Perfused group, tissues were harvested, cannulated, secured in the biochamber and cultured with perfusion. Both experimental groups were cultured in standard culture conditions (5% C02 and 37 °C) with media supplemented with 10% FBS for 48 hours, with media changed at 24 hours. After 48 hours in culture, tissues were perfused with FITC-conjugated albumin diluted in PBS (1 mg mL-l) to ensure microvascular networks maintained flow during culture.

Quantification of Angiogenesis

Vascular density and capillary sprouts were quantified from randomly selected microvascular networks per tissue from 10X montage images for both Static and Perfused experimental groups. Microvascular networks were defined as a branching capillary plexus with feeding arteriole and draining venule. Vascular density was defined as the number of lectin-positive endothelial cell segments between two branch points per vascularized area. Capillary sprouts were defined as the number of blind-ended lectin-positive endothelial cell segments originating from a vessel per vascularized area. Capillary sprouts were further divided into two categories, invading or introverting, based on their location within the microvascular networks, similar to previous descriptions of capillary phenotypes during angiogenesis. Introverting sprouts were defined as capillary sprouts enclosed within the microvascular networks and invading sprouts were defined as capillary sprouts entering the avascular space surrounding the microvascular networks.

Quantification of angiogenesis was analyzed for the following groups: 1) Static and 2) Perfused. Analysis was performed using the Cell Counter plugin with NIH Fiji open-source software version 2.0.0.

Immunostaining

Following perfusion culture experiments, mesentery tissues were removed from the biochambers and fixed in 100% methanol at -20 °C for 30 minutes. Tissues were then washed three times in PBS with 0.1% saponin for 10 minutes each followed by antibody labeling. Microvascular networks were visualized with Alexa-Fluor™ 647-conjugated lectin (1 : 100; Invitrogen,

Carlsbad, CA) and smooth muscle cells were stained with aSMA Cy3- conjugated primary antibody (1 :200; Sigma- Aldrich, St. Louis, MO). Additional tissues were labeled for endothelial cells and vascular pericytes by tagging with CD31 (1 :200; BD Pharmingen, San Jose, CA) and NG2 (1 : 100; Millipore, Burlington, MA) primary antibodies, followed by streptavidin Cy2- conjugated (1 :500; Jackson Immunoresearch, West Grove, PA) and goat anti-rabbit Cy3- conjugated (1 : 100; Jackson Immunoresearch, West Grove, PA) secondary antibodies. All antibodies were diluted in antibody buffer solution (PBS + 0.1% saponin + 2% bovine serum albumin + 5% normal goat serum). Tissues were washed three times in PBS with 0.1% saponin for 10 minutes each between primary and secondary antibody labeling incubations.

Image Acquisition

Fixed tissues immunolabeled for endothelial cells (CD31) and perivascular cells (aSMA and NG2) were imaged using a 20X (oil, NA=l.4) objective coupled with a 1.5X magnification. For quantification of angiogenesis, tissues labeled for lectin were imaged using a 10X (dry, NA=0.3) objective and montaged with NIS Elements software. All images were acquired on an inverted microscope Olympus 1X71 paired with a Photometries Cool SNAP EZ camera.

Statistical Analysis

Data are presented as mean ± standard deviation (SD). Two-sided student’s t-test was used to report the statistical significance of the angiogenesis quantification between Static and Perfused experimental groups. A p-value < 0.05 was considered statistically significant and all analysis was performed using GraphPad Prism version 8.0 software.

RESULTS

Demonstration of Blood Vessel Perfusion in Cultured Microvascular Networks

To perfuse blood vessels in microvascular networks, we developed a bioreactor system that features (i) freshly harvested mesentery tissue containing microvascular networks, (ii) open-loop perfusion through blood vessels entering the feeding arteriole and exiting the draining venule, and (iii) a biochamber that enables live tissue imaging. Mesentery, a highly vascularized thin connective tissue, was harvested from Wistar rats and the feeding arteriole was cannulated. The biochamber was designed to secure the cannulated mesentery tissue flat and enable live imaging during culture. The bioreactor system is open-loop where the main feeding arteriole of the mesentery tissue is cannulated and fluid is allowed to flow through the vasculature and exit out the draining venule. Flow is generated by a peristaltic pump from the perfusate reservoir and passes through the vasculature and exits into the waste reservoir.

Perfusion in freshly harvested mesentery tissue (Day 0) was confirmed by the presence of FITC-albumin in blood vessel lumens (Fig. 32). Topical labeling with Alexa-594 lectin visualized microvascular networks where arterioles, venules, and capillaries were identified based on morphology and network structure (Fig. 32C). Albumin was not detected in lymphatic vessels for all perfused tissues (Fig. 32A).

To measure the observed flow in capillaries of perfused microvascular networks, fluorescent microspheres (1 um diameter) were introduced into blood vessels via the cannulated arteriole. Microspheres were observed across the hierarchy of perfused microvascular networks including arterioles, venules, and capillaries (Fig. 33A, 33B and supplementary video capture). Microspheres were tracked as they traveled along the length of perfused capillaries and average velocities were measured (Table 1 and supplementary video capture). There was significant variation in the measured microsphere velocities across the tissues ranging from 0.1 mm sec-l up to 2.9 mm sec-l. The mean wall shear stress for perfused capillaries was calculated assuming Hagen-Poiseuille flow in a cylindrical pipe and approximate fluid viscosity of 0.006922 dyne-s cm-2. The mean velocity and shear stress for all observed capillaries (n = 28 from 4 tissues) was 0.9 ± 0.6 mm sec-l and 8.9 ± 6.9 dyne-s cm-2, respectively (Table 1).

Injection of FITC-albumin confirmed blood vessels remain perfused after 48 hours (Day 2) in culture with the bioreactor system (Fig. 34). Albumin was observed in microvascular networks including arterioles, venules, capillaries, and newly formed loops and blind-ended sprouts (Fig. 34). We also observed an apparent increase in vascular permeability along capillary vessels (Figs. 34A, 34B) and newly formed sprouts (Figs. 34C-34F), indicative of the angiogenic process.

SMA and NG2 labeling of microvascular networks cultured with perfusion for 48 hours identified smooth muscle cells and pericytes, respectively (Fig. 35 A and 35B). Smooth muscle cells were consistently observed to remain tightly wrapped along arterioles and venules (Fig.

35 A) and NG2 -positive pericytes were observed to wrap along capillaries (Fig. 35B).

Presence of Perfusion Influences Angiogenesis

Lectin labeling of mesentery tissues in Perfused and Static experimental groups identified endothelial cells along blood vessels in microvascular networks (Fig. 34A,B). Microvascular networks cultured with (Perfused) and without (Static) perfusion underwent angiogenesis, supported by a significant increase in capillary sprouting compared to freshly harvested unstimulated mesentery tissue (data not shown). Interestingly, comparison of the number of capillary sprouts per vascular density between Perfused (28.8 ± 10.6 sprouts/vascular area, n = 9) and Static (28.3 ± 8.81 sprouts/vascular area, n = 7) microvascular networks did not reveal a significant difference (p = 0.9269) between experimental groups (Fig. 34C). Qualitative observations of denser microvascular networks were identified in the Static culture group compared to the Perfused culture group, suggesting the presence of flow in blood vessels influences the density of angiogenic networks. Quantitative analysis of blood endothelial segments from perfusion cultured microvascular networks (94.9 ± 44.2 segments/vascular area, n = 9) revealed a significant decrease (p = 0.0027) in vascular density compared to static cultured networks (332 ± 192 segments/vascular area, n = 7) (Fig. 34D).

While the total number of capillary sprouts per vascular area was not significantly different between Perfused and Static experimental groups, additional analysis identified differences in both invasive and introverting sprout phenotypes (Fig. 35). Quantitative evaluation of the percent invasive sprouts per total capillary sprouts identified a significant increase (p = 0.0298) in microvascular networks cultured with perfusion (29.5 ± 11.0%, n = 9) compared to static culture (17.1 ± 8.83%, n = 7) (Fig. 35C). Additionally, quantitative analysis of the percent introverting sprouts per total capillary sprouts identified a significant decrease (p = 0.0298) in the Perfused group (70.5 ± 11.0%, n = 9) compared to the Static group (82.9 ± 8.83%, n = 7) (Fig. 35D).

The main contribution of this study is the establishment of a novel experimental platform that integrates whole mesentery microvascular networks with physiological vascular perfusion in a bioreactor system to evaluate the effects of flow during angiogenesis. To our knowledge, this is the first demonstration of ex vivo cultured mesentery tissue that remains viable with flow in the microvasculature. A major challenge for tissue engineering experimental models is matching the complexity of real tissues with physiological flow in an in vitro setting. More recently, advancements in cutting edge technologies such as microfluidics have helped bridge this gap.

For example, Moya et al. (2013) demonstrated the development of vascularized microtissues that anastomose with side channels to enable the evaluation of flow and shear rates in human capillary networks. Osaki et al. (2018) engineered microchannels that mimic the cooperative effects of sprouting interactions during angiogenesis and lymphangiogenesis to elucidate mechanisms of action that drive cancer metastasis and corneal implant rejection. The results of this current study establish the physiologic relevance of our bioreactor model as we demonstrate the presence of flow influences the spatial patterning of angiogenic microvascular networks. Here we introduce a new method for culturing mesentery tissue with flow in the microvascular networks through the development of a bioreactor system and supports its use as an experimental tool for microvascular research.

To incorporate perfusion into microvascular networks of mesentery tissue, we introduced flow via a cannulated feeding arteriole secured within an engineered bioreactor system using PDMS and acrylic that was designed, fabricated, and characterized. Our perfused bioreactor system enables: (i) velocity measurements in physiologically relevant microvascular networks, (ii) observation of angiogenesis at specific locations within a vascular tree, and (iii) evaluation of wall shear stress on capillary sprouting. The novelty of the perfused mesentery bioreactor system is the incorporation of multiple tissue systems and their respective cell types, extracellular matrix proteins, maintained microvascular architecture, and physiological flow.

In order to create a realistic in vitro microvascular model with physiologically relevant flow, perfusion of the vascular networks is required. In this study, perfusion of freshly harvested microvascular networks was demonstrated by injection with FITC-albumin following a vascular flush with heparinized PBS to remove blood (Fig. 32A). Microvascular networks maintained perfusion in culture up to 48 hours, demonstrated by perfusion of FITC-albumin. We also observed an apparent increase in vessel permeability in angiogenic microvascular networks, indicated by the leakage of albumin from capillaries and sprouts (Fig. 34).

Evaluation of blood velocity profiles in capillary networks is important from a microvascular physiological point of view. Capillary blood flow regulates the supply of oxygen, nutrients, and waste products. Velocity also influences the relative wall shear stress in microvessels, which has been shown to regulate angiogenesis during microvascular remodeling. Here we measured the velocity profiles in capillaries from four microvascular networks using fluorescent microbeads and live tissue imaging. By controlling peristaltic pump speed, we demonstrate a wide range of capillary velocities ranging from 100 um/sec to 2900 um/sec across four different microvascular networks (Fig. 33A and 33B). These velocities measurements are in the physiological range based on comparison with the literature using intravital microscopy to observe in vivo capillaries in mesentery tissue. Fluorescent microbeads in venules and arterioles were observed. We have demonstrated that capillary velocities in perfused microvascular networks are within physiological range compared to in vivo microvascular networks and supports the chosen rate for perfusion in further characterization studies. The importance of wall shear stress in microvascular remodeling has been implicated in numerous processes including vascular permeability, growth factor secretion, regulation of endothelial cell phenotype, and the onset of atherosclerosis. For example, Ueda et al. (2014) demonstrated that increased shear stress increased endothelial cell migration velocity on the surface of a collagen gel in vitro. In another study using an in vivo rabbit ear chamber, Ichioka et al. (1997) found that increased wall shear stress in microvessels improved wound healing angiogenesis. For the present study, wall shear stresses were estimated from velocity

measurements in perfused capillary networks assuming Poiseuille flow through a cylindrical tube using the following equation: t = p8(Vmean/d), where Vmean is the average velocity, d is the inner capillary diameter, and u represents viscosity. Our estimations demonstrate a wide range of shear stresses from 2.6 dyne/cm2 up to 27.3 dyne/cm2 calculated across four microvascular networks. This heterogeneity of shear stresses in perfused capillary networks is not surprising when considering that blood flow rate is dependent on capillary resistance as a function of network geometry. Since t is directly proportional to m and therefore influences shear stress calculations, it is important to note for this study we estimated perfusate viscosity to be water at 37C.

The effects of flow during angiogenesis in microvascular networks was examined by culturing mesentery tissue with and without perfusion while undergoing serum stimulation. Our finding that the presence of flow influences angiogenesis is supported by the decreased microvascular network density in perfusion cultured mesentery tissue compared to the static culture. Importantly, we found the amount of capillary sprouting between the perfused and static culture groups were comparable, indicating microvascular networks cultured with flow indeed underwent angiogenesis (Fig. 35). Considering the differences in vascular density between culture groups, our results suggest the lack of flow causes an enhanced rate of growth as increased density follows capillary sprouting. Other qualitative observations of perfusion effects during angiogenesis included decreased vessel tortuosity and the specific location and phenotype of capillary sprouts. For example, invasive an5 introverting sprouts phenotypes were evaluated in perfused and static cultured microvascular networks. Invasive sprouts are defined by their outward growth into the avascular tissue region of mesentery tissue and introverting sprouts grow within central vascular regions of microvascular networks. Analysis revealed the percent invasive sprouts per total number of sprouts in perfused microvascular networks was increased compared to static cultured tissues, indicating the presence of flow influences the outward growth of new capillary sprouts. Inversely, the percent introverting sprouts per total number of sprouts in static cultured microvascular networks was increased, suggesting an enhanced growth rate demonstrated by the vascular density differences.

In summary, we have demonstrated that microvascular networks in ex vivo mesentery tissue can be perfused during culture using a bioreactor system and undergo angiogenesis by serum stimulation. Our results suggest the presence of flow influences microvascular remodeling in perfusion cultured tissues and supports the novelty of our model as a physiologically relevant tissue engineered platform for evaluating microvascular remodeling dynamics.

Claims

1. A tissue culture system comprising:

(i) a solid substrate or vessel comprising at least one contiguous surface defining a volume;

(ii) a three-dimensional tissue from a mammalian mesentery or mammalian

mesometrium, the tissue comprising a plurality of endothelial cells and the tissue free of intestinal wall;

(iv) reservoir of cell medium.

(v) at least a first length of tubing in fluid connection with the reservoir and the three- dimensional tissue.

2. The tissue culture system of claim 1 further comprising a pump in operable connection to the tubing or the reservoir.

3. The tissue culture system of any of claims 1 - 2, wherein the three-dimensional tissue is from a rodent.

4. The tissue culture system of any of claims 1 - 3, wherein the three-dimensional tissue is derived from rat or mouse mesentery or mesometrium.

5. The tissue culture system of any of claims 1 - 4, wherein the solid substrate or vessel comprises at least one solid sterilizable material forming a flat or substantially flat surface relative and parallel to the ground and at least one or a plurality of sidewalls extending upward from the flat surface, the flat surface and the at least one or plurality of sidewalls defining a volume into which the three-dimensional tissue is positioned parallel to the flat or substantially flat surface such that the three-dimensional tissue has a single upper surface and a single bottom surface, wherein the bottom surface is in physical contact with the flat or substantially surface of the solid substrate.

6. The tissue culture system of claim 5 further comprising an insert comprising at least one flat surface positioned adjacent to or substantially adjacent to a top surface of the three- dimensional tissue, such that the three-dimensional tissue is positioned between the flat surface of the solid substrate and the flat surface of the insert, and wherein the single upper surface is in physical contact with at least a portion of the flat surface of the insert and the bottom surface is in physical contact with the flat surface of the solid substrate.

7. The tissue culture system of any of claims 1 through 6 further comprising a fluid linkage element operably linking the first length of tubing and a feeding arteriole within the three- dimensional tissue.

8. The tissue culture system of claim 7, wherein the fluid linkage element comprises or consists essentially of a cannula.

9. The tissue culture system of any of claims 1 through 8, wherein the three-dimensional tissue comprises one or a combination of extracellular matrix and live cells chosen from:

pericytes, immune cells, endothelial cells of blood vascular and lymphatic origins, blood cells, smooth muscle cells, interstitial cells, and nerve cells.

10. The tissue culture system of any of claims 1 through 9, wherein the three-dimensional tissue comprises (i) an upper and bottom surface with the least one vasculature positioned in between the upper and bottom surfaces; and (ii) a portion of the upper and bottom surfaces comprising a translucent connective tissue through which the at least one vasculature is positioned; wherein the vasculature comprises at least one arteriole and at least one venule pair positioned across at least a portion of the translucent connective tissue.

11. The tissue culture system of claim 10 further comprising a first and second fluid linkage element, the first fluid linkage element connecting the first portion of tubing to the three- dimensional tissue at one end of the vasculature and the second fluid linkage element connecting the three-dimensional tissue to a second length of tubing, such that the fluid linkage elements create a closed fluid system among the three-dimensional tissue, the reservoir and the first and second length of tubing.

12. The tissue culture system of any of claims 1 through 11, wherein the pump creates fluid flow of cell medium across a surface of the three-dimensional tissue in a rate and volume sufficient to maintain the viability of constitutive cells and extracellular matrix protein components in the vasculature.

13. The tissue culture system of claim 12, wherein the wherein the pump creates fluid flow of cell medium across a surface of the three-dimensional tissue in a rate and volume sufficient to maintain the viability of constitutive cells and extracellular matrix protein components in the vasculature for no less than about 5, 10, 60, 120, 240, 480 minutes.

14. The tissue culture system of claim 12, wherein the pump creates fluid flow of cell medium across a surface of the three-dimensional tissue in a rate and volume sufficient to maintain the viability of constitutive cells and extracellular matrix protein components in the vasculature for no less than about 12 hours.

15. The tissue culture system of claim 12, wherein the pump creates fluid flow of cell medium across a surface of the three-dimensional tissue in a rate and volume sufficient to maintain the viability of constitutive cells and extracellular matrix protein components in the vasculature for no less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 14, 15 days.

16. The tissue culture system of claim 12, wherein the pump creates fluid flow of cell medium across a surface of the three-dimensional tissue at a rate and in a volume sufficient to maintain the viability of constitutive cells and extracellular matrix protein components in the vasculature, immune cells in the tissue, and pericytes in the tissue for no less than about 5, 10,

60, 120, 240, 480 minutes.

17. The tissue culture system of claim 12, wherein the pump creates fluid flow of cell medium within an interior region of the three-dimensional tissue in at a rate and in a volume sufficient to maintain the viability of the constitutive cells and extracellular matrix protein components in the vasculature, immune cells in the tissue, and pericytes in the tissue for no less than about 12 hours.

18. The tissue culture system of claim 12, wherein the pump creates fluid flow of cell medium across a surface of the three-dimensional tissue at a rate and in a volume sufficient to maintain the viability of the constitutive cells and extracellular matrix protein components in the vasculature, immune cells in the tissue, and pericytes in the tissue for no less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 14, 15 days.

19. The tissue culture system of any of claims 1 through 18, wherein the solid substrate comprises a lid defining an interior chamber comprising the volume and into which the three- dimensional tissue is positioned.

20. The tissue culture system of claim 19 further comprising at least one gasket forming an airtight or semi-airtight seal between the interior chamber and the space outside the solid substrate.

21. The tissue culture system of any of claims 18 through 19, wherein the solid substrate comprises at least one inlet and at least one outlet, the inlet defining a space through which the first length of tubing and/or a first fluid linkage element connect the interior chamber to the reservoir in fluid communication and the outlet defining a space through which fluid exits the interior chamber.

22. The tissue culture system of claim 21, wherein the outlet defines a space through which a second length of tubing and / or a second fluid linkage element connects the interior chamber and a point exterior to the solid substrate in fluid communication.

23. The tissue culture system of claim 21, wherein the outlet defines a space through which a second length of tubing and / or a second fluid linkage element connects the interior chamber and the reservoir in a fluid circuit.

24. The tissue culture system of claims 22 or 23, wherein the first and second fluid linkage elements are cannulas.

25. The tissue culture system of any of claims 1 through 24 further comprising a valve and/or adapter in fluid communication with the first length of tubing, wherein the valve comprises at least a first and second operable condition; wherein, in a first operable condition the valve is closed preventing introduction of fluid into the three-dimensional tissue; and wherein, in the second operable condition, the valve is at least partially open allowing introduction of fluid into the three dimensional tissue.

26. The tissue culture system of any of claims 1 through 25 further comprising a valve and/or adapter in fluid communication with the volume and/or interior chamber, wherein the valve comprises at least a first and second operable condition; wherein, in a first operable condition the valve is closed preventing introduction of fluid into the volume and/or interior chamber of the solid substrate; and wherein, in the second operable condition, the valve is at least partially open allowing introduction of fluid into the volume and/or the interior chamber.

27. The tissue culture system of any of claims 1 through 26, wherein cell medium is pumped across the three-dimensional tissue at no less than about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 microliters per second.

28. The tissue culture system of any of claims 1 through 27 further comprising an incubator enclosing the solid substrate.

29. The tissue culture system of claim 28, wherein the three-dimensional tissue is maintained at about 37 degrees Celsius.

30 The tissue culture system of claim 28 or claim 29, wherein the three-dimensional tissue exposed to no more than about 5% carbon dioxide.

31. The tissue culture system of any of claims 1 through 30, wherein the volume or interior chamber is no less than about 50, 45, 40, 35, 30, 25, 20, 15 or 10 milliliters.

32. The tissue culture system of any of claims 1 through 31, wherein the three-dimensional tissue is exposed to fluid flow through an interior region of the three-dimensional tissue at a physiologically effective rate.

33. The tissue culture system of claim 32, wherein the three-dimensional tissue is free of steady shear stress or oscillatory shear stress of magnitudes greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 dynes per centimeter squared of tissue from fluid flow through the tissue.

34. The tissue culture system of any of claims 1 through 33, wherein the three-dimensional tissue comprises a plurality of pericytes and/or immune cells and/or blood cells.

35. The tissue culture system of any of claims 1 through 34, wherein the three-dimensional tissue is free of intestinal wall tissue

36. A method of measuring angiogenesis or capillary sprouting comprising:

(i) exposing a tissue of any of claims 1 through 35 in culture to an agent; and

(ii) measuring growth of vasculature or capillary sprouting in the tissue before and after exposure to the agent.

37. The method of claim 36 further comprising a step of harvesting the tissue of any of claims 1 through 35 prior to performing a step of exposing the tissue to a therapeutic.

38. The method of any of claims 36 or 37 further comprising a step of correlating the presence, absence, or quantity of growth of the vasculature or capillary to effect of the agent; and characterizing the agent as promoting angiogenesis if the agent stimulates vasoreactivity, growth or density of vasculature or if the agent stimulates sprouting of capillaries.

39. A method of evaluating the toxicity of an agent comprising: (a) culturing one or plurality of a mesenteric and/or a mesometrial tissue in any of the compositions described herein; (b) exposing at least one agent to the one or more cells; (c) measuring and/or observing one or more morphometric changes of the one or more mesenteric and/or a mesometrial tissue; and (d) correlating one or more morphometric parameters of the one or more mesenteric and/or a mesometrial tissues with the toxicity of the agent, such that, if the morphometric parameters are indicative of decreased cell viability, the agent is characterized as toxic and, if the morphometric parameters are indicative of unchanged or positive cell viability, the agent is characterized as non-toxic.

40. The method of claim 39, wherein at least one agent comprises a small chemical compound.

41. The method of claim 39, wherein the at least one agent comprises at least one

environmental pollutant.

42. The method of claim 39, wherein the at least one agent comprises one or a combination of small chemical compounds chosen from: chemotherapeutics, analgesics, cardiovascular modulators, cholesterol, neuroprotectants, neuromodulators, immunomodulators, anti inflammatories, and anti-microbial drugs.

43. A method of inducing growth of one or a plurality of cells in a three-dimensional tissue comprising: (a) contacting one or a plurality of isolated mesenteric or mesometrial tissues of any of claims 1 through 34 with the solid substrate, said solid substrate comprising at least one exterior surface, at least one interior surface and at least one interior volume defined by the at least one interior surface and accessible from a point exterior to the solid substrate through at least one opening; (b) positioning one or a plurality of isolated mesenteric or mesometrial tissues of any of claims 1 through 34 to the at least one interior volume; (c) applying a cell medium into the culture vessel with a volume of cell medium sufficient to cover the at least one interior volume; (d) affixing at least a one or a plurality of fluid linkage elements to at least the first vasculature.

44. The method of claim 43 further comprising exposing one or plurality of isolated mesenteric or mesometrial tissues with at least one agent.

45. The method of claim 44, wherein at least one agent comprises one or a combination of small molecules chosen from: chemotherapeutics, analgesics, cardiovascular modulators, cholesterol, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatories, and anti-microbial drugs.

46. The method of claim 44, wherein at least one agent is one or a plurality of stem cells or modified T cells.

47. The method of any of claims 44 through 46 further comprising monitoring growth of cells after exposure of the tissue to one or a plurality of agents.

48. A method of detecting and/or quantifying cell mobility in vitro comprising: (i) culturing one or a plurality of mesenteric or mesometrial tissues of any of claims 1 through 34 in any of the systems disclosed herein; and (ii) exposing the tissue or tissues to a known number of cells in the composition after a culturing for a time period sufficient to allow cell migration of the one or plurality of cells.

49. The method of claim 48 further comprising measuring a recording, distance of migration or cell-to-cell interaction between the cell or plurality of cells and the tissue after step (ii).

50. The method of claim 48 further comprising the step (iii) detecting an internal and/or external recording of such one or more cells after culturing one or more tissues and correlating the recording with a measurement of the same recording corresponding to a known or control number of cells.

51. The method of any of claims 48 through 50 further comprising contacting one or more tissues to one or more agents.

52. The method of any of claims 48 through 51, step (iii) comprises measuring an internal and/or external recording before and after the step of contacting the one or more tissues to one or more agents; and correlating the difference in the recording before contacting the one or more tissues to the one or more agents to the recording after contacting the one or more tissues to one or more agents.

53 The method of any of claims 49 through 52, wherein the recording is a distance between where the cell or cells were introduced or exposed into the system and where the cell or cells were positioned after allowing a time period sufficient to for the cell or cells to migrate through the tissue.

54. A method of measuring vasculature growth within a tissue comprising: (a) positioning one or a plurality of tissues in any of the compositions disclosed herein; (b) quantifying the one amount or density of vasculature in the one or plurality of tissues; (c) contacting the one or plurality of tissues to one or a plurality of agents; and (d) quantifying the number or density of vasculature after contacting the one or plurality of tissues to one or a plurality of agents; and (e) calculating the difference in the number or density of vasculature prior to the step (c) and after step (c).

55. The method of claim 54, wherein the step of quantifying comprises staining the one or plurality of tissues.

56. The method of any of claims 54 or 55 wherein, steps (b), (d), and/or (e) are performed via microscopy or digital imaging.

57. A method of measuring intracellular or extracellular recordings comprising: (a) culturing one or a plurality of tissues in any of the composition disclosed herein; (b) measuring a recording across the one or a plurality of tissues.

58. The method of claim 57, wherein the step of measuring comprises staining the one or plurality of tissues.

59. The method of any of claims 57 or 58, wherein, step (b) is performed via microscopy or digital imaging.

60. A method of real-time imaging of tissue comprising culturing tissue within the tissue culture system of any of claims 1 through 34; and exposing the tissue culture system of any of claims 1 through 34 to digital imaging.

61. The method of claim 60 further comprising exposing one or plurality of isolated mesenteric or mesometrial tissues with at least one agent.

62. The method of claim 61, wherein the at least one agent comprises one or a combination of small molecules chosen from: chemotherapeutics, analgesics, cardiovascular modulators, cholesterol, neuroprotectants, neuromodulators, immunomodulators, anti-inflammatories, and anti-microbial drugs.

63. The method of claim 61, wherein the at least one agent is one or a plurality of stem cells or modified T cells.

64. The method of any of claims 60 through 63 further comprising monitoring growth of cells in the tissue after exposure of the tissue to one or a plurality of agents.

65. A method of making a tissue culture system of any of claims 1 - 34 comprising: (i) forming an interior chamber within a solid substrate; (ii) affixing mesenteric or mesometrial tissue from a subject to the solid substrate; (iii) positioning at least one fluid linkage element into the tissue in fluid communication with a feeding arteriole; (iv) culturing the tissue in cell culture medium at about 37 degrees Celsius; (v) attaching at least a first length of tubing to the at least first fluid linkage element; (vi) placing a reservoir of cell medium in fluid connection with at least the first length of tubing; (vii) placing a pump in operable connection to the at first length of tubing; and, optionally (viii) sealing the tissue within the solid substrate, such that the tissue is positioned within an internal cavity of the solid substrate in fluid communication with the reservoir.

66. The method of claim 65 further comprising harvesting mesenteric or mesometrial tissue from a subject prior to step (ii).

67. The method of any of claims 65 or 66 further comprising (ix) positioning at least a second fluid linkage element into the tissue in fluid communication with an exiting venule in the tissue; (x) attaching at least a second length of tubing to the at least second fluid linkage element; and (xi) placing the second length of tubing in fluid communication with the reservoir and the tissue.

68. The method of any of claims 65 through 67, wherein the tissue culture system is an open or closed system.

69. The method of any of claims 65 through 68 further comprising introducing fluid flow through the tissue.

70. The method of any of claims 65 through 69 further comprising attaching one or more valve and/or adapters in fluid communication with the first length of tubing, wherein the valve comprises at least a first and second operable condition; wherein, in a first operable condition the valve is closed preventing introduction of fluid into the three-dimensional tissue; and wherein, in the second operable condition, the valve is at least partially open allowing introduction of fluid into the three dimensional tissue.

71. The method of any of claims 65 through 70 further comprising attaching a syringe to the adaptor or valve such that the syringe becomes part of a fluid circuit within the system.

72. A method for testing the efficacy of a test substance comprising: exposing a three- dimensional tissue comprising mesentery or mesometrial cells to the test substance, in which the three-dimensional cell culture comprises tissue secured to a solid substrate and in a culture chamber; and determining the effect of the test substance by measuring or observing a change in the three-dimensional cell culture.

73. The method of claim 72, wherein the tissue comprises rat or mouse mesentery or rat or mouse mesometrium.

74. A method of producing a tissue culture, in-vitro model of rat mesentery tissue wherein the tissue comprises blood and lymphatic microvascular networks, endothelial cells, smooth muscle cells, immune cells, neural cells, and pericytes, the method comprising harvesting rat mesentery tissue from a rat and securing it to a solid substrate.

75. The method of claim 74, wherein the step of harvesting comprises exteriorizing the mesentery from the gut of the animal and harvesting the mesenteric window.

76. The method of claim 74, wherein the rat is chosen from one or a combination of: a transgenic rat, an aged rat and an immunocompromised rat.

77. The method of claim 74 further comprising cannulating the tissue to allow perfusion through the tissue into an arteriole or vein while the mesentery is exteriorized and secured with sutures on the cannulated blood vessel.

78. The method of claim 74 further comprising securing the tissue in a bioreactor or incubator and attaching tubing to one or a plurality of blood vessels within the tissue.

79. The method of claim 78 further comprising placing the bioreactor in culture chamber, operably connecting the bioreactor and tubing to a pump; and operating the pump for a time sufficient to create physiologically effective perfusion of the tissue.

80. A method of printing cells on tissue culture system of any of claims 1 through 34; and evaluating the cells wherein the evaluating comprises tracking growth and/or interactions of the cells within the tissue culture system.

81. A method of producing a tissue culture, in-vitro model of mouse mesentery tissue comprising blood and lymphatic microvascular networks, endothelial cells, smooth muscle cells, immune cells, neural cells, and pericytes, the method comprising inducing vascularization of mouse mesentery by injecting said mouse with tamoxifen, sunflower oil, of VEGF for 5 consecutive days before harvesting the mouse mesentery.

82. The method of claim 81 wherein the mouse mesentery is free of the intestinal loop tissue comprising endothelial and epithelial cells of the mouse.

83. The method of claim 81 further comprising harvesting the mesentery tissue by exteriorizing the mesentery from the gut of the animal and harvesting the mesenteric window.

84. The method of claim 81 wherein the mouse is one or a combination of: a transgenic mouse, an aged mouse, and an immunocompromised mouse.

85. The method of claim 81 further comprising cannulating the tissue to allow perfusion through the tissue and securing a first and/or second cannula with sutures.

86. The method of claim 85 further comprising placing the tissue into a bioreactor and attaching tubing to the first and or second cannula.

87. A method of manufacturing a tissue culture, in-vitro model of mouse mesometrium tissue comprising blood and lymphatic microvascular networks, endothelial cells, smooth muscle cells, immune cells, neural cells, and pericytes, the method comprising harvesting the mouse mesometrium and securing the mouse mesometrium to a solid substrate.

88. The method of claim 87, wherein the harvesting comprises exteriorizing the mesentery from the gut of the animal and harvesting the mesenteric window.

89. The method of claim 87, wherein the mesometrium is free of uterine or ovarian cells.

90. The method of claim 87, wherein the mouse is one or a combination of mice chosen from: a transgenic mouse, an aged mouse, and an immunocompromised mouse.

91. The method of claim 87 further comprising cannulating the tissue by introducing a cannula into an arteriole or vein while the mesometrium is exteriorized and securing the cannula to the tissue with sutures.

92. The method of claim 87 further comprising placing the tissue of in a bioreactor and attaching at least a first length of tubing to the cannula.

93. The method of claim 87 further comprising operably attaching a pump to the tubing and perfusing cell culture medium through the tissue.

94. The method of claim 17, wherein the interior region of the three-dimensional tissue is the lumen and/or feeding arteriole of the three-dimensional tissue.

95. The method of claim 32, wherein the interior region of the three-dimensional tissue is chosen from one or a combination of: a lumen, a feeding arteriole, microvasculature, exiting venule.