CN117586876A - Vascularized organoid chip and vascularized organoid - Google Patents
Vascularized organoid chip and vascularized organoid Download PDFInfo
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Abstract
The invention provides a vascularized organoid chip and a vascularized organoid, wherein the vascularized organoid chip can enlarge the contact surface of cells and a culture medium based on a fluid limiting structure, and promote the spontaneous generation of a vascular network through fluid stimulation, so that a vascularized organoid model is obtained. The invention provides a feasible method for constructing the organoid vascularization in-vitro model, simulates a more bionic in-vivo microenvironment, and simultaneously the obtained model can be used for research of immune co-culture, angiogenesis and the like.
Description
Technical Field
The invention relates to the technical field of organoid culture, in particular to a vascularized organoid chip and a vascularized organoid.
Background
The organoid has self-renewing and self-organizing capabilities, shows organ functions similar to those of the original tissue, can be better used for simulating the occurrence process and physiological and pathological states of the organ tissue, and has wide application prospect in basic research and clinical diagnosis and treatment. The first intestinal organoids were cultivated from Hans Clevers laboratories of the netherlands scientist in 2009, and after more than ten years of development, organoids have been used nowadays as a high-quality in vitro model for the treatment detection of preclinical cancers and the drug efficacy and toxicity test, and gradually move to the central stage of basic research and clinical treatment of tumor cancers, and in addition, many laboratories and enterprises are devoted to the establishment of biological libraries of tumor organoids to enable physiologically relevant drug screening. However, compared with the physiological environment of normal tissue and organ of human body, the current organoid culture can not be cultured for a long time due to lack of vascular structures, and organoid central cell necrosis phenomenon caused by incapability of entering culture medium, oxygen, nutrient substances and the like can occur when the organoid volume is increased to a certain size. In addition, vascularization enables the organoid model to more mimic the microenvironment in vivo, and co-culture with immune cells mimics the immune microenvironment in vivo.
The reconstruction of the in-vitro vascular system can be realized by utilizing the biological 3D printing technology, the microfluidic chip and other technologies, and at present, a plurality of researchers realize the in-vitro three-dimensional vascular network construction based on hydrogel based on the two technologies. However, the vascular network constructed by the 3D printing technology is mainly designed by people and has a certain difference with the vascular network of the human body. The three-dimensional vascular network based on the micro-fluidic chip is formed by cells spontaneously and has high similarity with the morphological structure of blood vessels in vivo, but the existing chip basically adopts a micro-column structure to limit the spatial distribution of gel, and the micro-column structure occupies a part of space, so that the contact area between the cells and a culture medium is reduced, and in addition, the micro-column structure can also block the ports of the blood vessel structure which are formed spontaneously, so that the vascular perfusion is influenced. Meanwhile, the existing microfluidic vascularization chip is basically of a closed structure, and flow channels are separated by microcolumns, so that the circulation of a culture medium is not facilitated, and uneven or insufficient fluid stimulation is easily caused. In addition, the chip is basically of a closed structure, often only manual operation is relied on, the operation difficulty is high, and meanwhile, organ tissues cultured in the chip are difficult to take out, so that the subsequent analysis and detection means are limited. The closed chip cannot be matched with instruments and equipment such as automatic liquid adding and the like, and the requirements of automation and flux are not met. The existing open vascularized organ chip adopts PMMA and glass to manufacture a vascular chip in a double-sided adhesive bonding mode, and after the chip manufacture is finished, PDMS is smeared on the outer side of the PMMA, which is contacted with the glass, so that the tightness is improved. The gun head for the hydrogel is driven into the bottom of the circular ring structure through one side of the liquid storage tank, and the hydrogel is distributed at the bottom of the circular ring structure in the middle through capillary force to form a ring shape. The thickness of the hydrogel is determined by the height of the gap between the annular structure and the substrate, and the width of the hydrogel is determined by the width of the annular structure. However, this method has the following problems: 1, a cutting machine is needed to cut the double faced adhesive tape in a corresponding shape and then align and bond the double faced adhesive tape with the upper PMMA, the operation process is complicated, and errors are easy to generate in the alignment process to cause slight differences of chip structures; 2, because the hydrogel is injected by tilting the gun head, the requirement of an automatic instrument is difficult to meet, and the flux is not facilitated; 3, adding the cell ball into the middle hole when co-culturing with the cell ball, wherein the blood vessel is difficult to enter the cell ball due to distance so as to obtain the vascularized cell microsphere; and 4, the vascular structure is positioned at the bottom of the circular ring structure, and the sample is difficult to take out as the closed chip, so that the requirement of a subsequent diversity analysis and detection means cannot be met.
Therefore, there is a need to provide a vascularized organoid chip to solve the above technical problems.
Disclosure of Invention
The present invention aims to solve one of the technical problems in the related art at least to some extent. Therefore, the invention provides a vascularized organoid chip and a vascularized organoid, wherein the vascularized organoid chip can enlarge the contact surface of cells and a culture medium based on a fluid limiting structure, and promote the spontaneous generation of a vascular network through fluid stimulation, so that a vascularized organoid model is obtained. The invention provides a feasible method for constructing the organoid vascularization in-vitro model, simulates a more bionic in-vivo microenvironment, and simultaneously the obtained model can be used for research of immune co-culture, angiogenesis and the like.
To this end, a first aspect of the present invention provides a vascularized organoid chip comprising a liquid storage layer, a cell culture layer and a substrate layer arranged in sequence;
the liquid storage layer is provided with at least one through hole unit, each through hole unit comprises a first through hole, a second through hole and a third through hole which are sequentially arranged, and the diameters of the adjacent through holes are arranged differently;
the cell culture layer is provided with at least one culture unit, the culture units are in one-to-one correspondence with the through hole units, each culture unit comprises a first micropore, a second micropore and a third micropore which are sequentially arranged, the first micropore, the second micropore and the third micropore are respectively in one-to-one correspondence with the first through hole, the second through hole and the third through hole, gaps with target heights are formed at the bottom of the second micropore so as to form a channel, and the first micropore, the second micropore and the third micropore are communicated through the channel.
The invention provides a vascularized organoid chip based on a fluid limiting structure, which can enlarge the contact surface of cells and a culture medium, and promote the spontaneous generation of a vascular network through fluid stimulation to obtain a vascularized organoid chip model. The vascularized organoid chip solves the following problems:
1) The method solves the problem that most of the current chips adopt microstructures such as a micro-column array to control the space distribution of hydrogel, thereby causing non-uniform or insufficient fluid stimulation; 2) The problem that the existing closed chip sample is difficult to take out and the follow-up analysis means are limited is solved; 3) The problem that the closed chip can only be manually operated and is difficult to match with automatic sample adding equipment is solved, and 4) the chip structural design avoids chip structural difference caused by errors in alignment bonding.
According to an embodiment of the invention, the thickness ratio of the reservoir layer, the cell culture layer and the basal layer is (20-50): 2:1.
According to an embodiment of the invention, the thickness of the liquid storage layer is 5-10mm. Thereby meeting the demand of the culture medium.
According to an embodiment of the invention, the thickness of the cell culture layer is 0.2-1mm. Therefore, the filling of the hydrogel material is facilitated, and the situation that the gel material is adhered to the hole wall and cannot be paved on the bottom of the hole is avoided.
According to an embodiment of the invention, the thickness of the base layer is 0.1-0.5mm. Thereby facilitating subsequent viewing by microscopy.
According to an embodiment of the invention, the ratio of the thickness of the cell culture layer to the target height is (1.2-1.5): 1. It is thereby achieved that the vascularized cells and/or organoid cells and gel mixture in the second microwell do not spill over into the two side channels.
According to an embodiment of the invention, the target height is 200-500 μm.
According to an embodiment of the present invention, the peripheral bottom of the second micro-hole has a wedge structure to form a gap of a target height, and the width of the wedge structure is gradually reduced along the direction from the liquid storage layer to the substrate layer.
According to an embodiment of the invention, the diameter of the first and/or the third through hole is larger than the second through hole. Thereby meeting the demand of the culture medium.
According to an embodiment of the present invention, the diameters of the first through hole and the third through hole are the same.
According to an embodiment of the present invention, the diameter of the second micro-hole is smaller than the diameter of the second through-hole. Therefore, the vascularized cells and/or the organoid cells and hydrogel mixture are concentrated in the second micropores, and the phenomenon that gel adheres to the pore walls and cannot fill the whole micropores when the vascularized cells and/or the organoid cells and hydrogel mixture is added due to the influence of surface tension is avoided. Meanwhile, the use amount of the hydrogel material is reduced, and the cost is saved.
According to the embodiment of the invention, the diameter ratio of the first through hole to the second through hole to the third through hole is 8:5:8.
According to an embodiment of the invention, the diameter of the first through hole is 8mm, the diameter of the second through hole is 5mm, and the diameter of the third through hole is 8mm.
According to an embodiment of the invention, the diameter of the second microwells is 2mm.
According to an embodiment of the invention, the cell culture layer is in contact with the substrate layer.
According to an embodiment of the present invention, the material of the liquid storage layer is one or more of PMMA, PS, COC.
According to an embodiment of the present invention, the material of the cell culture layer is one or more of PMMA, PS, PC.
According to the embodiment of the invention, the material of the substrate layer is one or more of glass, PC and PS.
In a second aspect, the invention provides the use of a vascularised organoid chip according to the first aspect for the preparation of a vascular model or vascularised organoid.
The third aspect of the present invention provides a method for preparing a vascular model, wherein the method for preparing a vascularized organoid chip according to the first aspect comprises mixing vascularized cells with a hydrogel material, injecting the mixture into the second through hole, and injecting a culture medium into at least one of the first through hole and the third through hole and the second through hole for culturing.
According to an embodiment of the invention, the vascularized cells include human umbilical vein endothelial cells, human lung fibroblasts.
According to an embodiment of the invention, the hydrogel material comprises a material selected from gelatin or matrigel.
According to an embodiment of the invention, the method of preparing further comprises placing the vascularized organoid chip in a fluid shear cell culture automation device or a semi-automation device.
According to a fourth aspect of the present invention, there is provided a method for preparing a vascularized organoid, the method comprising mixing vascularized cells, organoids and hydrogel material and injecting the mixture into the second through-hole, and injecting a culture medium into at least one of the first through-hole and the third through-hole and the second through-hole for culturing.
According to an embodiment of the invention, the vascularized cells include human umbilical vein endothelial cells, human lung fibroblasts.
According to an embodiment of the invention, the organoids comprise at least one selected from the group consisting of tumor organoids, pancreatic organoids, liver organoids, kidney organoids, stomach organoids, intestinal organoids.
According to an embodiment of the present invention, the tumor organoid comprises at least one selected from the group consisting of intestinal cancer organoids, lung cancer organoids, gastric cancer organoids, pancreatic cancer organoids.
According to an embodiment of the invention, the organoid comprises a organoid selected from intestinal cancers.
According to an embodiment of the invention, the hydrogel material comprises a material selected from gelatin or matrigel.
According to an embodiment of the invention, the method further comprises placing the vascularized organoid chip in a fluid shear cell culture automation device or a semi-automation device.
In a fifth aspect, the present invention provides a vascular model obtained by the production method according to the third aspect.
In a sixth aspect the present invention provides a vascularised organoid obtainable by the preparation method according to the fourth aspect.
The seventh aspect of the present invention provides the use of a vascular model according to the fifth aspect or a vascularised organoid according to the sixth aspect in drug screening.
Compared with the prior art, the invention has the beneficial effects that:
1. the vascularized organoid chip provided by the invention has the flow channel with the target height at the bottom, so that vascularized cells and/or organoid cells in the middle through hole can be prevented from overflowing into the flow channels at the two sides, and compared with the traditional microcolumn design, the structure can greatly increase the contact area of cells and a culture medium, thereby realizing more uniform and sufficient fluid stimulation;
2. the upper liquid storage layer of the vascularized organoid chip provided by the invention is thicker, so that the demand of a culture medium can be met; the middle cell culture layer is thinner, and the second micropores are smaller, so that the hydrogel material is prevented from adhering to the wall due to the hydrophobic material of the chip, the consumption of the hydrogel material is reduced, and the cost is reduced; the substrate layer is thinner to facilitate subsequent observation under a microscope. Meanwhile, the chip design structure is concentrated on the middle cell culture layer, so that errors possibly occurring in alignment bonding and structure differentiation are avoided;
3. the vascularized organoid chip provided by the invention is of an open design, so that the chip sample adding and the automatic equipment are matched for use, and meanwhile, the open design is convenient for taking out the cultured tissues for subsequent multi-means analysis and test;
4. the vascularized organoid chip provided by the invention can be made of plastic materials, so that the adsorption of the traditional PDMS material to small molecular medicines can be avoided, and the result in the subsequent medicine sieve application is more accurate. Meanwhile, the chip can be manufactured in a mass production mode by an injection molding process, the defect that the traditional PDMS chip can only be manufactured manually but cannot be produced in batch can be avoided, and batch difference easily occurs in manual processing;
5. the vascularized organoid chip provided by the invention can adapt to a small amount of demands of scientific research work by adopting a modularized design, and the high-flux demands of drug screening can be met by adopting an orifice plate design.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows a schematic diagram of the structure of a vascularized organoid chip provided by the present invention;
FIG. 2 is a schematic cross-sectional view of a vascularized organoid chip according to the present invention;
FIG. 3 shows a schematic structural diagram of a vascularized organoid chip comprising a plurality of structural units provided by the present invention;
FIG. 4 shows a schematic diagram of a vascularized organoid chip of the present invention for vascular model culture;
FIG. 5 shows a physical diagram of a fluid shear cell culture automation device;
FIG. 6 shows a three-dimensional vascular network generated using vascularized organoid chips in example 1 of the present invention;
FIG. 7 shows a graph of the results of perfusion of a three-dimensional vascular network with 70kD FITC-Dextran in example 1 of the present invention;
FIG. 8 is a graph showing the results of the perfusion of a three-dimensional vascular network with fluorescent microspheres in example 1 of the present invention;
FIG. 9 shows a vascular network surrounding a organoid using vascularized organoid chips in example 2 of the present invention.
Reference numerals:
100-vascularized organoid chip;
101-a liquid storage layer; 1011-via units; 10111-a first via; 10112-a second via; 10113-a third via;
102-cell culture layer; 1021-a culture unit; 10211-first microwells; 10212-second microwells; 10213-third microwells; 103-substrate layer.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
The first aspect of the present invention provides a vascularized organoid chip 100, as shown in fig. 1 and 2, the chip 100 comprising a reservoir layer 101, a cell culture layer 102 and a substrate layer 103, arranged in this order. Wherein the liquid storage layer is provided with at least one through hole unit 1011, and each through hole unit 1011 includes a first through hole 10111, a second through hole 10112, and a third through hole 10113 which are sequentially arranged. The cell culture layer 102 is provided with at least one culture unit 1021, the culture units 1021 are in one-to-one correspondence with the through holes 1011, each culture unit 1021 comprises a first micro-hole 10211, a second micro-hole 10212 and a third micro-hole 10213 which are sequentially arranged, and the first micro-hole 10211, the second micro-hole 10212 and the third micro-hole 10213 are respectively in one-to-one correspondence with the first through holes 10111, the second through holes 10112 and the third through holes 10113. Wherein the first through hole 10111 and the third through hole 10113 in the through hole unit 1011 are used for injecting a culture medium, which will flow from the reservoir into the cell culture layer. The second through-hole 10212 in the through-hole unit 1011 is used for injecting vascularized cells and/or organoids. The bottom of the second through hole 10212 forms a gap of a target height to form a channel through which the first, second and third micro holes 10211, 10212 and 10213 communicate.
Wherein the thickness ratio of the reservoir layer, the cell culture layer and the basal layer may be (20-50): 2:1. The thickness of the liquid storage layer is set to be 5-10mm and meets the requirement of the culture medium. The thickness of the cell culture layer is set to be convenient for the subsequent pouring of the hydrogel material, the condition that the hydrogel is adhered to the hole wall and cannot be paved on the bottom of the hole is avoided, the thickness of the basal layer can be set to be 0.2-1mm, the thickness of the basal layer is set to be convenient for the subsequent observation through a microscope, and the thickness of the basal layer can be set to be 0.1-0.5mm. It should be noted, however, that the above selection of the thickness of the structure is exemplary and not intended to limit the scope of the invention. The diameters of the adjacent through holes in each through hole unit in the liquid storage layer are different, and the diameter of the second micropore is smaller than that of the second through hole corresponding to the second micropore. Preferably, the diameter of the first through hole and/or the third through hole is larger than the second through hole; more preferably, the diameters of the first through hole and the third through hole are the same. Specifically, the diameter of the first through hole is 8mm, the diameter of the third through hole is 8mm, the diameter of the second through hole is 5mm, and the diameter of the second micropore is 2mm. However, it should be noted that the diameter selection of the through-holes and the micro-holes is not limited to the above diameter range, and can be reasonably adjusted according to the actual organoid size preparation requirements.
According to a specific embodiment of the invention, the bottom of the second microwell in the cell culture layer is set with a gap of a target height to form a channel, so that three microwells in one culture unit are communicated. The target height should be such that the cells in the second microwells do not overflow into the two side flow channels, but do not affect the flow of the medium in the entire flow channels in the two side microwells, and the ratio of the thickness of the cell culture layer to the target height may be (1.2-1.5): 1. Specifically, the structure for achieving the target height may have various structural forms, for example, in a preferred case, as shown in fig. 2, the bottom of the periphery of the second micropore 10212 has a wedge-shaped fluid limiting structure, the width of which gradually decreases along the direction from the liquid storage layer to the basal layer, so as to reduce the distance between the cells and the culture medium, and avoid air bubbles generated in the middle runner when the culture medium is added into the through holes on two sides, thereby influencing the mobility of the culture medium. The target height may be selected to be reasonably selected according to the cell type in the second microwell, for example, 200-500 μm, but is not limited to this range of values.
According to a specific embodiment of the present invention, the vascularized organoid chip can be obtained by bonding a liquid storage layer, a cell culture layer and a basal layer, wherein the material of the liquid storage layer can be PMMA, PS, COC, the material of the cell culture layer can be PMMA, PS, PC, and the material of the basal layer can be glass, PC or PS to meet the requirement of observing the generation condition of organoids or blood vessels in the cell culture layer 10212.
According to a specific embodiment of the present invention, the vascularized organoid chip may be designed as a unit module comprising a plurality of structural units, as shown in fig. 3, comprising 32 through-hole units, each through-hole unit being capable of culturing a respective organoid, respectively, to meet different experimental requirements.
The invention also provides a preparation method of the blood vessel model, which is based on the vascularized organoid chip, and comprises the steps of mixing vascularized cells with a hydrogel material, injecting the mixture into a second through hole, and respectively injecting culture medium into at least one of the first through hole and the third through hole and the second through hole for culture. Wherein the vascularized cells can be selected from human umbilical vein endothelial cells, human lung fibroblasts, etc., and the hydrogel material can be selected from gelatin or matrigel, etc. Specifically, human Umbilical Vein Endothelial Cells (HUVEC) and human lung fibroblasts (NHLF) can be selected to be uniformly mixed in fibrinogen, thrombin and matrigel according to a certain proportion, then the mixture is injected into the second through holes, after the matrigel is solidified, a culture medium is injected from one side of the through holes at two sides, and a certain amount of culture medium is injected into the second through holes, and due to the difference of liquid level, the culture medium flows into the through holes at the other side through the channel until the liquid levels in the through holes at two sides are consistent, as shown in fig. 4. Due to surface tension, the extracellular matrix in the second through-hole will collect in the middle through-hole without overflowing into the two-sided flow channels.
The invention also provides a culture method of the vascularized organoid, which is based on the vascularized organoid chip, and comprises the steps of mixing vascularized cells, organoids and hydrogel materials, injecting the mixture into the second through hole, and respectively injecting culture mediums into at least one of the first through hole and the third through hole and the second through hole for culture. Wherein the vascularized cells can be selected from human umbilical vein endothelial cells, human lung fibroblasts, etc., the organoid comprises at least one selected from tumor organoids, pancreas organoids, liver organoids, kidney organoids, intestine organoids, stomach organoids, the tumor organoids comprise at least one selected from intestinal cancer organoids, lung cancer organoids, stomach cancer organoids, pancreatic cancer organoids, etc., and the hydrogel material can be gelatin or matrigel, etc. Specifically, organoids, human umbilical vein endothelial cells and human lung fibroblasts can be mixed uniformly in fibrinogen, thrombin and matrigel according to a certain proportion, then injected into a second through hole, and after extracellular matrix in the second through hole is solidified, culture medium is injected into the three through holes for culture. After a period of incubation, the chip may also be placed on a fluid shear cell culture automation or semi-automation device, as shown in fig. 5, the device is tilted to create a level difference, the medium will flow through the flow channel into the other side through-holes until the liquid levels in the two side through-holes are uniform, then tilted in opposite directions, and fluid shear stimulation is continued. During the culture process, the HUVEC cells spontaneously grow to form a three-dimensional vascular network by the stimulation of fluid shear force generated by the liquid level difference in the through holes at two sides. The vascular network was cultured under dynamic conditions for 10 days to form a perfusable vascular network.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1 vascularized organoid chip for generating three-dimensional vascular networks
Human umbilical vein endothelial cells and human lung fibroblasts were prepared and cultured in T25 cell culture flasks and when the cell confluence reached 100%, pancreatin was digested and counted. Respectively taking 8×10 6 Personal umbilical vein endothelial cells and 4 x 10 6 The individual lung fibroblasts were mixed in a 15ml centrifuge tube and centrifuged at 300 Xg for 5min. Carefully aspirate the supernatant, add fibrinogen gel at a concentration of 10mg/ml prepared in advance, mix well and place on ice for use. 10U/ml thrombin was diluted to 1U/ml with endothelial cell complete medium and dispensed in 10. Mu.L portions into 1.5ml EP tubes and placed on ice for use. Mixing 10 μl of fibrinogen gel containing cells with one part of complete culture medium containing thrombin, and pouring into chipAnd solidifying the two through holes in a cell culture box at 37 ℃ for 20min. After solidification, endothelial cell complete culture medium is added into the through holes at the two sides, the chip is placed on the matched equipment, the equipment is inclined to generate liquid level difference, the culture medium flows into the through holes at the other side through the runner until the liquid levels in the through holes at the two sides are consistent, and then the chip is reversely inclined to continuously generate fluid stimulation. After 10 days of culture, a perfusable three-dimensional vascular network was created, as shown in fig. 6. 70kD FITC-Dextran perfusion showed good perfusion of the vessels, as shown in FIG. 7. In addition, the perfusion is performed by using fluorescent microspheres, as shown in fig. 8, the fluorescent microspheres can reach from one side of the vascular network to the other side along the inner wall of the blood vessel, which indicates that the blood vessel has formed a closed tubular network structure, and the perfusion performance is good.
EXAMPLE 2 vascularized organoid chip for vascular organoid co-culture
Preparing 1000 intestinal cancer device tubes with the size of about 100 mu m, transferring to a 15ml centrifuge tube, adding 5ml of organoid recovery liquid, slightly shaking on ice for 30min to remove matrigel, adding DPBS to constant volume to 10ml, centrifuging at 300 Xg for 5min, discarding supernatant, adding 100 mu L matrigel, blowing, mixing, and placing on ice for later use. Vascular endothelial complete medium and intestinal cancer organoid medium were mixed according to 1:1, and mixing the materials according to the proportion for standby. 10mg/ml fibrinogen is mixed with organoid-containing matrigel in a ratio of 9:1 and placed on ice for use. 10U/ml thrombin was prepared and dispensed in 2. Mu.L portions into 1.5ml EP tubes on ice for use. Preparation of 8×10 6 Personal umbilical vein endothelial cells and 4 x 10 6 The individual lung fibroblasts were mixed in a 15ml centrifuge tube and centrifuged at 300 Xg for 5min. Carefully sucking the supernatant, adding the organoid-containing mixed gel prepared in advance, uniformly mixing, and placing on ice for later use. Mixing 8 μl of the mixed gel with the packaged thrombin for 2-3 times, rapidly pouring into the second through hole of the chip, and standing at 37deg.C for 10min for solidification. Adding mixed culture medium into the through holes at the two sides after solidification, placing the chip on matched equipment, tilting the equipment to generate liquid level difference, enabling the culture medium to flow into the through holes at the other side through a runner until the liquid levels in the through holes at the two sides are consistent, tilting reversely, and continuously generating fluid shear force stimulation. Can be used for 3 daysSee endothelial cells gradually form a vascular network surrounding the organoids (fig. 9).
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (15)
1. The vascularized organoid chip is characterized by comprising a liquid storage layer, a cell culture layer and a basal layer which are sequentially arranged;
the liquid storage layer is provided with at least one through hole unit, each through hole unit comprises a first through hole, a second through hole and a third through hole which are sequentially arranged, and the diameters of the adjacent through holes are arranged differently;
the cell culture layer is provided with at least one culture unit, the culture units are in one-to-one correspondence with the through hole units, each culture unit comprises a first micropore, a second micropore and a third micropore which are sequentially arranged, the first micropore, the second micropore and the third micropore are respectively in one-to-one correspondence with the first through hole, the second through hole and the third through hole, gaps with target heights are formed at the bottom of the second micropore so as to form a channel, and the first micropore, the second micropore and the third micropore are communicated through the channel.
2. The vascularized organoid chip of claim 1, wherein the thickness ratio of the reservoir layer, cell culture layer, and basal layer is (20-50): 2:1;
optionally, the thickness of the liquid storage layer is 5-10mm;
optionally, the cell culture layer has a thickness of 0.2-1mm;
optionally, the base layer has a thickness of 0.1-0.5mm;
optionally, the ratio of the thickness of the cell culture layer to the target height is (1.2-1.5): 1;
optionally, the target height is 200-500 μm;
optionally, the peripheral bottom of the second microwell has a wedge-shaped structure to form a gap of a target height, the width of the wedge-shaped structure gradually shrinking along the direction from the liquid storage layer to the basal layer.
3. The vascularized organoid chip of claim 1, wherein the first and/or third through holes have a larger diameter than the second through hole;
optionally, the diameters of the first through hole and the third through hole are the same;
optionally, the diameter of the second microwells is less than the diameter of the second through holes;
optionally, the diameter ratio of the first through hole, the second through hole and the third through hole is 8:5:8;
optionally, the diameter of the first through hole is 8mm, the diameter of the second through hole is 5mm, and the diameter of the third through hole is 8mm;
optionally, the diameter of the second microwells is 2mm.
4. The vascularized organoid chip of claim 1, wherein the cell culture layer is in contact with the base layer.
5. The vascularized organoid chip of claim 1, wherein the material of the reservoir layer is one or more of PMMA, PS, COC;
optionally, the material of the cell culture layer is one or more of PMMA, PS, PC;
optionally, the substrate layer is made of one or more of glass, PC and PS.
6. Use of the vascularized organoid chip of any of claims 1-5 for the preparation of a vascular model or vascularized organoid.
7. A method for preparing a vascular model, wherein the method for preparing the vascularized organoid chip according to any one of claims 1 to 5 comprises mixing vascularized cells with a hydrogel material, injecting the mixture into the second through hole, and injecting a culture medium into at least one of the first through hole and the third through hole and the second through hole, respectively, for culturing.
8. The method of claim 7, wherein the vascularized cells comprise human umbilical vein endothelial cells, human lung fibroblasts;
optionally, the hydrogel material comprises a material selected from gelatin or matrigel.
9. The method of claim 7, further comprising placing the vascularized organoid chip in a fluid shear cell culture automation device or a semi-automation device.
10. A method for preparing a vascularized organoid, wherein the method for preparing a vascularized organoid chip according to any one of claims 1 to 5 comprises mixing vascularized cells, organoids and hydrogel material and injecting the mixture into the second through-hole, and injecting a culture medium into at least one of the first through-hole and the third through-hole and the second through-hole, respectively, for culturing.
11. The method of claim 10, wherein the vascularized cells comprise human umbilical vein endothelial cells, human lung fibroblasts;
optionally, the organoids comprise at least one selected from the group consisting of tumor organoids, pancreatic organoids, liver organoids, kidney organoids, intestinal organoids, and stomach organoids;
optionally, the tumor organoid comprises at least one selected from the group consisting of intestinal cancer organoids, lung cancer organoids, gastric cancer organoids, pancreatic cancer organoids;
optionally, the organoids comprise a member selected from the group consisting of intestinal cancer organoids;
optionally, the hydrogel material comprises a material selected from gelatin or matrigel.
12. The method of claim 10, further comprising placing the vascularized organoid chip in a fluid shear cell culture automation device or a semi-automation device.
13. A vascular model, characterized in that it is obtained by the preparation method according to any one of claims 7-9.
14. A vascularized organoid, characterized in that it is obtained by the preparation method according to any one of claims 10-12.
15. Use of a vascular model as claimed in claim 13 or a vascularised organoid as claimed in claim 14 in drug screening.
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| CN109804057A (en) * | 2016-06-15 | 2019-05-24 | 米梅塔斯私人有限公司 | Cell culture apparatus and cell culture processes |
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