WO2015176653A1 - 一种适用于三维组织细胞灌注培养的生物反应器 - Google Patents

一种适用于三维组织细胞灌注培养的生物反应器 Download PDF

Info

Publication number
WO2015176653A1
WO2015176653A1 PCT/CN2015/079330 CN2015079330W WO2015176653A1 WO 2015176653 A1 WO2015176653 A1 WO 2015176653A1 CN 2015079330 W CN2015079330 W CN 2015079330W WO 2015176653 A1 WO2015176653 A1 WO 2015176653A1
Authority
WO
WIPO (PCT)
Prior art keywords
liquid
casing
static pressure
dimensional tissue
groove
Prior art date
Application number
PCT/CN2015/079330
Other languages
English (en)
French (fr)
Inventor
伍津津
Original Assignee
第三军医大学第三附属医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201420264095.3U external-priority patent/CN203855588U/zh
Priority claimed from CN201410218150.XA external-priority patent/CN103966095B/zh
Application filed by 第三军医大学第三附属医院 filed Critical 第三军医大学第三附属医院
Priority to US15/328,460 priority Critical patent/US9902929B2/en
Priority to EP15795352.2A priority patent/EP3147349B1/en
Publication of WO2015176653A1 publication Critical patent/WO2015176653A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/18—External loop; Means for reintroduction of fermented biomass or liquid percolate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/44—Means for regulation, monitoring, measurement or control, e.g. flow regulation of volume or liquid level

Definitions

  • the invention belongs to the field of cell culture devices, and relates to a bioreactor suitable for three-dimensional tissue cell perfusion culture, in particular to a bioreactor in which a plurality of tissues and organs are co-cultured.
  • the engineered tissue (or three-dimensional construct) that has been constructed needs to be cultured in vitro for a period of time before it can be transplanted or used for research.
  • the most common culture mode is to use the culture plate for regular use.
  • the static culture method of changing the liquid that is, removing the old culture solution and then adding the new culture solution, and then placing it in the incubator for static culture, the advantage is that it is suitable for small batch culture, and the liquid exchange period may vary with tissues and cells. And take the appropriate strategy.
  • the consumption of nutrient solution is progressively decreased, while the increasing metabolites cause some physiological damage to the cells; the second is to provide nutrition by means of diffusion, which has significant disadvantages of limited nutrient supply, and even leads to The cells inside the engineered tissue can not get nutrients and die; the third is the periodic fluid change to make the nutrients cyclically fluctuate, which is not conducive to the uniform growth of cells.
  • the rich capillary network framework acts as an effective mass transfer channel to provide sufficient oxygen and nutrients to the cells. Therefore, in order to ensure the supply of nutrients in vitro culture, it is necessary to establish a method for effective circulation of the culture solution.
  • roller bottle culture, rotary culture and perfusion culture and the advantages of perfusion culture are particularly remarkable. Since the culture solution continuously flows through or around the three-dimensional construct, the disadvantage of limited nutrient transfer can be overcome, and a reliable nutrient supply can be obtained even if the thickness of the construct exceeds 500 ⁇ m; on the other hand, the liquid flow during perfusion culture can be generated. A certain shear force, providing appropriate mechanical stimulation, is conducive to the development and maturation of the construct. Kalyanaraman et al.
  • the cell viability test showed that the cells were perfused at a rate of 5 ml/min. The viability of the cells was significantly better than that of the cells under static culture. The proliferation of cells by BrdU incorporation also showed that the cell proliferation at the perfusion rate of 5 ml/min or 15 ml/min was significantly better than that of the cells under static culture. The comparison also showed that only a small amount of cell proliferation was observed when the perfusion rate was 50 ml/min. Experimental studies have shown that low flow rate can increase cell viability and maintain the barrier of the epidermis, which is suitable for transplantation, while high flow rate produces high shear force, which can lead to cell degeneration. Necrosis.
  • the existing perfusion culture device simply realizes the circulation of the liquid, it does not consider the overall situation of the flow field.
  • the flow rate of the culture solution is unstable during the cycle, the change is frequent, and it cannot be guaranteed to flow through the plant to be cultivated.
  • the tissue engineering product is in a uniform flow field with low flow velocity, it is unfavorable for cell proliferation; on the other hand, the culture liquid at the edge is not easy to flow, the flow field in the culture device is not uniform, the nutrient transfer is uneven, and the cell growth is uneven; In the case of uneven flow rate, the shear force exerted by the liquid on the cells is different.
  • the present inventors disclosed a perfusion bioreactor, including culture, in the Journal of Third Military Medical University, Vol. 35, No. 7, "Design and Preliminary Simulation of a Novel Skin Tissue Engineering Perfusion Bioreactor Based on Quasi-Static Planar Flow Field".
  • Base bottle peristaltic pump, sterile silicone tube, buffer tank, overflow dam, culture chamber and drain groove.
  • the bioreactor realizes a quasi-static plane flow field by overflowing the culture liquid, and the culture liquid enters the buffer tank, overflows into the culture chamber through the high overflow dam, thereby feeding the tissue cells in the culture, and then the culture liquid After the low overflow dam overflows to the drain groove, the final culture liquid enters the next reactor buffer tank.
  • the flow velocity flowing into the buffer tank is not constant and the head pressure is unstable due to unevenness, and the discharge slope of the high overflow dam cannot be absolutely flat, so the culture solution
  • the buffer tank accumulates potential energy and flows through the vent slope of the high overflow dam, one or several uneven fluids are formed, and even more, some regions of the sloping slope are cut off at a lower flow rate.
  • the medium appears uneven flow field when entering the flow field of the culture section, causing cells in some areas to get no or little nutrients and cannot be eliminated in time.
  • Damage or death of metabolites; and existing screens are mostly fixed on the bottom wall of the reaction chamber, and certain brackets are required to fix the screen, thereby causing the screen frame to be fixed in the direction of the flow field.
  • the inability to form a uniform flow field on the back side of the pillars also has a certain effect on some of the culture areas.
  • the liquid flow field flowing through the tissue cells to be cultured is uniform, and the flow rate is always kept at a low flow rate, which is essential for improving the quality of tissue engineering products, shortening the cultivation time, and improving the cultivation efficiency.
  • the technical problem to be solved by the present invention is to provide a bioreactor suitable for three-dimensional tissue cell culture, which can realize a suitable flow rate of a culture liquid in a plane flow field, and the flow field is uniform and stable; the present invention also provides A laminated bioreactor with compact structure, good expandability and more optimization.
  • a bioreactor suitable for three-dimensional tissue cell culture includes a liquid storage tank, a peristaltic pump, and at least one reaction chamber including a rectangular parallelepiped box body and a lid body; a shut-off device is fixed at one end of the box body.
  • the box body constitutes a static pressure groove
  • the other end is fixed with an overflow inserting plate
  • the box body constitutes a drain groove
  • a sieve which is not in contact with the intercepting device and the overflow plug plate is fixed between the static pressure groove and the drain groove
  • the grid frame is fixed on the box body, the screen mesh is fixed in the screen frame; the reaction area is formed between the intercepting device and the overflow inserting plate; the reaction chamber is provided with a static pressure groove and can directly inject the liquid
  • the liquid inlet hole of the static pressure tank is provided with a liquid outlet hole communicating with the drain groove on the wall of the box body and capable of directly discharging the liquid out of the drain groove; the intercepting device is provided with a plurality of evenly arranged energy along the same horizontal section
  • the liquid in the static pressure tank is evenly introduced into the water permeable hole of the reaction zone; the side wall of the static pressure tank is provided with an overflow port, and the side wall of the box body is provided with an excess of the static pressure tank overflowing
  • Liquid is discharged through the inlet
  • the drainage trough of the flow trough; the intercepting device is equal to or lower than the depth of the casing, the screen frame is lower than the height of the intercepting device, the overflow inserting plate is higher than or equal to the height of the screen mesh on the screen frame, and the lower edge of the overflow opening Lower than the height of the upper surface of the intercepting device and higher than the height of the upper surface of the overflow inserting plate, the inlet opening and the upper edge of the water permeable opening to form a stepped differential liquid in the static pressure groove, the reaction zone and the drain groove
  • the infusion tube connects the liquid storage tank and the peristaltic pump in sequence between the liquid outlet hole and the liquid inlet hole.
  • the screen frame is fixed on the two side walls of the box body, and the intercepting device, the overflow plugging plate and the screen frame are respectively engaged with the box body, and the intercepting device and the overflow plugging plate are in contact with the box body.
  • a stepped clip is arranged on the surface of the screen frame contacting the side wall of the box body, and the side wall and the bottom wall of the box body are provided with engaging grooves for engaging with the clip.
  • the overflow port of the present invention comprises a U-shaped groove and a U-shaped groove provided by the U-shaped groove in the static pressure groove, which can be rotated by the positioning shaft and can adjust the static pressure groove by adjusting the height of the lower edge of the U-shaped groove.
  • a blank for the inner liquid level By providing an overflow port, the height of the liquid surface in the static pressure tank can be conveniently adjusted to control the uniform flow of the culture liquid through the water permeable hole of the intercepting device.
  • the water permeable holes are a plurality of identical strip holes, circular holes, semicircular holes or irregular holes uniformly arranged.
  • the mesh plane of the screen is provided with a specification mold having the same height as the bracket of the screen frame, and the specification mold may be a square, a circle, a triangle or a profiled structure.
  • the preferred overflow insert of the present invention has a screen height of 0 to 4 mm above the screen frame.
  • the shut-off device is a gate with a water-permeable hole, and the water-permeable hole is located at a middle and a lower part of the gate.
  • the intercepting device is a turbulent dam with a venting slope
  • the turbulent dam comprising an upper dam body and a lower dam body, the side of the lower dam body adjacent to the screen frame having a venting slope
  • the water permeable hole is located in the upper dam body of the turbulent dam, and the leakage slope is at an angle of 15° to 25° with the bottom surface of the box body, and the height of the bleed slope is 1/3 to 1/2 of the height of the turbulent dam.
  • the culture liquid can not only generate a uniform flow field at a uniform speed when passing through the water-permeable hole, but also is more convenient to disassemble and clean.
  • Another improvement of the present invention is to provide a laminated casing which can be mass-produced, which is formed by mutually opposingly fitting the casings together.
  • the outer side of the upper surface of the side wall of the casing is provided with a stepped flange, and the periphery of the bottom wall of the casing is provided with an object fitting recess which is fitted to the flange of the upper surface of the side wall.
  • the plurality of casings are mated to each other to form a laminated casing of the casing, and the cover is covered on the top casing of the laminated casing, and the casing and the upper casing constitute a reaction chamber, and the bottom wall and the phase of the upper casing
  • the box bodies in the lower part of the stack together form a reaction chamber; the liquid outlet holes of the upper box body in the stacking box body are connected to the liquid inlet holes of the lower box body through the infusion tube, and the liquid outlet holes of the box body at the bottom of the stacking box body pass through the infusion tube,
  • the liquid storage tank and the peristaltic pump are connected to the inlet hole of the reaction chamber at the top; the laminated box body and the cover body, the infusion tube, the liquid storage tank and the peristaltic pump together form a laminated bioreactor.
  • the liquid outlet hole of the upper casing in the laminated casing is disposed on the bottom wall of the drain groove, and communicates with the static pressure groove of the lower casing of the adjacent fitting body to constitute the lower portion.
  • the liquid inlet hole of the casing is such that liquid flows directly from the upper casing of the laminated casing into the static pressure groove of the lower casing.
  • the structure of the stacked bioreactor is simplified by the liquid outlet opening in the bottom wall.
  • the number of the casings in the preferred laminated casing is 2 to 20;
  • the laminated bioreactor can also be provided on the outside of the laminated box body, which is composed of a box body and a box cover and has a hole for the infusion tube to cooperate with the top box static pressure tank and the bottom box body liquid outlet hole.
  • the sterile reaction box is located in the box, and the liquid storage tank and the peristaltic pump are located outside the reaction box.
  • the above-mentioned external bioreactor with a reaction tank can also be provided with a circulating water jacket around the reaction tank, and the liquid storage tank and the peristaltic pump are located outside the circulating water jacket. So that the inside of the bioreactor is at a constant temperature.
  • the culture liquid when the culture liquid is injected into the static pressure tank, the culture liquid is accumulated in the static pressure tank, the height of the overflow port is adjusted, and excess liquid overflows from the overflow port to ensure stable pressure in the static pressure tank.
  • the water permeable hole on the intercepting device slowly flows into the bottom of the screen frame, and there is no barrier of the screen frame bracket in the flow field, so that a uniform steady-state plane flow field is formed in the middle of the reaction box body, and uniform nutrient diffusion is performed; It is naturally immersed into the bottom of the screen frame after forming a stable pressure in the static pressure tank, so the culture liquid flowing into the bottom of the screen frame is always maintained at a low uniform and stable flow rate, which can be on the screen frame.
  • the three-dimensional tissue organ or tissue engineering product to be cultured or produced provides uniform shear force for cell proliferation and tissue development.
  • the invention relates to a bioreactor suitable for perfusion culture of three-dimensional tissue organs, cells or tissue engineering products, and is particularly suitable for perfusion culture of tissue engineering skin or other membranous tissues such as cornea, heart valve and various tissues, organs and cells. Includes co-cultivation.
  • the working principle is as follows: firstly, the raw material solution of the three-dimensional tissue organ or tissue engineering product to be cultivated or produced is injected into the sieve frame, and the specification mold is placed on the sieve frame to make a specific shape specification, and the specification mold is taken out and covered.
  • the cover body is then injected into the static pressure tank, and the culture liquid is accumulated in the static pressure tank, and the excess liquid overflows from the overflow port to ensure a stable pressure in the static pressure tank, and then flows through the water permeable hole on the intercepting device to the screen frame.
  • a uniform steady-state planar flow field is formed in the middle of the reaction box body, and the three-dimensional tissue organ or tissue engineering product to be cultivated or produced on the screen frame is just immersed by the flowing nutrient solution, and the liquid level can be appropriately lowered to make the screen frame
  • the three-dimensional tissue to be cultured is always in the state of gas-liquid interface culture, providing sufficient nutrients and suitable shearing force for the three-dimensional tissue organ or tissue engineering product to be researched or produced, and the liquid level in the middle of the reaction box is high.
  • the culture liquid flows into the drain tank through the top of the overflow plug plate, and then flows into the liquid storage tank from the liquid outlet hole, and then pumps the liquid into the static pressure tank through the peristaltic pump. Now closed cycle training.
  • the present invention has the following advantages: 1. By providing an overflow port and a shut-off device with a water-permeable hole capable of uniformly introducing the liquid in the static pressure tank into the reaction zone uniformly, the culture fluid easily breaks through when it flows through the water-permeable hole.
  • the tension of the self forms a uniform flow path, so that the culture liquid always maintains a low stable uniform flow rate when flowing through the screen frame, and a stable and uniform plane flow field is formed in the reaction zone of the reaction box, which is favorable for cell proliferation;
  • the bioreactor of the invention can be used independently or in series or in multiple layers, and has good expandability, can maintain the biological characteristics of the three-dimensional tissue and organs, ensure the quality of the three-dimensional tissue organ or product, and improve the quality. The efficiency of basic research or production; 3.
  • the shut-off device, specification mold, screen frame and overflow insert of the invention can be removed and replaced according to actual needs, and is not only suitable for tissue engineering skin culture, but also for film-like materials such as cornea, Culture of heart valves, nerves, blood vessels, mesentery, or other tissues and organs, and can simultaneously culture different types of tissues and organs, dismantle Characteristics of the grid can be used for culturing adherent cells grown seed having versatility, wide application range.
  • Figure 1 is a perspective view of a reaction chamber of the present invention
  • Figure 2 is a perspective view of the body wall of the casing of the present invention.
  • FIG. 3 is a schematic view showing the structure of the bioreactor of the present invention.
  • Figure 4 is a perspective view of the gate of the present invention.
  • Figure 5 is a perspective view of a turbulent dam in the present invention.
  • Figure 6 is a perspective view of the screen and the screen frame of the present invention.
  • Figure 7 is a schematic structural view of a laminated bioreactor of the present invention.
  • Figure 8 is a schematic structural view of Embodiment 3 of the present invention.
  • Figure 9 is a schematic view of the flow field of the present invention.
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • a bioreactor suitable for three-dimensional perfusion culture of tissue cells including a liquid storage tank 28, a peristaltic pump 29, and at least one including an open top a reaction chamber composed of a rectangular parallelepiped box body 1 and a lid body 2; a shut-off device 3 is fixed at one end of the box body 1, and a static pressure groove 7 is formed with the box body 1, and an overflow plug plate 8 is fixed at the other end, and the box body 1 is formed Between the sump 9, the static pressure tank 7 and the sump 9, a screen frame 20 which is not in contact with the shut-off device 3 and the overflow plug 8 is fixed; the screen frame 20 is stuck on both sides of the casing 1 On the wall, the screen 21 is fixed in the screen frame 20; the intercepting device 3 is provided with a plurality of evenly arranged horizontally at the same level to uniformly level the liquid in the static pressure tank 7.
  • the water permeable hole 6 is a plurality of uniformly arranged strip holes, circular holes, semicircular holes or special-shaped holes;
  • the static pressure groove 7 is provided with an overflow on the side wall shared with the casing 1
  • the mouth 13 and the overflow port 13 include a U-shaped groove and a blocking piece 15 disposed in the static pressure groove 7 and capable of rotating the U-shaped groove by the positioning shaft 14 and adjusting the height and size of the U-shaped groove, in the casing 1
  • the side wall is provided with a draining liquid 16 capable of discharging the excess liquid overflowing the static pressure tank 7 through the overflow port 13 into the drain tank 9 through the inlet port 17;
  • the reaction chamber is provided with a static pressure tank 7 and can be connected
  • the liquid is directly injected into the liquid inlet hole 18 of the static pressure tank 7, and a liquid passage hole 19 communicating with the drain groove 9 and discharging the liquid directly from the drain groove 9 is provided on the wall of the casing 1;
  • the shut-off device 3 is directly
  • the shut-off device 3 in this embodiment is a gate 4, and the peristaltic pump 29 injects the culture liquid in the liquid storage tank 28 into the static pressure tank 7, and injects it higher than the water-permeable hole 6.
  • the height of the culture liquid overflows from the overflow port 13 and flows into the drain tank 9 through the overflow tank and the inlet port 17, and the culture liquid passes through the water permeable hole 6 in the gate 4 and slowly flows into the bottom of the screen frame 20.
  • a uniform planar flow field is formed in the middle of the casing 1 (see FIG. 9), uniform nutrient diffusion is performed, and the pseudo-production tissue engineering product on the screen 21 is subjected to uniform shear force to avoid nutrient diffusion due to the culture solution. Cell death caused by uneven or excessive shear forces.
  • the side walls and the bottom wall of the casing 1 have engaging recesses 24, and the gate 4, the screen frame 20 and the overflow insert 8 are capable of being inserted into the
  • the fixed latch 23 is engaged in the engaging recess 24, and the shutter 4, the screen frame 20 and the overflow insert 8 are detachably fixed to the casing 1.
  • the overflow insert 8 is higher than the screen 21 on the screen frame 20 by 0 to 4 mm, and the liquid level in the middle of the box 1 can be adjusted by replacing the overflow insert 8 to provide a gas-liquid interface culture environment for the tissue engineering skin.
  • the screen frame 20 may be a whole or a plurality of mutually independent screen frames 20 arranged at a certain interval.
  • the size mold 22 is engaged on the screen 21 of the screen frame 20 and is equal to the height of the frame of the screen frame 20.
  • the size mold 22 can be designed as a square, circular, triangular or profiled structure depending on the product variety.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • the function of the gate 4 can also be realized by the turbulent dam 5, which includes the upper dam body. 10 and the lower dam body 11, the side of the lower dam body 11 adjacent to the screen frame 20 has a venting slope 12, and the water permeable hole is located at the upper dam body 10 of the turbulent dam, and the venting slope 12 is opposite to the bottom surface of the box body
  • the angle of the sloping slope 12 is 1/3 to 1/2 of the height of the turbulent dam at an angle of 15° to 25°.
  • the height of the discharge slope 12 can be adjusted to control the flow rate of the liquid to the screen frame 20 at a low flow rate. status.
  • Embodiment 3 is a diagrammatic representation of Embodiment 3
  • the outer side of the upper surface of the side wall of the casing 1 of the bioreactor in the first embodiment or the second embodiment is provided with a stepped flange 25, and the casing 1 is provided.
  • the periphery of the bottom wall is provided with an object fitting recess 26 that is fitted to the flange 25 of the upper surface of the side wall.
  • the plurality of casings 1 are butt-joined by reverse fitting to form a laminated casing 30, and the cover 2 is covered on the casing 1 at the top; the bottom wall and the lower casing of the casing 1 in the upper portion of the adjacent laminated casing 30 1 constituting a reaction chamber, the top case 1 and the cover 2 constitute a reaction chamber; wherein the liquid outlet 19 of the case 1 is located on the bottom wall of the case 1, and the liquid outlet 19 formed on the bottom wall of the case 1 constitutes
  • the liquid inlet hole 18 of the lower casing 1 can directly input the liquid in the sump 9 of the upper casing 1 into the static pressure tank 7 of the lower casing 1, and the liquid inlet 18 of the top casing 1 is located therein.
  • the liquid discharged from the liquid outlet 19 of the bottom casing 1 is directly pumped into the static pressure tank 7 of the top casing 1 by the peristaltic pump 29; the number of the casings 1 in the laminated casing 30 may be 2 to 20, laminated box 30 Together with the lid body 2, the infusion tube 27, the reservoir tank 28, and the peristaltic pump 29, a laminated bioreactor is constructed.
  • the culture solution flows from the static pressure tank 7 of the top casing 1 through the intercepting device 3, the screen frame 20, the overflow inserting plate 8, and then flows into the draining tank 9, and the excess liquid flows in from the overflow port 13 through the draining groove 16.
  • the drain tank 9 is then directly injected into the static pressure tank 7 of the lower casing 1 through the liquid outlet hole 19, and then flows into the liquid storage tank 28 from the liquid outlet hole 19 of the casing 1 at the bottom, and then the liquid storage tank is passed through the peristaltic pump 29.
  • the culture solution in 28 is pumped into the static pressure tank 7 of the top case 1 to effect a closed cycle of the culture solution.
  • the number of the casings 1 installed in the middle can be increased or decreased as needed.
  • the case 1 in the laminated case 30 of the present embodiment can also use the case 1 as shown in Figs. 1 and 3, and in use, the liquid discharge hole 19 of the case 1 and the adjacent lower case of the upper part of the case 30 are laminated.
  • the inlet holes 18 of the body 1 can also be connected by an infusion tube 27.
  • Embodiment 4 is a diagrammatic representation of Embodiment 4:
  • Figure 8 is a view showing the laminated case 30 of the third embodiment placed in a sterile reaction box.
  • the reaction box is composed of a case 31 and a cover 32.
  • the reaction case is opened with the top case 1 of the laminated case 30.
  • the liquid hole 18 and the liquid outlet hole 19 of the bottom case 1 are engaged with a hole 34 through which the infusion tube 27 can pass, the laminated case 30 is located in the case 31, and the liquid storage tank 28 and the peristaltic pump 29 are located outside the case 31.
  • a circulating water jacket 33 may also be provided at the periphery of the reaction tank, and the liquid storage tank 28 and the peristaltic pump 29 are located outside the circulating water jacket 33.
  • the reaction box can make the laminated box 30 in a sterile environment
  • the circulating water jacket can keep the laminated box at a constant temperature
  • the culture liquid circulates inside and outside the reaction box
  • the liquid flow rate flowing through the middle of the box 1 is always at
  • the low flow rate state provides sufficient nutrients and suitable shearing force for the intended production of tissue engineering products or three-dimensional tissue organs to be cultured in the reaction chamber.
  • the circulating water jacket 33 disposed outside the reaction tank ensures a constant temperature reaction state in the reaction chamber.
  • the present invention cooperates with the overflow port 13 by providing the gate 4 or the turbulent dam 5, and the culture fluid can form a stable pressure in the static pressure tank 7 at any perfusion rate, flowing through the screen frame 20
  • the flow rate is always kept low, and a uniform steady-state plane flow field is formed in the middle of the casing 1 to overcome the existing perfusion type.
  • the uneven flow field and uneven nutrient transfer in culture can cause cell necrosis, improve product quality and shorten cultivation time, and can also be used for co-culture research and drug screening of three-dimensional constructs or tissues and organs.
  • the invention also has the characteristics of good expansibility, strong versatility and wide application range, realizes standardized cultivation of tissue engineering products, greatly reduces labor intensity, improves production efficiency, and has good promotion and application value.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Sustainable Development (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Clinical Laboratory Science (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Mechanical Engineering (AREA)
  • Cell Biology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

提供了一种适用于三维组织细胞灌注培养的生物反应器，通过输液管依次将反应室、储液罐和蠕动泵连接；反应室包括侧壁、底壁、截流装置、溢流插板、筛网架、静压槽、泄流槽、引流槽等构成的盒体和盖体，截流装置为闸门或堰流坝并且沿其纵向截面同一水平上开设有若干均匀排列的透水孔，静压槽侧壁设有与引流槽相配合的溢流口,筛网架固定在盒体的两侧壁上。还提供了一种层叠结构的生物反应器。

Description

一种适用于三维组织细胞灌注培养的生物反应器 技术领域
本发明属于细胞培养装置领域，涉及一种适用于三维组织细胞灌注培养的生物反应器，尤其是多种组织器官共培养的生物反应器。
背景技术
通常情况下已构建好的工程化组织(或称三维构建物)需要在体外进行一段时间的培养，使其成熟后才能移植或供研究用，而目前最常用的培养模式是采用培养板进行定期换液的静态培养法，即先移去旧培养液再添加新培养液，然后放入培养箱中予以静置培养，其优点是适合小批量的培养，换液周期可随组织、细胞的不同而采取相应的策略。但其缺点显而易见：一是营养液的消耗呈进行性下降，而代谢产物不断增加，会对细胞造成一些生理性损伤；二是依靠弥散方式提供营养，存在营养供应受限的显著缺点，甚至导致工程化组织内部的细胞得不到营养而死亡；三是周期性换液使营养成分呈周期性波动，不利于细胞的均匀生长。
在体内，丰富的毛细血管网构架起一个有效的传质通道，为细胞提供充足的氧和营养物。因此体外培养时为了保证营养物的供应，须建立培养液有效循环的方法，目前有以下几个策略：滚瓶培养、旋转培养和灌注式培养，其中灌注式培养的优点尤为显著，一方面，由于培养液连续地流经三维构建物内部或周围，能够克服营养物质传递受限的缺点，即使构建物厚度超过500μm也能得到可靠的营养供应；另一方面，灌注培养时的液流能够产生一定的剪切力，提供适当的机械刺激，有利于构建物的发育和成熟。Kalyanaraman等(Kalyanaraman BM，Dorothy MS，Steven TB.Medium Flow Rate Regulates Viability and Barrier Function of Engineered Skin Substitutes in Perfusion Culture.Tis Eng：Part A.2008，14(5)：583-93)已设计了一种生物反应室，进、出水口管是从培养室的底面两侧分别接入培养室，通过设置出液管的高度来控制培养室液面的高度，即培养室液面高于出水管后液体将溢进管子而流出，通过对构建的组织工 程皮肤进行熟化培养，结果表明在灌注速度为5ml/min或15ml/min培养条件下，组织学结构好于静态培养，其中，通过细胞活力检测结果表明，在5ml/min的灌注速度下的细胞活力明显好于静态培养下的细胞活力；采用BrdU掺入法检测细胞的增生情况也表明，在灌注速度5ml/min或15ml/min时的细胞增生情况明显好于静态培养下的细胞增生情况。对比还发现灌注速度50ml/min时只显示少量细胞增生，实验研究证明了低流速能够增加细胞活力和维持表皮的屏障，适合于移植，而高流速产生高的剪切力则可导致细胞的变性坏死。
尽管现有的灌注式培养装置简单地实现了液体的循环，但却没有考虑流场的整体情况，一方面，因培养液在循环过程中流速不稳定，变化频繁，无法保证在流经待培植组织工程产品时处于低流速的均匀流场，对细胞增生不利；另一方面，处于边缘的培养液不易流动，培养装置内流场不均一，营养传递不均，容易造成细胞生长不均匀；况且，在流速不均的情况下液体对细胞所施加的剪切力大小不同，流速快时剪切力过大而使细胞易受到损伤，流速过低(接近静止)时又不利于细胞增生。本发明人在第三军医大学学报第35卷第7期《基于准静态平面流场的新型皮肤组织工程灌注式生物反应室设计与初步模拟》中公开了一种灌注式生物反应器，包括培养基瓶、蠕动泵、无菌硅胶管、缓冲槽、溢流坝、培养室和泄流沟。该生物反应器是培养液通过溢流的方式来实现准静态平面流场，培养液进入缓冲槽，经高溢流坝溢流进入培养室，从而在培养中对组织细胞进行供养，然后培养液经低溢流坝溢出至泄流沟，最后培养液进入到下一层反应器缓冲槽中。利用这种生物反应器，由于培养液存在表面张力，流入缓冲槽的流速不恒定和不均匀所造成的压头不稳定，加之高溢流坝的泄流斜面不可能处于绝对平整，所以培养液在缓冲槽积蓄势能后流经高溢流坝的泄流斜面时，形成一股或几股不均匀的流液，更有甚者，在较低流速下泄流斜面的某些区域形成了断流以致培养基在进入培养段流场时出现了不均匀的流场，造成某些区域的细胞得不到或得到很少的营养物质以及不能及时排除 代谢产物而出现损伤或者死亡；而且现有的筛网大多固定在反应室的底壁上，必定需要一定的支架用来固定筛网，由此就会造成在流场方向上，固定筛网架的支柱后侧不能形成均匀的流场也会对部分培养区域造成一定的影响。
由此可见，在灌注式培养中，使流经待培育组织细胞的液体流场均匀，流速始终保持在低流速，对提高组织工程产品质量、缩短培植时间、提高培育效率至关重要。
发明内容
针对上述问题，本发明要解决的技术问题是提供一种适用于三维组织细胞培养的生物反应器，该生物反应器能够实现平面流场的培养液流速合适，流场均匀稳定；本发明还提供了一种结构紧凑、扩展性好、更加优化的层叠生物反应器。
为了解决上述技术问题，适用于三维组织细胞培养的生物反应器，包括储液罐、蠕动泵和至少一个包括顶部敞口的长方体盒体和盖体组成的反应室；盒体一端固定一截流装置，与盒体构成静压槽，另一端固定一溢流插板，与盒体构成泄流槽，静压槽和泄流槽之间固定有与截流装置和溢流插板均不接触的筛网架；筛网架固定在盒体上，筛网固定在筛网架当中；截流装置与溢流插板之间形成反应区域；反应室上设置一与静压槽连通并能将液体直接注入静压槽的进液孔，在盒体的壁上设置一与泄流槽连通并能将液体直接排出泄流槽的出液孔；截流装置沿纵向截面同一水平上开设有若干均匀排列的能将静压槽内的液体匀速均匀引入反应区域的透水孔；静压槽的侧壁上设置有溢流口，在盒体的侧壁上设置有能将静压槽经溢流口溢出的多余液体通过进流口排入泄流槽的引流槽；截流装置等于或低于盒体的深度，筛网架低于截流装置的高度，溢流插板高于或等于筛网架上筛网的高度，溢流口的下缘低于截流装置的上表面的高度并高于溢流插板上表面、进流口和透水孔上缘的高度，以在静压槽、反应区域和泄流槽中形成阶梯状的差位液面；输液管将储液罐、蠕动泵依次连接在出液孔与进液孔之间。
本发明中筛网架固定在盒体的两侧壁上，截流装置、溢流插板和筛网架分别与盒体通过卡合连接，截流装置、溢流插板与盒体接触的面上以及筛网架与盒体侧壁接触的面上设有台阶状的卡子，盒体的侧壁与底壁上设置有与卡子相卡合的卡合凹槽。通过卡合连接方便反应室的拆卸与清洗，而且将筛网架固定在盒体的侧壁上与以前的筛网架固定在底壁上相比，减少了筛网架支架对反应区域内培养液的阻挡，形成了均匀的流场。
优选的本发明中的溢流口包括U型槽以及在静压槽内U型槽旁设置的能通过定位轴转动盖合U型槽并能通过调节U型槽下缘高度来调节静压槽内液面高度的挡片。通过设置溢流口，能够方便的调节静压槽内液面的高度以控制培养液均匀地流过截流装置的透水孔。
本发明中透水孔为若干个均匀排列的相同的条形孔、圆形孔、半圆形孔或异形孔。通过设置均匀排列的透水孔，可以实现培养基进入反应区域时形成均匀的流道，以实现均匀的流场。
本发明中，筛网的网平面上设置有与筛网架的支架等高的规格模具，规格模具可以为方形、圆形、三角形或异形结构。
本发明中优选的溢流插板高出筛网架上筛网高度为0～4mm。通过设置略高于筛网高度的溢流插板，可以使培养液与筛网上的培养物形成良好的接触。
本发明的改进方案中，截流装置为带有透水孔的闸门，透水孔位于闸门的中下部。
本发明的另一改进方案中，截流装置为带有泄流斜面的堰流坝，堰流坝包括上坝体和下坝体，下坝体与筛网架相邻的一侧具有泄流斜面，透水孔位于该堰流坝的上坝体，该泄流斜面与盒体底面呈15°～25°夹角，泄流斜面的高度为堰流坝高度的1/3～1/2。
通过上述两种改进的截流装置，与现有的截流装置相比，培养液通过透水孔时不仅能够产生匀速均匀的流场，而且拆卸、清洗更加方便。
本发明的另一种改进方案是提供了一种能够批量生产的层叠盒体，该层叠盒体是通过盒体相互反向嵌合在一起构成。
本发明中盒体的侧壁的上表面的外侧设有台阶状的凸缘，盒体的底壁的四周设有与侧壁上表面的凸缘相嵌合的对象嵌合凹槽。多个盒体通过嵌合对接构成盒体的层叠盒体，盖体盖合在层叠盒体的顶部盒体上，盖体与上部的盒体构成反应室，上部的盒体的底壁与相邻下部的盒体共同组成一个反应室；层叠盒体中上部盒体的出液孔通过输液管与下部盒体的进液孔连接，层叠盒体底部的盒体的出液孔通过输液管、储液罐、蠕动泵与顶部的反应室的进液孔相连接；层叠盒体与盖体、输液管、储液罐、蠕动泵共同组成层叠生物反应器。
优选的上述改进方案中层叠盒体中的上部的盒体的出液孔设置在泄流槽的底壁上，并与其相邻嵌合的下部的盒体的静压槽相连通，构成了下部盒体的进液孔，以便液体从层叠盒体的上部盒体直接流入下部盒体的静压槽当中。通过开设在底壁上的出液孔，简化了层叠生物反应器的结构。
优选的层叠盒体中的盒体的数目为2到20个；
上述层叠生物反应器还可以在层叠盒体的外部设有由箱体和箱盖组成并带有与顶部盒体静压槽和底部盒体出液孔相配合的能使输液管通过的孔道的无菌反应箱，层叠盒***于箱体中，储液罐和蠕动泵位于反应箱外。通过设置反应箱，可以使层叠生物反应器在无菌密闭的环境下进行反应。
上述外部带有反应箱的生物反应器还可以在反应箱***设有循环水套，储液罐和蠕动泵位于循环水套外。以便生物反应器内部处于恒温状态。
在上述技术方案中，当向静压槽内注入培养液，培养液在静压槽内积蓄，调节溢流口的高度，多余液体从溢流口溢出，保证静压槽内形成稳定压力后随截流装置上的透水孔缓慢地流入筛网架底部，而且流场中没有了筛网架支架的阻挡，使反应盒体中部形成均匀的稳态平面流场，进行均匀的营养扩散；由于培养液是从静压槽内形成稳定压力后自然浸流入筛网架底部的，故流入筛网架底部的培养液始终保持在较低的均匀稳定流速，该流速能够为筛网架上的 拟培养或生产的三维组织器官或组织工程产品提供均匀一致的剪切力，有利于细胞增生和组织发育。
本发明是一种适用于三维组织器官、细胞或组织工程产品灌注培养的生物反应器，尤其适用于对组织工程皮肤或其他膜状组织如角膜、心瓣膜以及多种组织器官、细胞进行灌注培养包括共培养。其工作原理为：先将拟培养或生产的三维组织器官或组织工程产品的原料溶液注入筛网架上，在筛网架上放入规格模具制成特定形状的规格，取出规格模具，盖上盖体；然后向静压槽内注入培养液，培养液在静压槽内积蓄，多余液体从溢流口溢出，保证静压槽内形成稳定压力后经截流装置上的透水孔流向筛网架，使反应盒体中部形成均匀的稳态平面流场，筛网架上的拟培养或生产的三维组织器官或组织工程产品恰好被流动的营养液浸没，也可适当降低液面使筛网架上待培养的三维组织始终处于气液界面培养状态，为拟研究或生产的三维组织器官或组织工程产品提供充足的营养和适宜的剪切力，当反应盒体中部的液面蓄水高度高于溢流插板高度时，培养液经溢流插板顶部流入泄流槽，进而从出液孔流入储液罐内，通过蠕动泵再将液体泵入静压槽内，实现封闭式的循环培养。
由于以上结构，本发明具有以下优点：1.通过设置溢流口和带有能将静压槽内的液体匀速均匀引入反应区域的透水孔的截流装置，培养液流过透水孔时，容易突破自身的张力形成均匀的流道，使培养液在流经筛网架时始终保持较低的稳定的均匀流速，反应盒体反应区域内形成稳定均匀的平面流场，有利于细胞增生；2、本发明的生物反应器可以单个独立使用，也可以多个串联或层叠使用，具有良好的扩展性，既能维持三维组织器官的生物学特性，保证了三维组织器官或产品的质量，又提高了基础研究或生产的效率；3.本发明的截流装置、规格模具、筛网架和溢流插板可根据实际需要拆取更换，不仅适合组织工程皮肤的培养，也适合膜状材料如角膜、心瓣膜、神经、血管、肠系膜，或其它组织器官等的培养，而且可同时培养不同类型的组织器官，拆除筛网架后还可用于培养贴壁生长的种子细胞，具有通用性强、适用范围广的特点。
附图说明
图1是本发明反应室的立体示意图；
图2是本发明盒体体壁的立体示意图；
图3是本发明生物反应器的结构示意图；
图4是本发明中闸门的立体示意图；
图5是本发明中堰流坝的立体示意图；
图6是本发明中筛网及筛网架的立体示意图；
图7是本发明层叠生物反应器的结构示意图；
图8是本发明实施例三的结构示意图；
图9是本发明流场的示意图；
图中：1、盒体；2、盖体；3、截流装置；4、闸门；5、堰流坝；6、透水孔；7、静压槽；8、溢流插板；9、泄流槽；10、上坝体；11、下坝体；12、泄流斜面；13、溢流口；14、定位轴；15、挡片；16、引流槽；17、进流口；18进液孔；19、出液孔；20、筛网架；21、筛网；22、规格模具；23卡子；24、卡合凹槽；25、凸缘；26、嵌合凹槽；27、输液管；28、储液罐；29、蠕动泵；30、层叠盒体；31、箱体；32、箱盖；33、循环水套；34、孔道。
具体实施方式
下面结合附图对本发明做进一步说明。
实施例一：
参见图1、图2、图3、图4、图6、图9，本发明一种适用于组织细胞三维灌注培养的生物反应器，包括储液罐28、蠕动泵29和至少一个包括顶部敞口的长方体盒体1和盖体2组成的反应室；盒体1一端固定一截流装置3，与盒体1构成静压槽7，另一端固定一溢流插板8，与盒体1构成泄流槽9，静压槽7和泄流槽9之间固定有与截流装置3和溢流插板8均不接触的筛网架20；筛网架20卡和在盒体1的两侧壁上，筛网21固定在筛网架20当中；截流装置3沿纵向截面同一水平上开设有若干均匀排列的能将静压槽7内的液体匀速均匀 引入反应区域的透水孔6，透水孔6为若干个均匀排列的条形孔、圆形孔、半圆形孔或异形孔；静压槽7与盒体1共用的侧壁上设置有溢流口13，溢流口13包括U型槽以及在静压槽7内设置的能通过定位轴14转动盖合U型槽并能调节U型槽高度和大小的挡片15，在盒体1的侧壁上设置有能将静压槽7经溢流口13溢出的多余液体通过进流口17排入泄流槽9的引流槽16；反应室上设置一与静压槽7连通并能将液体直接注入静压槽7的进液孔18，在盒体1的壁上设置一与泄流槽9连通并能将液体直接排出泄流槽9的出液孔19；截流装置3等于或低于盒体1的深度，筛网架20低于截流装置3的高度，溢流插板8高于或等于筛网架20上筛网21的高度，溢流口13的下缘低于截流装置3的上表面的高度并高于溢流插板8上表面、进流口17和透水孔6上缘的高度；输液管27将储液罐28、蠕动泵29依次连接在出液孔19与进液孔18之间。
如图2、图3所示，本实施例中的截流装置3为闸门4，蠕动泵29将储液罐28中的培养液泵向静压槽7内的过程中，注入高于透水孔6高度的培养液，多余液体从溢流口13溢出，通过溢流槽、进流口17流入泄流槽9中，培养液通过闸门4上的透水孔6，并缓慢地流入筛网架20底部，使盒体1中部形成均匀的平面流场(参见图9)，进行均匀的营养扩散，并使筛网21上的拟生产组织工程产品受到均匀一致的剪切力，避免因培养液营养扩散不均匀或剪切力过高而造成的细胞死亡。
为使本发明适用范围更广、拆装清洗更便捷，盒体1侧壁和底壁上具有卡合凹槽24，闸门4、筛网架20和溢流插板8上具有能够***所述卡合凹槽24内卡合固定的卡子23，闸门4、筛网架20和溢流插板8可分离的固定在盒体1上。溢流插板8高于筛网架20上筛网21为0～4mm，通过更换溢流插板8可以调节盒体1中部的液面高度，不仅能够为组织工程皮肤提供气液界面培养环境，而且也可为膜状材料如角膜、心瓣膜、神经、血管、肠系膜等提供浸没培养环境，通过更换闸门4、筛网架20、溢流插板8以及滑动溢流口13旁的挡片15，可调节流场的剪切力大小及液面高度，用于其它三维组织器官如 肝、肾、肺、骨、软骨等组织及细胞如干细胞、癌细胞的培养，而且在移除筛网架20时，还可用于培养贴壁生长的种子细胞。
所述筛网架20可为一整体，也可为多个相互独立的筛网架20，相隔一定的间距排列。所述规格模具22卡合在筛网架20的筛网21上并与筛网架20的支架等高，规格模具22根据产品品种需求可以设计为方形、圆形、三角形或异形结构。
(1)将拟研究三维构建物或拟生产组织工程产品的原料溶液注入筛网架20上，在筛网21上放入规格模具22制成特定形状的规格，取出规格模具22，盖上盖体2；然后以400ml/min的高灌注速度向静压槽7内灌注培养液，当培养液的液面高度积蓄到一定程度时，滑动溢流口13的挡片15以调节溢流口13高度及大小，使培养液有少量液体刚要从溢流口13溢出，产生稳定的静压，培养液从闸门4下透水孔6流向筛网架20；实验对比表明，在该400ml/min的低流速条件下，每天灌注2次，每次半小时，培养10天，细胞增生是静态培养条件下相同天数细胞增生的2.37倍，可缩短组织工程皮肤培育时间5天。
(2)将拟研究三维构建物或拟生产组织工程产品的原料溶液注入筛网架20上，在筛网21上放入规格模具22制成特定形状的规格，取出规格模具22，盖上盖体2；然后以200ml/min的中等灌注速度向静压槽7内灌注培养液，当培养液的液面高度积蓄到一定程度时，滑动溢流口13的挡片15以调节溢流口13高度及大小，使培养液有少量液体刚要从溢流口13溢出，产生稳定的静压，培养液从闸门4下透水孔6流向筛网架20；实验对比表明，在该200ml/min的低流速条件下，每天灌注2次，每次半小时，培养10天，细胞增生是静态培养条件下相同天数细胞增生的2.48倍，可缩短组织工程皮肤培育时间5天。
(3)将拟研究三维构建物或拟生产组织工程产品的原料溶液注入筛网架20上，在筛网21上放入规格模具22制成特定形状的规格，取出规格模具 22，盖上盖体2；然后以100ml/min的低灌注速度向静压槽7内灌注培养液，当培养液的液面高度积蓄到一定程度时，滑动溢流口13的挡片15以调节溢流口13高度及大小，使培养液有少量液体刚要从溢流口13溢出，产生稳定的静压，培养液从闸门4下透水孔6流向筛网架20；实验对比表明，在该100ml/min的低流速条件下，每天灌注2次，每次半小时，培养10天，细胞增生是静态培养条件下相同天数细胞增生的2.75倍，缩短组织工程产品培植时间5天。
实施例二：
如图1、图2、图3、图5、图6、图9所示，上述实施例一中，闸门4的功能也可以通过堰流坝5实现，所述堰流坝5包括上坝体10和下坝体11，下坝体11与筛网架20相邻的一侧具有泄流斜面12，透水孔位于该堰流坝的上坝体10，该泄流斜面12与盒体底面呈15°～25°夹角，泄流斜面12的高度为堰流坝高度的1/3～1/2。因泄流斜面12具有一定高度，为避免流经泄流斜面12的培养液对筛网架20造成过大冲击，可调整泄流斜面12的高度控制流向筛网架20的液体流速处于低流速状态。
实施例三：
参见图1、图3、图7，第一个实施例或第二个实施例中的生物反应器的盒体1的侧壁的上表面的外侧设有台阶状的凸缘25，盒体1的底壁的四周设有与侧壁上表面的凸缘25相嵌合的对象嵌合凹槽26。多个盒体1通过反向嵌合对接构成层叠盒体30，盖体2盖合在顶部的盒体1上；相邻的层叠盒体30上部的盒体1的底壁与下部的盒体1构成反应室，顶部的盒体1与盖体2构成反应室；其中盒体1的出液孔19位于盒体1的底壁上，开设在盒体1底壁上的出液孔19构成了下部盒体1的进液孔18并能将上部盒体1的泄流槽9中的液体直接输入下部盒体1的静压槽7当中，顶部盒体1的进液孔18位于其反应室上，并通过蠕动泵29将底部盒体1出液孔19排出的液体直接泵入到顶部盒体1的静压槽7当中；层叠盒体30中的盒体1的数目可以是2到20个，层叠盒体30 与盖体2、输液管27、储液罐28、蠕动泵29共同构成层叠生物反应器。
使用时，培养液自顶部盒体1的静压槽7流经截流装置3、筛网架20、溢流插板8后流入泄流槽9，多余液体从溢流口13经引流槽16流入泄流槽9，然后通过出液孔19直接注入下层盒体1的静压槽7，再从底部的盒体1的出液孔19流入储液罐28，再经蠕动泵29将储液罐28中的培养液泵入到顶部盒体1的静压槽7中，来实现培养液的封闭式循环。其中，安装在中部的盒体1的数量可根据需要增加或减少。如此循环流动，所有流经盒体1的中部的液体流速始终处于低流速状态，使筛网架20上的拟生产组织工程产品或拟培养的三维组织器官都能得到充足的营养和适宜的剪切力，保证了产品质量。
本实施方式中的层叠盒体30中的盒体1也可使用如图1、图3当中的盒体1，使用时层叠盒体30上部的盒体1的出液孔19和相邻下部盒体1的进液孔18也可通过输液管27连接。
实施例四：
附图8是将实施例三中的层叠盒体30放入无菌反应箱中，反应箱由箱体31和箱盖32组成，反应箱上开设有与层叠盒体30顶部盒体1的进液孔18和底部盒体1的出液孔19相配合的能使输液管27通过的孔道34，层叠盒体30位于箱体31中，储液罐28和蠕动泵29位于箱体31外。
在反应箱***还可以设一循环水套33，储液罐28和蠕动泵29位于循环水套33外。使用时，反应箱可使层叠盒体30处于无菌环境下，循环水套可以使层叠盒体处于恒温状态，培养液在反应箱内外循环流动，流经盒体1的中部的液体流速始终处于低流速状态，为反应箱内的拟生产组织工程产品或拟培养的三维组织器官提供了充足的营养和适宜的剪切力。在反应箱外面设置的循环水套33，保障了反应箱内处于恒温的反应状态。
本发明通过设置闸门4或堰流坝5，与溢流口13相配合，无论在何种灌注速度下，都能使培养液在静压槽7内形成稳定的压力，流经筛网架20时始终保持较低流速，盒体1中部形成均匀的稳态平面流场，克服了现有灌注式 培养中存在的流场不均一、营养传递不均匀容易造成细胞坏死的问题，提高了产品质量、缩短了培育时间；还可用于三维构建物或组织器官的共培养研究和药物筛选。本发明还具有扩展性好、通用性强、适用范围广的特点，实现了对组织工程产品的标准化培养，大幅降低了劳动强度，提高了生产效率，具有很好的推广和应用价值。
显然，上述描述的所有实施例是本发明的一部分实施例，而不是全部的实施例。基于本发明的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范畴。

Claims (14)

  1. 一种适用于三维组织细胞灌注培养的生物反应器，包括储液罐(28)、蠕动泵(29)以及盖体(2)和至少一个顶部敞口的长方体盒体(1)组成的反应室；所述盒体(1)一端设置有截流装置(3)，与盒体(1)构成静压槽(7)，另一端设置有溢流插板(8)，与盒体(1)构成泄流槽(9)，静压槽(7)和泄流槽(9)之间设置有与截流装置(3)和溢流插板(8)均不接触的筛网架(20)；所述反应室上设置有与静压槽(7)连通并能将液体直接注入静压槽(7)的进液孔(18)，在盒体(1)的壁上设置有与泄流槽(9)连通并能将液体直接排出泄流槽(9)的出液孔(19)；所述截流装置(3)的高度等于或低于盒体(1)的深度，筛网架(20)的高度低于截流装置(3)的高度，溢流插板(8)的高度高于或等于筛网架(20)上筛网(21)的高度；所述反应室的出液孔(19)、进液孔(18)、储液罐(28)与蠕动泵(29)通过输液管(27)依次连接；其特征在于：所述静压槽(7)的侧壁上设置有能够调节静压槽(7)内液面高度以形成稳定静压的溢流口(13)，在盒体(1)的侧壁上设置有能将静压槽(7)经溢流口(13)溢出的多余液体通过进流口(17)排入泄流槽(9)的引流槽(16)；所述截流装置(3)沿纵向截面同一水平上开设有若干均匀排列的能将静压槽(7)内的液体匀速均匀引入反应区域的透水孔(6)；调节进液孔(18)中液体的流速与溢流口(13)的高度与大小以在静压槽(7)、反应区域和泄流槽(9)中形成阶梯状的差位液面。
  2. 根据权利要求1所述的一种适用于三维组织细胞灌注培养的生物反应器，其特征在于：所述筛网架(20)固定在盒体(1)的两侧壁上；所述截流装置(3)、溢流插板(8)与盒体(1)接触的面上以及筛网架(20)与盒体(1)侧壁接触的面上设有卡子(23)，盒体(1)的侧壁 与底壁上设置有与卡子(23)相卡合的卡合凹槽(24)。
  3. 根据权利要求1所述的一种适用于三维组织细胞灌注培养的生物反应器，其特征在于：所述溢流口(13)包括U型槽以及在静压槽内U型槽旁设置的能通过定位轴(14)转动盖合U型槽并能通过调节U型槽下缘高度来调节静压槽(7)内液面高度的挡片(15)。
  4. 根据权利要求1所述的一种适用于三维组织细胞灌注培养的生物反应器，其特征在于：所述透水孔(6)为相同的条形孔、圆形孔、半圆形孔或其他异形孔。
  5. 根据权利要求1所述的一种适用于三维组织细胞灌注培养的生物反应器，其特征在于：所述溢流插板(8)的高度高于筛网架(20)上的筛网(21)的高度为0～4mm。
  6. 根据权利要求1所述的一种适用于三维组织细胞灌注培养的生物反应器，其特征在于：所述截流装置(3)为长方体的闸门(4)，透水孔(6)位于闸门(4)的中下部。
  7. 根据权利要求1所述的一种适用于三维组织细胞灌注培养的生物反应器，其特征在于：所述截流装置(3)为包括上坝体(10)与下坝体(11)的堰流坝(5)，堰流坝(5)在静压槽(7)一侧为长方体，下坝体(11)与筛网架(20)相邻一侧为与堰流坝(5)底面齐平的泄流斜面(12)；所述泄流斜面(12)与盒体(1)底面呈15°～25°夹角，泄流斜面(12)的高度为堰流坝(5)高度的1/3～1/2；所述透水孔(6)位于上坝体(10)上。
  8. 根据权利要求1所述的一种适用于三维组织细胞灌注培养的生物反应器，其特征在于：所述筛网(21)上设置有细胞附着的规格模具(22)，规格模具(22)为方形、圆形、三角形或异形结构。
  9. 根据权利要求1至8任一项所述的一种适用于三维组织细胞灌注培养的生物反应器，其特征在于：多个盒体(1)通过反向层叠对接固定 构成层叠盒体(30)，盖体(2)盖合在顶部的盒体(1)上并与顶部盒体(1)构成顶部反应室，层叠盒体(30)的相邻盒体(1)的出液孔(19)与进液孔(18)相连，储液罐(28)和蠕动泵(29)通过输液管(27)连接在层叠盒体(30)底部盒体(1)的出液孔(19)和顶部反应室的进液孔(18)之间。
  10. 根据权利要求9所述的一种适用于三维组织细胞灌注培养的生物反应器，其特征在于：所述层叠盒体中盒体的数量为2到20个。
  11. 根据权利要求9所述的一种适用于三维组织细胞灌注培养的生物反应器，其特征在于：所述盒体(1)的侧壁上表面的外侧设有台阶状的凸缘(25)，盒体(1)底壁上开设有能与凸缘(25)相嵌合的嵌合凹槽(26)，多个盒体(1)反向并通过凸缘(25)与嵌合凹槽(26)的对接构成层叠盒体(30)。
  12. 根据权利要求9所述的一种适用于三维组织细胞灌注培养的生物反应器，其特征在于：所述层叠盒体(30)中底部盒体(1)的出液孔(19)位于盒体(1)的侧壁上，其他盒体(1)的出液孔(19)位于盒体(1)的底壁上并能将泄流槽(9)中的液体直接输入下部盒体(1)静压槽(7)内。
  13. 根据权利要求9所述的一种适用于三维组织细胞灌注培养的生物反应器，其特征在于：所述层叠盒体(30)的外部设有由箱体(31)和箱盖(32)组成的反应箱，反应箱上开设有能使输液管(27)通过的孔道(34)，储液罐(28)和蠕动泵(29)位于反应箱外。
  14. 根据权利要求13所述的一种适用于三维组织细胞灌注培养的生物反应器，其特征在于：所述反应箱***设有循环水套(33)，储液罐(28)和蠕动泵(29)位于循环水套(33)外。
PCT/CN2015/079330 2014-05-22 2015-05-20 一种适用于三维组织细胞灌注培养的生物反应器 WO2015176653A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US15/328,460 US9902929B2 (en) 2014-05-22 2015-05-20 Bioreactor for three-dimensional tissue perfusion culture
EP15795352.2A EP3147349B1 (en) 2014-05-22 2015-05-20 Bioreactor for three-dimensional tissue perfusion culture

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201420264095.3 2014-05-22
CN201420264095.3U CN203855588U (zh) 2014-05-22 2014-05-22 一种适用于三维组织细胞灌注培养的生物反应器
CN201410218150.XA CN103966095B (zh) 2014-05-22 2014-05-22 一种适用于三维组织细胞灌注培养的生物反应器
CN201410218150.X 2014-05-22

Publications (1)

Publication Number Publication Date
WO2015176653A1 true WO2015176653A1 (zh) 2015-11-26

Family

ID=54553429

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/079330 WO2015176653A1 (zh) 2014-05-22 2015-05-20 一种适用于三维组织细胞灌注培养的生物反应器

Country Status (3)

Country Link
US (1) US9902929B2 (zh)
EP (1) EP3147349B1 (zh)
WO (1) WO2015176653A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2017158777A1 (ja) * 2016-03-16 2018-12-27 株式会社島津製作所 細胞培養容器
CN109762718A (zh) * 2019-03-18 2019-05-17 金婧菲 一种黏菌培养盒及使用方法
CN112877210A (zh) * 2021-02-25 2021-06-01 孔祥玲 一种pH值可控的干细胞培养灌流装置
US11566215B2 (en) 2016-08-27 2023-01-31 3D Biotek Llc Bioreactor with scaffolds

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102017213923A1 (de) * 2017-08-10 2019-02-14 Robert Bosch Gesellschaft Für Medizinische Forschung Mbh Vorrichtung zur Kultivierung von Gewebeschnitten
CN107723236B (zh) * 2017-10-31 2024-02-02 广州迈普再生医学科技股份有限公司 动态灌流培养***
KR102019437B1 (ko) * 2018-05-21 2019-09-09 주식회사 다나그린 세포 배양액 자동공급시스템
US11447729B2 (en) * 2018-12-21 2022-09-20 Global Life Sciences Solutions Usa Llc Apparatus and method for bioprocessing
EP4073233A4 (en) * 2019-12-11 2024-04-03 Humacyte, Inc. SYSTEMS, DEVICES AND METHODS FOR TRANSPORT AND CONTAINMENT OF A GENETICALLY ENGINEERED TISSUE CONSTRUCTION
CN111748475A (zh) * 2020-07-14 2020-10-09 兰州大学第一医院 一种流体细胞培养装置
CN116068148B (zh) * 2023-03-21 2023-06-16 中国地质调查局成都地质调查中心（西南地质科技创新中心） 降雨条件下滑坡堆积体形成泥石流运动过程模拟装置

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1560225A (zh) * 2004-03-01 2005-01-05 华东理工大学 双层活性皮肤体外构建的灌注式生物反应器***
CN103966095A (zh) * 2014-05-22 2014-08-06 中国人民解放军第三军医大学第三附属医院 一种适用于三维组织细胞灌注培养的生物反应器
CN203855588U (zh) * 2014-05-22 2014-10-01 中国人民解放军第三军医大学第三附属医院 一种适用于三维组织细胞灌注培养的生物反应器

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7456019B2 (en) * 2004-06-30 2008-11-25 Regenetech, Inc. Three-dimensional cell to tissue development process
CN107988072B (zh) * 2008-07-16 2022-11-29 儿童医疗中心有限公司 具有微通道的器官模仿装置及其使用和制造方法
WO2012118799A2 (en) * 2011-02-28 2012-09-07 President And Fellows Of Harvard College Cell culture system
WO2012163368A1 (en) * 2011-05-30 2012-12-06 Atech System Solutions Ltd. Bioreactor system for continuous cell cultivation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1560225A (zh) * 2004-03-01 2005-01-05 华东理工大学 双层活性皮肤体外构建的灌注式生物反应器***
CN103966095A (zh) * 2014-05-22 2014-08-06 中国人民解放军第三军医大学第三附属医院 一种适用于三维组织细胞灌注培养的生物反应器
CN203855588U (zh) * 2014-05-22 2014-10-01 中国人民解放军第三军医大学第三附属医院 一种适用于三维组织细胞灌注培养的生物反应器

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG, YANG ET AL.: "DESIGN AND PRELIMINARY SIMULATION OF A NEW PERFUSION BIOREACTOR FOR TISSUE-ENGINEERED SKIN BASED ON QUASI-STATIC PLANE FLOW FIELD", ACTA ACADEMIAE MEDICINAE MILITARIS TERTIAE, vol. 35, no. 7, 15 April 2013 (2013-04-15), pages 622 - 626, XP055360959 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2017158777A1 (ja) * 2016-03-16 2018-12-27 株式会社島津製作所 細胞培養容器
EP3431578A4 (en) * 2016-03-16 2019-11-27 Shimadzu Corporation CONTAINER OF CELL CULTURE
US11186810B2 (en) 2016-03-16 2021-11-30 Shimadzu Corporation Cell culture vessel
US11566215B2 (en) 2016-08-27 2023-01-31 3D Biotek Llc Bioreactor with scaffolds
US11926810B2 (en) 2016-08-27 2024-03-12 3D Biotek, Llc Bioreactor with scaffolds
CN109762718A (zh) * 2019-03-18 2019-05-17 金婧菲 一种黏菌培养盒及使用方法
CN112877210A (zh) * 2021-02-25 2021-06-01 孔祥玲 一种pH值可控的干细胞培养灌流装置
CN112877210B (zh) * 2021-02-25 2022-07-26 上海揽微赛尔生物科技有限公司 一种pH值可控的干细胞培养灌流装置

Also Published As

Publication number Publication date
EP3147349A1 (en) 2017-03-29
US20170226462A1 (en) 2017-08-10
EP3147349A4 (en) 2018-04-25
US9902929B2 (en) 2018-02-27
EP3147349B1 (en) 2019-07-10

Similar Documents

Publication Publication Date Title
WO2015176653A1 (zh) 一种适用于三维组织细胞灌注培养的生物反应器
CN103966095B (zh) 一种适用于三维组织细胞灌注培养的生物反应器
JP6719387B2 (ja) 自動細胞培養及び回収装置
CN203855588U (zh) 一种适用于三维组织细胞灌注培养的生物反应器
US5516691A (en) Module for culturing and using metabolisms and/or for maintaining microorganisms
CN206783688U (zh) 一种多级细胞扩增***
JP2022501003A (ja) 相互接続されたウェルを有するマイクロプレートを備えた流体デバイス
CN101611133A (zh) 高效装置及培养细胞的方法
US20170067019A1 (en) Method of Continuous Mass Production of Progenitor Stem-like Cells Using a Bioreactor System
CN111269830B (zh) 一种基于微流控技术的多器官芯片及其应用
CN103614296B (zh) 一种双腔三维灌注生物反应器***
KR20140014494A (ko) 세포 배양 유닛, 세포 배양 장치 및 세포 배양 시스템
CN105695328A (zh) 细胞培养和细胞间相互作用的共培养装置及其使用方法
CN109825437A (zh) 一种用于细胞培养的微流控芯片及培养方法
CA2387549A1 (en) Method for culturing and/or treating cells
KR101075032B1 (ko) 세포 배양기 및 이를 포함하는 세포 배양장치
CN103421682A (zh) 一种胚胎自动换液培养皿
US8039252B2 (en) Multi-chamber cell culture assembly
WO2023246341A1 (zh) 一种细胞培养方法、细胞培养容器及细胞培养装置
US11584906B2 (en) Cell culture vessel for 3D culture and methods of culturing 3D cells
CN205473829U (zh) 细胞培养和细胞间相互作用的共培养装置
CN215828801U (zh) 胚胎长时程培养***
CN204162724U (zh) 一种用于生物样品灌注式培养的灌注盖
CN210458217U (zh) 一种微负压细胞培养装置
CN103266182A (zh) 一种塔板型灌注式生物反应器的调节方法及其使用装置

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15795352

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

REEP Request for entry into the european phase

Ref document number: 2015795352

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2015795352

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 15328460

Country of ref document: US