Array type cell culture chip as well as construction method and application thereof

Abstract

The invention provides an array type cell culture chip as well as a construction method and application thereof, and belongs to the technical field of tissue engineering. The invention provides an array type cell culture chip. The array type cell culture chip comprises an upper-layer chip, a first sealing gasket, a porous membrane, a second sealing gasket and a lower-layer chip which are sequentially combined, the invention further provides a tissue culture method based on the array type cell culture chip, vascularized skin tissue is obtained in a dynamic culture mode, and the vascularized skin tissue is closer to human skin tissue and has a more complete skin function. The array type cell culture chip disclosed by the invention adopts a detachable structure, is low in processing difficulty, easy to assemble and high in reusability; each layer structure is convenient to disassemble, so that the internal area is convenient to clean and fully disinfect; the skin tissue cultured by using the chip has a vascularized tissue, forms an endothelial barrier, and can better simulate the transportation of drugs along with blood vessels; and a precise and controllable fluid microenvironment is provided, and a platform is provided for large-scale drug screening.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering, and in particular to an array cell culture chip and a construction method and application thereof. Background Art

[0002] As the organ with the largest surface area in the human body, the skin has the characteristics of large area, heterogeneity and multiple layers. The main function of human skin is to act as a physiological barrier to protect organs and tissues from physical, mechanical, chemical and pathogenic microbial invasion. The skin is exposed to many chemicals and biological agents on a daily basis, including cosmetics, skin cleansers, environmental pollutants and microbial pathogens. The rapid increase of these factors can cause various skin reactions, such as skin inflammation, inflammation, allergies and even cancer. The skin has the functions of protection, sensation, excretion, respiration and secretion in the human body. Protecting the health of the skin plays an extremely important role in maintaining the homeostasis of the human body.

[0003] The quality evaluation of cosmetics mainly focuses on their safety and efficacy. Since 2009, the European Commission has been emphasizing new regulations on cosmetics and establishing testing and marketing bans: it is prohibited to test finished cosmetics or ingredients on animals, and it is prohibited to commercialize any cosmetics or ingredients tested on animals in the EU. Therefore, the cosmetics industry has been greatly affected by restrictions on animal testing. Therefore, in vitro alternative technology has become an inevitable trend in the safety evaluation of cosmetics, and it is particularly important to develop physiologically relevant skin models to replace traditional inefficient methods.

[0004] At present, the most common method is in vitro cell culture, usually a single layer of epidermal keratinocytes and dermal fibroblasts. Although it can maintain certain structural functions in vitro, and has the advantages of high throughput, simple operation, and stable culture, there are also some shortcomings or defects, such as a single cell layer, easy aging of cells, and certain differences in the administration method from the human body. Only a single-layer cell culture is performed at a two-dimensional level, and the extrapolation of the results is uncertain. Recombinant human skin analogs developed by combining tissue engineering technology with cell culture technology are also called 3D skin models. This model can be assembled into complex structures to simulate more physiologically relevant conditions. 3D cell culture technology has solved the limitations of ex vivo skin and in vitro skin cell culture to a certain extent, and is closer to the actual situation of human skin in terms of administration methods. With the development of microfluidics, microfabrication, tissue engineering and other technologies, organ chips have become one of the best tools for large-scale drug testing. Organ chips simulate the organ functions in the human body in vitro, have a high degree of biomimetic and precisely controllable microenvironment, and significantly improve the ability to predict drug responses. The human skin microphysiological system derived from primary cells extracted from fresh skin tissue has become an in vitro model for skin biological mechanism research, physiology, pharmacology research and beauty evaluation to replace traditional animal experiments.

[0005] However, current skin chips still have limitations: First, existing skin chips can only culture a piece of skin tissue, which makes it difficult to conduct large-scale chemical testing. Second, the construction of vascularized tissue in vitro is a prerequisite for accurate drug testing, and the construction of vascularized tissue has always been a difficult point in research. Existing on-chip tissues often lack vascular structures. In fact, the dermis of the skin has abundant capillaries.

[0006] Therefore, it is necessary to propose a cell culture chip with vascularized tissue to better simulate the skin microenvironment and conduct accurate drug screening and testing. Summary of the invention

[0007] The main purpose of the present invention is to provide an array cell culture chip and a construction method and application thereof, through which vascularized skin tissue is cultured to facilitate accurate drug screening and testing.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is: An array cell culture chip comprises a stacked upper chip and a lower chip, the upper chip comprises a plurality of culture areas arranged side by side, adjacent culture areas are spaced apart, each culture area comprises a liquid inlet and a liquid outlet arranged at both ends, a blood channel is formed between the liquid inlet and the liquid outlet, a plurality of culture cavities are arranged between the blood inlet and the blood outlet, the liquid inlet, the culture cavities and the liquid outlet are arranged in a straight line, the lower chip comprises a plurality of parallel and spaced connecting grooves, each connecting groove is located below each culture area, the liquid inlet and the liquid outlet are connected via the connecting groove, the lower end of each culture cavity is connected to the connecting groove, a plurality of porous membranes are further arranged between the upper chip and the lower chip, each porous membrane covers the lower end of each culture cavity in the culture area.

[0009] Preferably, a first sealing pad and a second sealing pad are respectively provided on the upper and lower sides of the porous membrane, the upper chip and the first sealing pad are folded and clamped to clamp the porous membrane, a plurality of second through holes are provided on the first sealing pad, each second through hole is aligned with each culture chamber, a through waist groove is provided on the second sealing pad, and a first through hole and a third through hole are also provided on the first sealing pad and the second sealing pad, and the first through hole and the third through hole are respectively aligned with the blood inlet and the blood outlet; a sinking groove is provided at the through waist groove, and the porous membrane is placed in the sinking groove.

[0010] Preferably, a plurality of top columns are provided in the connecting groove, a flow groove is provided at the upper end of the top column, and the upper end of the top column abuts against the lower end of the porous membrane; the top columns are provided in multiple rows, and a plurality of rotatable adjustment columns arranged in a straight line are provided in the center of the connecting groove, the adjustment columns are provided with strip baffles, a rotating shaft is provided at one end of the adjustment column, the rotating shaft is rotatably connected to the lower chip and passes through the lower chip, an annular groove is provided at the end of the rotating shaft, and a plurality of disc springs are also provided, the disc spring is provided with a U-shaped slot, a clamping portion is provided at the bottom end of the U-shaped slot, the clamping portion is clamped with the annular groove, and the outer edge of the disc spring abuts against the bottom end of the lower plate.

[0011] Preferably, a partition partition is also provided at the upper end of the upper chip, and the partition partition includes a plurality of parallel and spaced strip partition grooves, each of which is arranged above each culture area, and a plurality of label grooves are provided on one side of the strip partition groove, each of which is provided with a magnetic block, and a plurality of label sheets are also provided, the label sheets are arranged in the label grooves and adsorbed by the magnetic blocks, and one end of the label sheets points to the culture chamber.

[0012] Preferably, the pore size of the porous membrane is 0.4-1 μm.

[0013] The present invention also provides a method for constructing the array cell culture chip, comprising the following steps: Use computer-aided design software to draw the microchannel structure and microscopic graphics of the upper chip and the lower chip, and process them on the surface of the upper chip and the lower chip substrate using micromachining technology; The upper chip, the first sealing gasket, the porous membrane, the second sealing gasket and the lower chip are aligned and sealed in sequence to form an array cell culture chip.

[0014] Preferably, in the construction of the array cell culture chip, sterile operation is adopted throughout the process.

[0015] Preferably, the materials of the upper chip and the lower chip include polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE) or polylactic acid (PLA); non-toxic and non-irritating materials are selected to make organ chips to reduce adverse effects on cell growth.

[0016] Preferably, the layers of chips are sealed together by extrusion, bonding or threaded assembly.

[0017] Preferably, the processing method of the upper chip and the lower chip is at least one of a 3D printing method, a laser etching method, a CNC milling method and a hot pressing method.

[0018] The present invention also provides a method for tissue culture using the array cell culture chip, comprising the following steps: Sterilizing the array cell culture chip; The corresponding cell-matrix mixed solution is added to the tissue culture area of ​​the cell culture chip, the culture solution is added to the blood channel, and the cells in the tissue culture area are co-cultured to obtain the corresponding tissue model.

[0019] Preferably, the method for preparing the cell-matrix mixture comprises the following steps: (1) Cell culture: Normal umbilical vein endothelial cells, fibroblasts, and keratinocytes were placed in a 37°C, 5% CO2 incubator for routine culture; (2) Cell suspension: After the adherent cells in the culture flask are digested into single cells, the culture medium is added to each cell, and after being thoroughly pipetted and mixed, the culture medium is added to dilute the cell suspension to a suitable concentration, and then the cell suspension is suspended in the matrix solution to obtain a cell-matrix and cell-culture medium mixture; The concentration of umbilical vein endothelial cells suspension was 1×10 6 -5×10 6 The concentration of fibroblast suspension was 0.5×10 6 -3×10 6 / mL, the concentration of keratinocyte suspension was 1×10 6 -2×10 6 Pieces / mL.

[0020] Preferably, the matrix solution is selected from at least one of type I rat tail collagen, bovine fibrin, collagen gel, hyaluronic acid, laminin or a mixture of type I rat tail collagen and bovine fibrin.

[0021] Preferably, in order to facilitate the generation of vascular tissue, the umbilical vein endothelial cells are suspended in matrix solution I to obtain a vascular tissue cell-matrix mixed solution.

[0022] Preferably, the matrix solution I is type I rat tail collagen or bovine fibrin solution.

[0023] Preferably, the fibroblast suspension is suspended in matrix solution II, and the keratinocytes are suspended in culture solution.

[0024] Further preferably, the matrix solution II is at least one of type I rat tail collagen solution and bovine fibrin solution (the main component of solidification into gel, collagen is an important component in human tissue, and fibrin is an extracellular matrix that is conducive to angiogenesis), and a small amount of hyaluronic acid and laminin are added to mix them to increase biomimetic properties. Type I rat tail collagen can achieve better results in the construction of liver and blood vessels at a concentration of 1.5~5mg / mL. If it is too thin, it cannot solidify or loosen. If it is too concentrated, the hardness is too high, which neither meets the physical and chemical conditions of the liver and blood vessels in the body nor is conducive to cell stretching and connection; type I rat tail collagen can achieve better results in the construction of dermal tissue at a concentration of 2~6mg / mL; bovine fibrin is conducive to the construction of vascularized tissue in vitro, and can provide support for endothelial cells to stretch into a three-dimensional vascular network, and the concentration can achieve better results at 1~6mg / mL.

[0025] More preferably, the concentration of hyaluronic acid is in the range of 0.5-4 mg / mL, and the concentration of laminin is in the range of 1-2 mg / mL; a single cell matrix is ​​difficult to meet all requirements for tissue regeneration in vitro, so the present invention mixes the cell matrix into a composite material to utilize the advantages of each component.

[0026] Preferably, the method for preparing the fibrin gel comprises: separately preparing a fibrin stock solution and a thrombin stock solution, and then mixing the two evenly, and the final thrombin concentration is 3U / mL, so as to obtain the fibrin gel.

[0027] Through the above steps, a cell-matrix mixture containing different types of cells can be prepared to provide the necessary materials for the subsequent construction of tissue and organ models.

[0028] Specifically, the method for performing tissue culture using the array cell culture chip comprises the following steps: S1. Pretreatment: sterilize the constructed array cell culture chip; S2. Vascular tissue construction: inject the umbilical vein endothelial cell-matrix mixture into the tissue culture area and let it stand for a certain period of time to allow the extracellular matrix to solidify and form a vascular layer; S3. Skin tissue construction: inject the fibroblast-matrix mixture into the tissue culture area and let it stand for a certain period of time to allow the extracellular matrix to solidify and form the dermis layer; then inject the keratinocyte-culture solution into the tissue culture area and allow it to adhere to the dermis layer to form the epidermis layer; S4. Tissue culture: slowly add culture medium from the liquid inlet, exhaust the gas in the blood channel, and perform dynamic culture on the rocker arm or connect to the peristaltic pump system; S5. Epidermal differentiation: The culture medium in the tissue culture area is aspirated, and the culture medium in the blood channel is replaced with new culture medium. After several days of cultivation on a rocker arm or connected to a peristaltic pump system, skin tissue with a stratum corneum can be formed.

[0029] Preferably, in S1, the sterilization treatment is to sterilize the cell culture chip with ultraviolet light for 30-60 minutes to ensure a sterile environment.

[0030] Preferably, in S2, the amount of the vascular tissue cell-matrix mixture added is 30-50 μL.

[0031] Preferably, in S3, the amount of the fibroblast cell-matrix mixture added is 50-100 μL, and the amount of the keratinocyte cell-matrix mixture added is 80-120 μL, to ensure that the cells are evenly distributed and adsorbed in the cell culture chip.

[0032] Preferably, in S4, culture solution I is slowly added from the liquid inlet to discharge the gas in the channel, and culture solution II is added to the skin tissue culture area at the same time, and the height of the culture solution at the liquid inlet and the liquid outlet is controlled by adjusting the regulating column so as not to exceed the horizontal line of the upper surface of the skin tissue; at the same time, a rocker arm or a peristaltic pump system is used to drive the culture solution to circulate in the blood channel to promote stable growth and reproduction of cells and maintain cell vitality and function.

[0033] More preferably, the culture medium I is a high-glucose DMEM culture medium with 0.1% antibiotics, 30-50 ng / ml endothelial growth factor EGF and 10% fetal bovine serum added; The culture medium II is based on high-glucose DMEM culture medium, supplemented with 0.2% commercial keratinocyte growth additive, 0.1% antibiotics and 10% fetal bovine serum to promote the proliferation of keratinocytes.

[0034] Preferably, in S4, when dynamic culture is carried out using a rocker arm, the seesaw amplitude is 15°, the shaking frequency is 15-50 rpm, and the motor power is 25W; when the peristaltic pump system is connected for dynamic culture, the two ends of the peristaltic pump are connected to the liquid inlet and outlet of the chip to directly form a liquid circulation, or a circulation is formed with the help of a culture bottle, the speed of adding the culture solution is 100 μL / min, the amount of the culture solution added is 1000-2000 μL, and the fluidity of the culture solution during the operation of the culture is 1 μL / min; the amount of culture solution added is related to the number of culture chambers. The more culture chambers there are, the more culture solution is needed.

[0035] Preferably, in the dynamic culture, the fluid shear force is controlled to be 0.01-10 dyn / cm 2 Within this range, the dynamic environment of tissue cell growth in the human body is simulated, and the nutrient replenishment and metabolic mechanism of the entire layer of skin are dynamically maintained in order to obtain a highly bionic tissue and organ model.

[0036] Preferably, in S5, in order to facilitate the formation of an air-liquid interface, the culture medium in the skin tissue culture area is sucked out, and the culture medium in the blood channel is replaced with a new culture medium for cell culture; during the culture process, the culture medium in the blood channel is replaced every 2-3 days, which can not only construct a skin tissue with an air-liquid interface culture microenvironment, but also discharge metabolites produced by the cells by replacing the new culture medium, and provide nutrients required for cell metabolism, thereby promoting cell growth.

[0037] Further preferably, the new culture medium is an epidermal differentiation culture medium, containing at least one of metal ions, ceramide and tissue plasminogen activator; these components play a key role in epidermal differentiation, and selecting appropriate concentrations can promote epidermal differentiation and shorten the time to form skin tissue with a stratum corneum.

[0038] The ceramides include C2-ceramide and C6-ceramide.

[0039] More preferably, the metal ion is a calcium ion. More preferably, the concentration of the calcium ion is 1-5 mmol / L. Epidermal differentiation can be well promoted in this concentration range.

[0040] More preferably, the new culture medium is based on high-glucose DMEM culture medium, supplemented with 0.1% antibiotics, 1.8 mM calcium chloride, 20-50 ng / ml endothelial growth factor EGF and 10% fetal bovine serum.

[0041] Preferably, in S5, the tissue cells in the skin tissue culture area are cultured for 10-15 days to form skin tissue having a stratum corneum.

[0042] The present invention also provides application of the array cell culture chip in cosmetic and drug testing.

[0043] Furthermore, the specific method of using the cell culture chip for cosmetic and pharmaceutical testing is: using systemic administration (oral, injection) and local (transdermal) administration methods to test drugs. For systemic drugs, the fluid enters the fluid channel in the chip through the fluid inlet through hole, and the skin tissue is tested after dynamic culture for a certain period of time; for transdermal drugs, they are added from the top of the culture area of ​​the skin tissue culture, and the skin tissue is tested after dynamic culture for a certain period of time.

[0044] The mechanical strength of the matrix material is a key condition for promoting cell differentiation in a specific direction. The present invention suspends different tissue cells in different matrix solutions according to their growth characteristics to promote cell growth and reproduction. In order to promote the production of vascular tissue, umbilical vein endothelial cells are suspended in a bovine fibrin solution with a concentration of 2.5 mg / mL to solidify the extracellular matrix and form a vascular layer; fibroblasts are suspended in a solution with a concentration of 2.5 mg / mL type I rat tail collagen and a solution with a concentration of 2.5 mg / mL bovine fibrin ( V 1 :V 2=1:1) in a mixed solution to solidify the extracellular matrix and form a dermis layer, and then keratinocytes are inoculated for culture to make the cells adhere to the dermis layer and form an epidermis layer; at the same time, a certain concentration of calcium ions is added to the culture medium during epidermal differentiation to promote the differentiation of epidermal cells and shorten the time for culturing skin tissue with a stratum corneum structure, thereby providing a guarantee for large-scale drug screening.

[0045] The arrayed cell culture chip provided by the present invention simulates the in vivo microenvironment through a dynamic culture method, thereby obtaining a highly bionic tissue and organ model, and can realize the construction of multiple tissue and organ models. The cell culture chip construction method of the present invention is simple, can meet the construction of different tissues and organs, and has a strong barrier function.

[0046] The array cell culture chip provided by the present invention can accurately evaluate the efficacy of drugs under two drug administration modes: transdermal administration (adding drugs above the culture area) and systemic administration such as oral injection (dosage at the entrance of the fluid channel).

[0047] Compared with the prior art, the present invention has the following beneficial effects: 1. The array cell culture chip of the present invention can be provided with multiple skin tissue culture areas on a single chip. This design is suitable for large-scale skin adverse reaction testing of chemicals, significantly improving testing efficiency and saving time.

[0048] 2. The array cell culture chip of the present invention adopts a detachable structure, has low processing difficulty, is easy to assemble, and has high reusability; each layer of the board is easy to disassemble, which is convenient for cleaning the internal area and fully disinfecting.

[0049] 3. The cell culture chip of the present invention is provided with multiple groups of liquid inlets and outlets, which can meet the needs of large-scale drug testing; multiple tissue culture areas are provided, which can not only meet the needs of multiple groups of repeated experiments during drug testing, but also analyze the effects of different concentrations of drugs on skin tissue, thereby realizing high-throughput drug screening and accurate drug efficacy evaluation.

[0050] 4. The array cell culture chip of the present invention can provide gas-liquid culture interface conditions and blood flow conditions for cultured skin tissue, truly simulating the fluid microenvironment of the human body; traditional skin models are difficult to simulate the process of drug transport through blood vessels. The present invention cultures vascular tissue under the skin tissue to form an endothelial barrier, which can better simulate the transport of drugs through blood vessels.

[0051] 5. The array cell culture chip of the present invention adopts an open design, which is convenient for adding samples to the chip. The open design also facilitates the removal of cultured tissues for subsequent multi-method analysis and testing.

[0052] 6. The present invention constructs a vascularized full-thickness skin tissue by suspending umbilical vein endothelial cells in a bovine fibrin solution to solidify the extracellular matrix and form a vascular layer; suspending fibroblasts in a mixture of rat tail collagen and fibrin solution to solidify the extracellular matrix and form a dermis layer; then inoculating keratinocytes for culture to make the cells adhere to the dermis layer and form an epidermis layer, thereby providing a platform for cosmetic testing and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The present invention will be further described below in conjunction with the accompanying drawings and embodiments: Figure 1 is an exploded view of the array cell culture chip of Example 1 of the present invention; Figure 2 This is a diagram of the internal structure of the array cell culture chip of Example 1 of the present invention; Figure 3 is a schematic diagram of a microcolumn array of an array-type cell culture chip in Example 1 of the present invention; Figure 4 Schematic diagram of the regulating column of the array cell culture chip in Example 1 of the present invention; Figure 5 is a cross-sectional view of the array cell culture chip of Example 1 of the present invention; Figure 6 Schematic diagram of the disc spring of the array cell culture chip in Example 1 of the present invention; Figure 7 is a schematic diagram of an array cell culture chip according to Example 1 of the present invention; Figure 8 This is a structural diagram of partition plates of an array cell culture chip according to Example 1 of the present invention; Fig. 9 The dermis and epidermis of the skin tissue constructed in Example 3 of the present invention; Fig.10 The vascular tissue constructed in Example 3 of the present invention; Fig.11 This is a result diagram of the FITC-dextran test of the skin barrier properties in vitro according to an embodiment of the present invention; Fig.12 1 is a graph showing the fluorescence staining results of Calcein-AM, Hoechst 33342 and PI in the control group and the drug-added group of the embodiment of the present invention; Fig.13 is a diagram of cell proliferation results of a drug test according to an embodiment of the present invention; Fig.14 : is a graph showing the results of DiI and DiO fluorescence staining of the control group and the drug-added group in an embodiment of the present invention; In the figure: upper chip 1, liquid inlet 101, culture chamber 102, liquid outlet 103, bolt hole 104, first sealing gasket 2, first through hole 201, second through hole 202, third through hole 203, porous membrane 3, second sealing gasket 4, through waist groove 401, sinking groove 402, lower chip 5, connecting groove 501, top column 502, flow groove 503, third sealing gasket 504, adjusting column 6, strip baffle 601, rotating shaft 602, annular groove 603, disc spring sheet 7, clamping part 701, U-shaped clamping groove 702, cutting groove 703, partition partition 8, strip partition groove 801, label groove 802, magnetic block 803, label sheet 9, culture area 10. DETAILED DESCRIPTION

[0054] Embodiment 1: like Figure 1-8 As shown, an array cell culture chip includes an upper chip 1 and a lower chip 5 stacked together, the upper chip 1 includes a plurality of culture areas 10 arranged side by side, and a spacing is provided between adjacent culture areas 10, each culture area 10 includes a liquid inlet 101 and a liquid outlet 103 arranged at both ends, a blood channel is formed between the liquid inlet 101 and the liquid outlet 103, and a plurality of culture chambers 102 are provided between the liquid inlet 101 and the liquid outlet 103, The cavity 102 and the liquid outlet 103 are arranged in a straight line, and the lower chip 5 includes a plurality of connecting grooves 501 arranged in parallel and spaced apart, each connecting groove 501 is located below each culture area 10, and the liquid inlet 101 and the liquid outlet 103 are connected through the connecting groove 501, and the lower end of each culture cavity 102 is connected to the connecting groove 501, and a plurality of porous membranes 3 are also provided between the upper chip 1 and the lower chip 5, and each porous membrane 3 covers the lower end of each culture cavity 102 in the culture area 10.

[0055] Each culture area 10 can work independently and is equipped with a liquid inlet 101 , a liquid outlet 103 , and a plurality of culture chambers 102 .

[0056] The liquid inlet 101, the connecting groove 501, and the liquid outlet 103 constitute a U-shaped channel. The culture chamber 102 is located on the line connecting the liquid inlet 101 and the liquid outlet 103 and is located inside the U-shaped structure. Skin or liver cells can be cultured in different culture chambers 102. The connecting groove 501 simulates blood vessels. The culture chamber 102 gradually approaches the connecting groove 501 in the depth direction. In other words, the distances between the cells at different height layers in the culture chamber 102 and the connecting groove 501 are different, which more realistically simulates the positional relationship between blood vessels and cells.

[0057] The porous membrane 3 is a microporous structure that can block cell penetration. Under the action of the delivery pressure, nutrients penetrate from the bottom end of the culture chamber 102 to the upper end through the connecting channel 501, which is closer to the environment of cells in the human body.

[0058] Preferably, the porous membrane 3 is provided with a first sealing gasket 2 and a second sealing gasket 4 on the upper and lower sides respectively, the upper chip 1 and the first sealing gasket 2 are folded and clamped with the first sealing gasket 2 and the second sealing gasket 4 to clamp the porous membrane 3, the first sealing gasket 2 is provided with a plurality of second through holes 202, each second through hole 202 is aligned with each culture chamber 102, the second sealing gasket 4 is provided with a through waist groove 401, the first sealing gasket 2 and the second sealing gasket 4 are also provided with a first through hole 201 and a third through hole 203, the first through hole 201 and the third through hole 203 are respectively aligned with the liquid inlet 101 and the liquid outlet 103.

[0059] A plurality of bolt holes 104 aligned in groups are provided on the upper chip 1, the first sealing gasket 2 and the second sealing gasket 4, and the corresponding positions of the bolt holes 104 on the lower chip 5 are threaded holes. The bolts are inserted from the upper chip 1, through the first sealing gasket 2 and the second sealing gasket 4, and the ends are threadedly connected to the lower chip 5, locking the upper chip 1 to the lower chip 5 to improve the sealing effect.

[0060] Preferably, a sinking groove 402 is provided at the through waist groove 401 , and the porous membrane 3 is placed in the sinking groove 402 .

[0061] The area of ​​the through waist groove 401 is slightly smaller than the porous membrane 3. The sink groove 402 plays a role in positioning the porous membrane 3 to avoid misalignment of the porous membrane 3 during installation. At the same time, it can press the outer edge of the porous membrane 3 to avoid horizontal movement.

[0062] Preferably, a plurality of top columns 502 are provided in the connecting channel 501 , and a flow channel 503 is provided at the upper end of the top column 502 , and the upper end of the top column 502 abuts against the lower end of the porous membrane 3 .

[0063] Since the porous membrane 3 is generally soft, the top pillars 502 at both ends can support the lower end of the second sealing gasket 4 near the outer edge of the porous membrane 3 to press it tightly, and the inner top pillar 502 directly supports the lower end of the porous membrane 3 to prevent the porous membrane 3 from falling and deforming after being filled with cells, gel and other nutrients.

[0064] The flow channel 503 allows the nutrient solution to flow through the contact point between the top of the top column 502 and the porous membrane 3.

[0065] Preferably, the top column 502 is provided with multiple rows, and a plurality of rotatable adjustment columns 6 arranged in a straight line are provided in the center of the connecting groove 501. The adjustment column 6 is provided with a strip baffle 601. A rotating shaft 602 is provided at one end of the adjustment column 6. The rotating shaft 602 is rotatably connected to the lower chip 5 and passes through the lower chip 5. An annular groove 603 is provided at the end of the rotating shaft 602, and a plurality of disc spring sheets 7 are also provided. The disc spring sheet 7 is provided with a U-shaped groove 702. A clamping portion 701 is provided at the bottom end of the U-shaped groove 702. The clamping portion 701 is clamped with the annular groove 603, and the outer edge of the disc spring sheet 7 rests against the bottom end of the lower chip 5.

[0066] A plurality of grooves 703 are axially arranged on the outer edge of the disc spring 7 to improve the deformation capability of the disc spring 7 .

[0067] The bottom of the connecting groove 501 is provided with a plurality of threaded holes and light holes. The threaded holes are used to install each top column 502, and the light holes are used to install each adjustment column 6. The third sealing gasket 504 is coated with glue and covers each hole. After the glue solidifies, a small hole is poked at each hole, and the top column 502 and the adjustment column 6 are installed to play a sealing role. The disc spring 7 is elastic. After clamping the adjustment column 6, the adjustment column 6 rotates and presses the third sealing gasket 504, taking into account the sealing.

[0068] Another annular groove can be processed on the rotating shaft 602 and a sealing ring can be sleeved thereon.

[0069] The end of the rotating shaft 602 can be processed with concave and convex stripes to increase muscle friction and facilitate finger rotation.

[0070] By adjusting the regulating column 6 below a culture chamber 102 to change the flow area at this point in the connecting channel 501, the liquid level of the culture solution in the culture chamber 102 can be adjusted. For example, by reducing the flow area, according to the Bernoulli principle, the flow rate of the connecting channel 501 at the lower end of a culture chamber 102 increases, and the pressure here decreases. The pressure is not enough to support the original liquid level, so the liquid level of the culture solution in the upper culture chamber 102 drops. Since the upper chip 1 and the lower chip 5 are made of transparent materials, the liquid level in the culture chamber 102 can be visually observed, and the end of the bottom regulating column 6 can be rotated until the required height is reached. It is also possible to engrave an arrow on the lower end of the regulating column 6 and engrave a scale line on the bottom end of the lower plate 5 to quantitatively adjust the rotation angle of the regulating column 6.

[0071] Preferably, a partition partition 8 is also provided at the upper end of the upper chip 1, and the partition partition 8 includes a plurality of parallel and spaced strip partition grooves 801, each of which is arranged above each culture area 10, and a plurality of label grooves 802 are provided on one side of the strip partition groove 801, each of which is provided with a magnetic block 803, and a plurality of label sheets 9 are also provided, the label sheets 9 are arranged in the label grooves 802 and adsorbed by the magnetic blocks 803, and one end of the label sheets 9 points to the culture chamber 102.

[0072] Since there are many culture areas 10 and many culture chambers 102 in the culture areas 10, the residual liquid adhering to the upper surface of the upper chip 1 when adding cells and reagents may flow into the adjacent culture areas 10, causing contamination. Therefore, partition partitions 8 are added and the different culture areas 10 are separated by partition grooves 801, and label sheets 9 are provided to label each culture chamber 102 or add text marks to avoid confusion.

[0073] Embodiment 2: The method for constructing the array cell culture chip of this embodiment comprises the following steps: Use computer-aided design software to draw the microchannel structure and microscopic graphics of the upper chip 1 and the lower chip 5, and process them on the surface of the substrate of the upper chip 1 and the lower chip 5 by micromachining technology; Align the second sealing pad 4 to the upper surface of the lower chip 5 and seal it, and use sterile tweezers to slowly remove the bubbles so that the two fit tightly together; Align the first sealing pad 2 to seal the lower surface of the upper chip 1, and use sterile tweezers to slowly remove the bubbles so that the two fit tightly; The upper chip 1 sealed with the first sealing gasket 2, the porous membrane 3 and the lower chip 5 sealed with the second sealing gasket 4 are aligned and fitted in sequence, screws are inserted into the screw holes, nuts are tightened, and the assembly is completed to form an array cell culture chip.

[0074] In this construction method, the porous membrane 3 is sealed between the upper chip 1 and the lower chip 5, the porous membrane 3 and the lower chip 5 enclose a lower fluid channel, and the porous membrane 3 and the upper chip 1 enclose a skin tissue culture area, forming a cell culture chip with upper and lower flow channels.

[0075] Example 3: Tissue culture This embodiment provides a method for tissue co-culture using an array cell culture chip, comprising the following steps: S1. Pretreatment: After sterilizing the chips at different layers, assemble them and place the assembled chips under UV irradiation for 30 minutes; S2. Cell culture: Normal umbilical vein endothelial cells, fibroblasts, and keratinocytes were placed in a 37°C, 5% CO2 incubator for routine culture; S3. Cell suspension: Digest the adherent cells in the culture flask into single cells, add fresh culture medium to each cell, mix thoroughly by pipetting, and then add fresh culture medium to dilute to a cell suspension of appropriate concentration. Then, add umbilical vein endothelial cells (5×10 6 / mL) were suspended in bovine fibrin solution (concentration of 2.5 mg / mL, final thrombin concentration of 3 U / mL) to form a concentration of 5×10 6 umbilical vein endothelial cells-fibrin solution; fibroblasts (1×10 6 / mL) was suspended in a solution of 2.5mg / mL type I rat tail collagen and 2.5mg / mL bovine fibrin in a volume ratio of 1:1. Under ice bath conditions, the pH was adjusted to about 7.4, and thrombin was added dropwise to a final concentration of 3U / mL (prepared immediately before use) to form a concentration of 1×10 6 fibroblasts / mL of collagen solution; S4. Vascular tissue construction: Add 30 μL of umbilical vein endothelial cell-bovine fibrin solution to the skin tissue culture area, place in an incubator, and let stand for 30 minutes at 37°C and 5% CO2 to allow the extracellular matrix to solidify and form a vascular layer; S5. Skin tissue construction: 80 μL of fibroblast-collagen solution was injected into the skin tissue culture area, placed in an incubator, and allowed to stand at 37°C, 5% CO2 for 30 minutes to allow the extracellular matrix to solidify and form the dermis layer; then 100 μL of keratinocyte suspension was inoculated thereon, and allowed to stand at 37°C, 5% CO2 for 1 hour to allow the cells to adhere to the dermis layer and form the epidermis layer; S6. Tissue culture: Add culture medium (using high-glucose DMEM culture medium as the base liquid, adding 0.1% antibiotics, 30-50ng / ml endothelial growth factor EGF and 10% fetal bovine serum) from the liquid inlet at a rate of 100μL / min until all gases are discharged. The amount of culture medium added is 1200μL. Add 50μL of culture medium (using high-glucose DMEM culture medium as the base liquid, adding 0.2% commercial keratinocyte growth additive, 0.1% antibiotics and 10% fetal bovine serum) to the skin tissue culture area to support keratinocyte proliferation. Connect the peristaltic pump system for dynamic culture, and control the height of the culture medium at the liquid inlet and outlet through the adjustment column so that it does not exceed the horizontal line of the upper surface of the skin tissue. S7. Induce epidermal differentiation: Aspirate the culture medium in the skin tissue culture area, replace the culture medium in the blood channel with epidermal differentiation culture medium containing calcium ions (high-glucose DMEM culture medium as the base liquid, supplemented with 0.1% antibiotics, 1.8mM calcium chloride, 20~50ng / ml endothelial growth factor EGF and 10% fetal bovine serum), connect the peristaltic pump system and culture in a cell culture incubator at 37°C and 5% CO2 for 14 days to form skin tissue with a stratum corneum.

[0076] The preparation of the fibrin gel comprises: (1) dissolving fibrin powder with PBS solution to obtain a fibrinogen solution with a solid content of 10 mg / mL; (2) preparing thrombin into a 100 U / mL stock solution with PBS solution, filtering with a 0.22 μm sterile filter membrane in an ultra-clean workbench to obtain a fibrin solution and a thrombin solution respectively; (3) quickly mixing the fibrin solution and the thrombin solution, wherein the final concentration of the fibrin is 2.5 mg / ml and the final concentration of the thrombin is 3 U / mL, and the mixture is mixed evenly to generate a transparent fibrin gel.

[0077] The preparation of the collagen gel comprises: adding 300 μL rat tail collagen type I into a centrifuge tube placed on ice, then adding into 18 μL 0.1 mol / L NaOH, adjusting the pH to about 7.4, and immediately mixing before use.

[0078] This embodiment also provides the application of the array cell culture chip in cosmetic and drug testing, using systemic administration (oral, injection) and local (transdermal) administration to perform drug testing: For systemically administered drugs, they enter the fluid channel in the chip through the fluid inlet through-hole, and the skin tissue is tested after dynamic culture for a certain period of time; For transdermal drugs, they are added from above the culture chamber of skin tissue culture, and the skin tissue is tested after dynamic culture for a certain period of time.

[0079] Evaluation of the skin tissue-organ co-culture model constructed in Example 3: 1. The biomimetic properties of the skin tissue-organ co-culture model constructed in Example 3 were evaluated using the following method: 1.1 Analysis of constructed skin tissue interface: The skin tissue in the chip was removed, sliced ​​and stained with H&E. Fig. 9 As shown, the interface between dermis and epidermis tissue can be seen, which is highly bionic.

[0080] 1.2 Vascularization assessment: The distribution, density, orientation, length and branching of the microvascular network in the vascularized skin tissue and vascularized liver tissue in Example 3 were quantitatively analyzed under a confocal microscope to evaluate the biomimetic degree of vascularization.

[0081] Fig.10 Shown is a fluorescent image of the vascular structure of skin tissue, showing that endothelial cells are connected to each other to form microvessels.

[0082] It can be seen from the above tests that vascularized skin tissue can be successfully cultured using the chip provided by the present invention and the method for constructing skin tissue in vitro by linkage therewith.

[0083] 2. The drug test of the skin tissue-organ co-culture model constructed in Example 3 was evaluated by the following method: 2.1 Skin permeability test experiment: To the skin chip with vascularized skin tissue constructed in Example 3, a fluorescent compound (such as FITC-dextran, MW70000) of corresponding concentration was added from above the skin tissue culture area, and then a peristaltic pump system was connected for dynamic infiltration for 2 hours. Samples were taken from the blood channel at 0.5 h, 1 h, and 2 h, respectively, and placed in a centrifuge tube, and the fluorescence intensity was measured using a multifunctional microplate reader or a fluorescence microscope.

[0084] In this experiment, three groups were set up in the skin tissue culture area: (1) chip without cultured skin tissue; (2) chip with skin tissue; (3) chip with skin tissue + 10% retinoic acid. Fig.11 shown.

[0085] The experimental results showed that compared with the chip without skin tissue, the presence of skin tissue reduced the amount of FITC-dextran that penetrated into the blood channel below, indicating that the skin tissue constructed in vitro is similar to human skin and has a significant barrier effect, which can be used for drug delivery research. Compared with the chip with skin tissue, the skin barrier properties were destroyed after adding 10% retinoic acid.

[0086] 2.2 Cell activity assay: From the skin chip with vascularized skin tissue constructed in Example 3, 10 μL of 1% diclofenac sodium (experimental group) and no diclofenac sodium drug (control group) were added to the skin tissue culture area for dynamic culture. After 7 days, the skin tissue was taken out and placed on a 24-well plate, and the cells were washed with PBS; then, PBS solutions containing 2 μM Calcein-AM, 10 μg / mL Hoechst 33342 and 10 μg / mL PI were added, respectively, and stained in a 37°C incubator for 30 minutes, followed by washing three times with PBS; finally, under a microscope, the cytoplasm of living cells showed green fluorescence, the nuclei of dead cells showed red fluorescence, and the nuclei of all cells showed blue fluorescence. (Among them: apoptotic cells were stained with PI, live cells were stained with Calcein-AM, and nuclei were stained with Hoechst 33342). ImageJ software was used to statistically analyze the number of dead cells (red) and all cells (blue), and calculate the cell survival rate. The experimental results are as follows Fig.12 shown.

[0087] As shown in the figure, the survival of cells was observed. The majority of cells were alive in the control group, and the number of apoptotic cells increased significantly after drug addition. ImageJ software was used for statistical analysis and the cell survival rate in the skin tissue was calculated: 85% in the control group and 58% in the drug addition group. It can be seen that the drug affects the survival of cells. Therefore, the skin chip of the present invention can be used to screen drugs and evaluate the damage of drugs to the skin.

[0088] 2.3 Cell proliferation assay: From the skin chip with vascularized skin tissue constructed in Example 3, 1, 5 and 10 μL of 1% diclofenac sodium (experimental group) and no diclofenac sodium (control group) were added to the fluid channel for dynamic culture. The skin tissue was taken out and placed in a 24-well plate, and CCK8 test solution was added. After 2 hours, 100 μL of the test solution was taken out and placed in a 96-well plate, and the absorbance value was detected by an ELISA instrument. The experimental results are shown in Figure 2. Fig.13 shown.

[0089] As can be seen from the figure, compared with the control group, as the amount of diclofenac sodium added increases, the cell proliferation becomes worse and the survival rate becomes lower. Therefore, the skin chip constructed according to the present invention can be used to test safe drug dosages.

[0090] 2.4 Cell distribution experiment: First, before cell inoculation, keratinocytes and fibroblasts were digested into single cells, and the supernatant was removed after centrifugation at 1500rpm; then, the above cells were incubated with 2.5μM DiI (red) or 2.5μM DiO (green) fluorescent dye at 37°C for 30 minutes; finally, centrifuged at 1500rpm for 3 minutes, the dye was removed and the cells were resuspended with PBS, and repeated 3 times. After completion, fluorescently labeled cells were obtained. The labeled cells were then cultured according to the steps of Example 3 to obtain a vascularized skin tissue chip. Finally, 10μL of 10% retinoic acid (experimental group) and no retinoic acid (control group) were added to the skin tissue culture area for dynamic culture. After culture, the skin tissue was removed and the cell distribution was observed under a microscope. The results are as follows Fig.14 As shown (keratinocytes were stained red with DII dye, fibroblasts were stained green with DIO dye).

[0091] As can be seen from the figure, the cells in the control group formed a tissue interface structure between the epidermis and dermis; while after the drug was added to the experimental group, it was observed that the epidermal structure of the cells was destroyed.

[0092] The above embodiments are only preferred technical solutions of the present invention and should not be regarded as limiting the present invention. The protection scope of the present invention shall be the technical solutions recorded in the claims, including equivalent replacement solutions of the technical features in the technical solutions recorded in the claims. That is, equivalent replacement improvements within this scope are also within the protection scope of the present invention.

Claims

1. An array cell culture chip, characterized in that: The invention comprises a stacked upper chip (1) and a lower chip (5), wherein the upper chip (1) comprises a plurality of culture areas (10) arranged side by side, and a spacing is provided between adjacent culture areas (10), and each culture area (10) comprises a liquid inlet (101) and a liquid outlet (103) arranged at two ends, a blood channel is formed between the liquid inlet (101) and the liquid outlet (103), and a plurality of culture chambers (102) are provided between the liquid inlet (101) and the liquid outlet (103), and the liquid inlet (101), the culture chamber (102) and the liquid outlet (103) are connected to each other. 03) are arranged in a straight line, the lower chip (5) includes a plurality of parallel and spaced connecting grooves (501), each connecting groove (501) is located below each culture area (10), the blood inlet (101) and the blood outlet (103) are connected through the connecting groove (501), the lower end of each culture cavity (102) is connected to the connecting groove (501), and a plurality of porous membranes (3) are provided between the upper chip (1) and the lower chip (5), and each porous membrane (3) covers the lower end of each culture cavity (102) in the culture area (10).

2. The array cell culture chip according to claim 1, characterized in that: A first sealing pad (2) and a second sealing pad (4) are respectively provided on the upper and lower sides of the porous membrane (3); the upper chip (1) and the first sealing pad (2) are closed and clamped to clamp the first sealing pad (2) and the second sealing pad (4) to clamp the porous membrane (3); a plurality of second through holes (202) are provided on the first sealing pad (2); each second through hole (202) is aligned with each culture chamber (102); a through waist groove (401) is provided on the second sealing pad (4); a first through hole (201) and a third through hole (203) are further provided on the first sealing pad (2) and the second sealing pad (4); the first through hole (201) and the third through hole (203) are respectively aligned with the blood inlet (101) and the blood outlet (103); a sink (402) is provided at the through waist groove (401); the porous membrane (3) is placed in the sink (402).

3. The array cell culture chip according to claim 1, characterized in that: A plurality of top columns (502) are arranged in the connecting groove (501), a flow groove (503) is arranged at the upper end of the top column (502), and the upper end of the top column (502) abuts against the lower end of the porous membrane (3); the top columns (502) are arranged in multiple rows, and a plurality of rotatable adjustment columns (6) arranged in a straight line are arranged in the center of the connecting groove (501), the adjustment column (6) is provided with a strip-shaped baffle (601), one end of the adjustment column (6) is provided with a rotating shaft (602), the rotating shaft (602) is rotatably connected to the lower chip (5) and passes through the lower chip (5), the end of the rotating shaft (602) is provided with an annular groove (603), and a plurality of disc springs (7) are also provided, the disc spring (7) is provided with a U-shaped clamping groove (702), the bottom end of the U-shaped clamping groove (702) is provided with a clamping portion (701), the clamping portion (701) is clamped with the annular groove (603), and the outer edge of the disc spring (7) abuts against the bottom end of the lower chip (5).

4. The array cell culture chip according to claim 1, characterized in that: A partition partition (8) is also provided at the upper end of the upper chip (1). The partition partition (8) comprises a plurality of parallel and spaced strip-shaped partition grooves (801). Each strip-shaped partition groove (801) is provided above each culture area (10). A plurality of label grooves (802) are provided on one side of the strip-shaped partition groove (801). Each label groove (802) is provided with a magnetic block (803). A plurality of label sheets (9) are also provided. The label sheets (9) are provided in the label grooves (802) and are adsorbed by the magnetic blocks (803). One end of the label sheet (9) points toward the culture chamber (102).

5. The array cell culture chip according to claim 1, characterized in that: The pore size of the porous membrane (3) is 0.4-1 μm.

6. The method for constructing the array cell culture chip according to claim 1, characterized in that: The following steps are involved: The microchannel structure and microscopic pattern of the upper chip (1) and the lower chip (5) are drawn using computer-aided design software, and the microchannel structure and microscopic pattern of the upper chip (1) and the lower chip (5) are processed on the substrate surface of the upper chip (1) and the lower chip (5) using micromachining technology; The upper chip (1), the first sealing gasket (2), the porous membrane (3), the second sealing gasket (4) and the lower chip (5) are aligned and sealed in sequence to form an array cell culture chip.

7. The method for constructing an array cell culture chip according to claim 6, characterized in that: The layers of chips are sealed together by extrusion, bonding or threaded assembly.

8. The method for constructing an array cell culture chip according to claim 6, characterized in that: The processing method of the upper chip and the lower chip is at least one of a 3D printing method, a laser etching method, a CNC milling method and a hot pressing method.

9. The method for tissue culture using the array cell culture chip according to claim 1, characterized in that: The following steps are involved: Sterilizing the array cell culture chip according to any one of claims 1 to 4; A corresponding cell-matrix mixture is added to the tissue culture area (10) of the cell culture chip, a culture solution is added to the blood channels (101, 103), and the cells in the tissue culture area (10) are co-cultured to obtain a corresponding tissue model.

10. The method for tissue culture using the array cell culture chip according to claim 9, characterized in that: The method for preparing the cell-matrix mixed solution comprises the following steps: (1) Cell culture: Normal umbilical vein endothelial cells, fibroblasts, and keratinocytes were placed in a 37°C, 5% CO2 incubator for routine culture; (2) Cell suspension: After the adherent cells in the culture flask are digested into single cells, culture medium is added to each of them, mixed thoroughly, and then culture medium is added to dilute them into a cell suspension, which is then suspended in a matrix solution to obtain a cell-matrix and cell-culture medium mixture.

11. The method for tissue culture using the array cell culture chip according to claim 10, characterized in that: The matrix solution is selected from at least one of type I rat tail collagen solution, bovine fibrin, fibrin gel, hyaluronic acid, laminin or a mixture of type I rat tail collagen and bovine fibrin.

12. The method for tissue culture using the array cell culture chip according to claim 10, characterized in that: The concentration of the umbilical vein endothelial cell suspension is 1×10 6 -5×10 6 The concentration of fibroblast suspension was 0.5×10 6 -3×10 6 / mL, the concentration of keratinocyte suspension was 1×10 6 -2×10 6 Pieces / mL.

13. The method for tissue culture using the array cell culture chip according to claim 10, characterized in that: The following steps are involved: S1. Pretreatment: sterilizing the array cell culture chip; S2. Vascular tissue construction: The umbilical vein endothelial cell-matrix mixture is injected into the skin tissue culture area and allowed to stand for a certain period of time to allow the extracellular matrix to solidify and form a vascular layer; S3. Skin tissue construction: inject the fibroblast cell-matrix mixture into the skin tissue culture area and let it stand for a certain period of time to allow the extracellular matrix to solidify and form the dermis layer; Then, the keratinocyte-culture fluid is injected into the skin tissue culture area to make it adhere to the dermis layer and form the epidermis layer; S4. Tissue culture: slowly add culture medium from the liquid inlet, discharge the gas in the blood channel, and perform dynamic culture on the rocker arm or connect to the peristaltic pump system; S5. Epidermal differentiation: The culture medium in the skin tissue culture area is aspirated, and the culture medium in the blood channel is replaced with new culture medium. After several days of cultivation on a rocker arm or connected to a peristaltic pump system, skin tissue with a stratum corneum can be formed.

14. The method for tissue culture using the array cell culture chip according to claim 13, characterized in that: In S2, the amount of the umbilical vein endothelial cell-matrix mixture added is 30-50 μL.

15. The method for tissue culture using the array cell culture chip according to claim 13, characterized in that: In S3, the amount of the fibroblast cell-matrix mixture added is 50-100 μL; The amount of the keratinocyte-culture solution added is 80-120 μL.

16. The method for tissue culture using the array cell culture chip according to claim 13, characterized in that: In S4, the fluid shear stress was controlled at 0.01-10 dyn / cm during dynamic culture. 2 .

17. The method for tissue culture using the array cell culture chip according to claim 13, characterized in that: In S5, the new culture medium is an epidermal differentiation culture medium, which contains at least one of metal ions, ceramide and tissue plasminogen activator.

18. The method for tissue culture using the array cell culture chip according to claim 17, characterized in that: The metal ions are calcium ions, and the concentration of the calcium ions is 1-5 mmol / L.

19. Use of the array cell culture chip according to any one of claims 1 to 5 in cosmetic and pharmaceutical testing.

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