WO2024113486A1 - Super-hydrophobic microplate for three-dimensional cell culture, multi-organ micro-fluidic chip, and use thereof - Google Patents
Super-hydrophobic microplate for three-dimensional cell culture, multi-organ micro-fluidic chip, and use thereof Download PDFInfo
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- WO2024113486A1 WO2024113486A1 PCT/CN2023/074873 CN2023074873W WO2024113486A1 WO 2024113486 A1 WO2024113486 A1 WO 2024113486A1 CN 2023074873 W CN2023074873 W CN 2023074873W WO 2024113486 A1 WO2024113486 A1 WO 2024113486A1
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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
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
-
- 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
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
-
- 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
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
- C12M3/02—Tissue, human, animal or plant cell, or virus culture apparatus with means providing suspensions
-
- 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
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
- C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
Definitions
- the invention relates to the technical field of three-dimensional cell culture and organ chip, and in particular to a super-hydrophobic well plate for three-dimensional cell culture, a multi-organ microfluidic chip and applications thereof.
- Three-dimensional cell culture is more bionic than traditional two-dimensional adherent culture and has gradually become the mainstream mode of cell culture in biological research.
- Three-dimensional cell culture can be divided into two categories: three-dimensional culture without scaffold and three-dimensional culture with scaffold. Among them, the scaffold-free culture method is more flexible for cell manipulation.
- the three-dimensional culture without scaffold relies on three modes: Hanging drop microplate, Micropatterned surface microplate and Ultra-low adhesion microplate (low adhesion plate), among which the low adhesion plate is more commonly used and convenient.
- the principle of traditional low-adhesion well plates is to modify the surface of the wells with a chemical coating.
- This layer of chemical coating can prevent cells from adhering to the bottom of the wells, so that the cells can be suspended in the culture medium and grow spontaneously in clusters to form three-dimensional cell spheres, primary tissue blocks or organoids.
- the problem with traditional low-adhesion well plates is that the original cells, primary tissues or the final aggregated cell spheres and organoids will continuously secrete extracellular matrix and proteins into the culture medium, and the chemical coating is not resistant to these extracellular matrix and proteins. These extracellular matrix and proteins will settle and adhere to the chemical coating, making the chemical coating ineffective, so that the cells, primary tissue blocks or generated cell spheres and organoids will adhere to the bottom of the wells, thus affecting the three-dimensional culture.
- the technical problem to be solved by the present invention is to provide a super hydrophobic low adhesion well plate for three-dimensional cell culture, which solves the above-mentioned problems of traditional low adhesion well plates.
- the present invention provides the following technical solutions:
- the present invention provides a super-hydrophobic well plate for three-dimensional cell culture, wherein the super-hydrophobic well plate has one or more culture wells, wherein the bottom surface of the culture well is a super-hydrophobic surface with a micro-nano morphology, and the contact angle between the bottom surface of the culture well and the aqueous solution is greater than 120°; and the side surface of the culture well is a hydrophobic surface or a super-hydrophobic surface, and the contact angle between the bottom surface of the culture well and the aqueous solution is greater than 90°.
- the micro-nano super-hydrophobic surface with self-cleaning function is utilized to manufacture low-adhesion orifice plate.
- aqueous phase solution such as cell culture fluid
- the solution can not completely cover the super-hydrophobic bottom surface, can only partially contact or even not contact, thereby exists in a semi-suspended or even suspended state, therefore, reduces the contact area between cell and culture well bottom surface.
- this micro-nano super-hydrophobic surface does not adhere to extracellular matrix and protein, therefore these materials (such as protein, extracellular matrix etc.) deposited thereon are easy to clean, thereby can realize repeated use and long-term use of super-hydrophobic orifice plate.
- the material for making the super-hydrophobic orifice plate can be a polymer material, such as polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polystyrene (PS), polycarbonate (PC), etc.; it can also be a material such as metal, ceramic, glass, quartz, silicon, etc.; or a combination of one or more of the above materials.
- PMMA polymethyl methacrylate
- PDMS polydimethylsiloxane
- PS polystyrene
- PC polycarbonate
- the number of culture holes on the super-hydrophobic orifice plate is unlimited, for example, it can be 1-2000.
- the bottom surface shape and flatness of the culture hole are also not limited, for example, it can be circular, square, rectangular, triangular or other irregular shapes, or it can be an uneven shape.
- the size and depth of the culture hole are also not limited, and can be set as needed.
- the depth of the culture hole can be 0.5-20cm, and the diameter can be 0.5-20cm.
- the method for making the super-hydrophobic orifice plate includes method 1 and method 2:
- the method 1 comprises the following steps:
- the second method comprises the following steps:
- the inner surface of the culture hole is super-hydrophobic modified to obtain the super-hydrophobic well plate for three-dimensional cell culture
- the methods for forming a super-hydrophobic surface include surface etching, MEMS processing, surface enrichment of silica micro-nanoparticles, surface spraying of super-hydrophobic coatings and secondary molding.
- the method for surface enrichment of silica micro-nanoparticles is:
- Nano-silicon dioxide particles, n-hexane and chloroform are mixed and then ultrasonically treated to disperse the nano-silicon dioxide particles in the mixed solution; then, a PMMA plate is immersed in the mixed solution, taken out and dried, thereby obtaining a super-hydrophobic surface with a micro-nano structure.
- the method for enriching the silicon dioxide micro-nano particles on the surface is as follows: weigh 0.25-2g of hydrophobic nano-silicon dioxide particles with a diameter of 2-200nm, measure 20-500ml of n-hexane and 0.5-50ml of chloroform. Mix the above materials and perform ultrasonic treatment to completely disperse the nano-silicon dioxide particles in the mixed solution. Immerse the PMMA plate in the above solution for about 5-300 seconds, take it out and dry it, and a PMMA surface with a surface super-hydrophobic micro-nano structure is obtained.
- the surface etching method is:
- the PDMS sheet is immersed in tetraethyl orthosilicate to make it swell, and then the swollen PDMS sheet is taken out and placed in an ethylenediamine aqueous solution; then the PDMS sheet is taken out, rinsed and heat-treated to obtain a super-hydrophobic surface with a micro-nano structure.
- the method for enriching the surface of silicon dioxide micro-nano particles is specifically as follows: immersing the PDMS sheet in tetraethyl orthosilicate at 30-70° C. for 10-200 minutes, then taking out the swollen PDMS sheet from the tetraethyl orthosilicate solution and immediately floating it in a 5%-40% ethylenediamine aqueous solution. After 3-24 hours, taking it out and rinsing it with deionized water for 3-5 times, and heat-treating it in an oven for 0.5-5 hours to obtain a PDMS sheet with a surface super-hydrophobic micro-nano structure.
- the secondary mold remodeling method is:
- a pouring a prepolymer of an elastic resin (eg, a PDMS prepolymer) onto a template having a surface super-hydrophobic micro-nanostructure, polymerizing the elastic resin prepolymer into a first elastic solid, and then peeling the first elastic solid from the template;
- an elastic resin eg, a PDMS prepolymer
- silanization modification of the surface of the first elastic solid and using the silanization modification of the first elastic solid as a template, and then pouring a prepolymer of an elastic resin to polymerize the elastic resin prepolymer into a second elastic solid;
- the template with the surface super-hydrophobic micro-nano structure is a natural material (such as cicada wings, lotus leaves, etc.) or is prepared by artificial methods, and the artificial methods include surface etching, MEMS processing, surface enrichment of silica micro-nano particles, and surface spraying of super-hydrophobic coatings.
- the above-mentioned super-hydrophobic orifice plate provided by the present invention after the aqueous phase solution is injected into the culture hole, the solution cannot completely cover the super-hydrophobic bottom surface, and can only partially contact or even not contact, so as to exist in a semi-suspended or even suspended state.
- the super-hydrophobic bottom surface is self-cleaning, and the adsorption rate of proteins, nucleic acids, cells, etc. is extremely low, and the surface does not adhere to the extracellular matrix and protein, which can effectively prevent the cells from crawling out.
- the super-hydrophobic orifice plate can be used for the three-dimensional culture of cells such as cell spheres, primary tissue blocks, and organoids, and can also be used for the culture of suspended cells such as blood cells and immune cells. In addition, it can also be used for drug formulation evaluation, including efficacy, pharmacokinetic and toxicity.
- the present invention also provides a multi-organ microfluidic chip, comprising the super-hydrophobic low-adhesion well plate, a porous membrane and an upper substrate; the upper substrate is covered on the super-hydrophobic well plate, and the porous membrane is located between the super-hydrophobic well plate and the upper substrate; a microchannel is provided on the upper substrate, the microchannel is connected to the culture well on the super-hydrophobic well plate through the porous membrane, and the upper substrate is provided with an inlet and an outlet connected to the microchannel;
- the culture wells are used for culturing suspended cells, cell spheres, primary tissue blocks or organoids, and the microchannels are used for adding drugs or exogenous stimulants; the drugs or exogenous stimulants in the microchannels can enter the culture wells below through the porous membrane.
- the material of the upper substrate can be a polymer (polymethyl methacrylate, polydimethylsiloxane, polystyrene, polycarbonate, etc.), metal, ceramic, glass, quartz, silicon and other materials, and it can adopt the same or different material as the low adhesion orifice plate.
- the material of the porous membrane includes polycarbonate, polydimethylsiloxane, polyethylene film, PES (polyether sulfone), cellulose and its derivatives, polyvinyl chloride, polyvinylidene fluoride PVDF, polysulfone, polyacrylonitrile, polyamide, polysulfoneamide, sulfonated polysulfone, cross-linked polyvinyl alcohol, modified acrylic polymer, polytetrafluoroethylene (PTFE) porous film, porous polyurethane film, hollow fiber ultrafiltration membrane, quantifoil copper mesh porous membrane, quantifoil silica support membrane, quantifoil carbon film, porous alumina membrane or inorganic ceramic membrane.
- PTFE polytetrafluoroethylene
- vascular endothelial cells and/or immune cells may be cultured on the porous membrane.
- the top of the culture well is connected to the microchannel, so the culture solution in the culture well can be updated in real time through the culture solution flowing in the microchannel, thereby realizing the long-term culture of micro-tissues, and different tissues in multiple wells can communicate in real time through the upper microchannel, thus laying the foundation for the construction of multi-organ chips.
- micro-tissues can be used to construct multi-organ chips, which can greatly increase the number of integrated organs in multi-organ chips.
- a through hole is provided on the bottom plate or side wall of the culture well, and the through hole is used to extend a micro stirring paddle or a sensor into the culture well, so that the culture liquid in the culture well can be stirred or some parameters of the culture liquid can be detected, and oxygen can also be delivered to the culture liquid in the culture well to maintain the vitality of the tissue.
- the aperture of the through hole is preferably between 0.5 mm and 4 mm.
- the liquid in the microchannel is connected to the liquid in the culture well through a porous membrane, and the drug or exogenous stimulant flowing in the microchannel can pass through the porous membrane and interact with the micro-tissue, suspended cells, organoids or cell spheres in the well, so that the drugability of the drug can be evaluated, including efficacy, pharmacokinetic and toxicity evaluation. Therefore, the present invention also provides an application of the multi-organ microfluidic chip in drug drugability evaluation.
- the present invention has the following beneficial effects:
- the solution In the super-hydrophobic well plate of the present invention, after the aqueous solution is injected into the culture wells containing the super-hydrophobic micro-nano structure, the solution cannot completely cover the super-hydrophobic bottom surface, but can only partially contact or even not contact it, so that it exists in a semi-suspended or even suspended state, thereby reducing the contact area between the cells and the bottom surface of the well, thereby reducing the probability of the cells adhering to the bottom surface.
- the super-hydrophobic orifice plate of the present invention has a micro-nano super-hydrophobic surface to which extracellular matrix and protein do not adhere, so these substances deposited thereon can be easily washed off, thereby enabling repeated and long-term use of the orifice plate.
- the micro-nano super-hydrophobic surface can be realized on a variety of materials, such as glass, polymer, ceramic, etc., including materials that can be used to process microfluidic chips. Therefore, the micro-nano super-hydrophobic surface can be used to integrate low-adhesion holes and microfluidic chips to construct an integrated new platform for cell analysis and drug screening.
- FIG1 is a diagram showing the adhesion and disintegration of primary renal tissue blocks on the surface of a traditional low-adhesion plate
- FIG2 is a diagram showing the adhesion and disintegration of primary brain tissue blocks on the surface of a traditional low-adhesion well plate
- FIG3 is an electron microscope photograph of the surface of polydimethylsiloxane after it has been molded twice through a lotus leaf;
- FIG4 shows the state of a droplet on a super-hydrophobic PDMS surface
- FIG5 shows the steps of making a polydimethylsiloxane silicon sheet with through holes
- FIG6 is a schematic diagram of the principle of a super hydrophobic low adhesion orifice plate based on polydimethylsiloxane
- FIG7 shows the state of the aqueous solution in the super-hydrophobic pores of PDMS
- FIG8 is a bright field photograph of tumor spheres in super-hydrophobic pores of PDMS
- FIG9 shows the change in contact angle of the superhydrophobic pore after repeated use for 12 times
- FIG10 is a surface electron microscope photograph of a polydimethylsiloxane sheet of silicon dioxide microbubbles
- FIG11 is an electron microscope photograph of a superhydrophobic PMMA surface
- FIG12 is a diagram showing the state of the aqueous solution in the PMMA-PDMS super-hydrophobic pores
- FIG13 is a bright field photograph of tumor spheres in PMMA-PDMS super-hydrophobic pores
- FIG14 shows the state of a droplet in a PMMA super-hydrophobic hole
- FIG15 is a bright field photograph of tumor spheres in PMMA superhydrophobic pores
- FIG16 is a design diagram of a drug screening microfluidic chip based on superhydrophobic pores
- FIG17 is a photo of the chip (without the porous membrane).
- FIG18 is a bright field photograph of cardiac cell spheroids
- FIG19 is a bright field photograph of hepatocyte spheroids
- FIG20 is a bright field photograph of a neurosphere
- FIG21 is a live-death staining image of liver cell spheroids (A), neural cell spheroids (B) and cardiac cell spheroids (C) 3 hours after adding doxorubicin, where red represents dead cells and green represents live cells;
- FIG22 is a design diagram of a kidney tissue chip
- FIG23 is a diagram showing the disintegration process of kidney microtissues in a conventional PDMS chip
- FIG24 shows the state of kidney tissue in the superhydrophobic PDMS chip on the third day
- FIG25 is a cross-sectional view of a multi-organ chip
- FIG26 is a top view of a multi-organ chip
- FIG27 is a real picture of the multi-organ chip (without the porous membrane).
- Figure 28 is a bright field photograph of 15 primary tissues
- Figure 29 shows the toxic effects of cisplatin on 15 primary tissues on day 3 (green represents living cells, red represents Dead Cells).
- Comparative Example 1 Adhesion and disintegration of primary kidney and brain tissue blocks on the bottom surface of a traditional low-adhesion plate
- the primary tissue blocks of mouse kidney and brain were cultured in Corning's commercial low-adhesion well plates. Under normal circumstances, due to the low adhesion of the bottom of the well plate, the primary tissue blocks of kidney and brain are in a suspended culture state and their morphology can be well maintained. However, it was actually observed that on the fifth day of culture, the primary tissue blocks of kidney and brain adhered to the bottom of the well plate, and the cells crawled out and the tissue blocks disintegrated, as shown in Figures 1 and 2.
- Example 1 Using natural materials as templates to make super-hydrophobic orifice plates
- the preparation method of the super hydrophobic orifice plate is as follows:
- the lotus leaf is fixed on a glass plate, and liquid polydimethylsiloxane is poured on its surface, and overnight, the liquid polydimethylsiloxane is polymerized into a solid, and the solidified polydimethylsiloxane sheet is peeled off from the lotus leaf as the template for the next step.
- the polydimethylsiloxane template surface is then silanized, and liquid polydimethylsiloxane is poured on the template, and the temperature is raised for polymerization, and the newly solidified polydimethylsiloxane sheet is peeled off from the polydimethylsiloxane sheet template of silanization modification.
- the electron microscope image on the polydimethylsiloxane sheet surface is as shown in Figure 3, and it has a super-hydrophobic nanostructure, and the contact angle>120 ° (Fig. 4), and the super-hydrophobic polydimethylsiloxane base plate is finished.
- the template is prepared by 3D printing technology to make a polydimethylsiloxane plate with holes. The specific process is shown in Figure 5.
- the square holes on the super-hydrophobic plate have a side length of 4 mm and a depth of 4 mm.
- the plate was used for a tumor cell spheroidization experiment, and human embryonic lung fibroblasts (MRC-5) and human lung adenocarcinoma cells (NCI-H1792) were mixed and cultured in RPMI 1640 + 10% FBS.
- the culture ratio of fibroblasts to cancer cells was 1:1, and the culture environment was 37°C, 5% CO 2 .
- tumor cells successfully spheroidized in the super-hydrophobic plate (Figure 8), achieving an effect similar to that of a traditional low-adhesion plate.
- the test repeatability showed that the plate was added with various cell culture media for 48 h, then poured out, cleaned, and dried. The experiment was repeated 12 times, and the contact angle of the aqueous solution was measured again, which was still greater than 120° (Figure 9), indicating that the plate can be reused at least 12 times, with a continuous use time of more than 24 days.
- Example 2 Making a super-hydrophobic orifice plate using polydimethylsiloxane and polymethyl methacrylate materials
- the preparation method of the super hydrophobic orifice plate is as follows:
- a polydimethylsiloxane plate was immersed in tetraethyl orthosilicate at 50°C for 20 minutes. Then, the polydimethylsiloxane plate swollen with tetraethyl orthosilicate was taken out from the tetraethyl orthosilicate solution and immediately floated in an aqueous solution of 10% ethylenediamine, and silica microbubbles gradually formed on the surface of the polydimethylsiloxane plate. After 10 hours, the polydimethylsiloxane plate with silica microbubbles was taken out and rinsed with deionized water 3 times. Finally, the polydimethylsiloxane plate with silica microbubbles was heat treated in an oven for 1 hour. The electron microscope photo of the prepared superhydrophobic surface is shown in Figure 10.
- the superhydrophobic polydimethylsiloxane plate and the superhydrophobic polymethyl methacrylate plate with through holes are pressed together to obtain the final cell three-dimensional culture low-adhesion well plate.
- the bottom of the well is polydimethylsiloxane with a surface superhydrophobic micro-nano structure, and the side is superhydrophobic polymethyl methacrylate.
- the morphology of the aqueous solution in the well is shown in Figure 12, showing an obvious droplet morphology.
- the diameter of the circular hole of the super hydrophobic plate is 3 mm and the depth is 4 cm.
- the plate was used for the tumor cell sphering experiment, and the experimental conditions were the same as those in Example 1. After 3 days of culture, tumor sphering was successful in the super hydrophobic plate (Figure 13), reaching the same level as the traditional low-lying Adhesive well plates have similar effects.
- test repeatability showed that the well plate was added with cell culture medium (1640 culture medium + 10% fetal bovine serum) for 48 hours, then poured out, washed, dried, and the above experiment was repeated 12 times. Finally, the contact angle of the aqueous solution was measured to be 135.8°, which was still greater than 120°, indicating that the well plate can be reused at least 12 times, and the continuous use time exceeds 24 days.
- cell culture medium (1640 culture medium + 10% fetal bovine serum
- Example 3 Making a super-hydrophobic orifice plate using polymethyl methacrylate material
- the preparation method of the super hydrophobic orifice plate is as follows:
- a polymethyl methacrylate plate with holes was made by laser engraving, wherein the holes were square and the inner wall of the holes was super-hydrophobicly modified as follows: (1) 0.25 g of hydrophobic nano-silica particles with a diameter of 20 nm were weighed, 50 ml of n-hexane and 2.5 ml of chloroform were measured, and the mixture was ultrasonically treated to completely disperse the nano-silica particles in the mixed solution; (2) the polymethyl methacrylate plate with through holes was immersed in the above solution for about 15 seconds, and then taken out and dried. Another polymethyl methacrylate plate was modified by the same method.
- the superhydrophobic polymethyl methacrylate plate and the superhydrophobic polymethyl methacrylate plate with through holes are pressed together by bolts to obtain the final cell three-dimensional culture low-adhesion well plate.
- the bottom of the well is a polymethyl methacrylate plate with a surface superhydrophobic micro-nano structure, and the side is also a superhydrophobic polymethyl methacrylate.
- the morphology of the aqueous solution in the well is shown in Figure 14, showing an obvious droplet morphology.
- the square hole of the super hydrophobic plate has a side length of 4 mm and a depth of 4 cm.
- the plate was used for a tumor cell spheroidization experiment under the same experimental conditions as in Example 2. After 3 days of culture, tumor spheroidization was successful in the super hydrophobic plate (Figure 15), achieving an effect similar to that of a conventional low-adhesion plate.
- test repeatability showed that the well plate was added with cell culture medium (1640 culture medium + 10% fetal bovine serum) for 48 hours, then poured out, washed, dried, and the above experiment was repeated 12 times. Finally, the contact angle of the aqueous solution was measured to be 132.6°, which was still greater than 120°, indicating that the well plate can be reused at least 12 times, and the continuous use time exceeds 24 days.
- cell culture medium (1640 culture medium + 10% fetal bovine serum
- Example 4 Drug screening microfluidic chip based on super-hydrophobic well plate
- Cell spheroids have good biomimetic properties and are very important cell models for drug screening. Microfluidic chips are automated and intelligent and are also good carriers for drug screening. Many studies load cell spheroids on microfluidic chips for automated drug evaluation. However, this type of research usually produces cell spheroids through traditional commercial low-adhesion well plates, then transfers the cell spheroids to microfluidic chips, and then evaluates drugs, which is very cumbersome.
- This embodiment designs an integrated cell ball chip.
- the chip design is shown in FIG16 , and the actual picture is shown in FIG17 .
- the square is a three-hole PMMA super-hydrophobic low-adhesion plate, and the side length of the hole is 4mm and the depth is 4mm.
- the cardiomyocytes and fibroblasts induced by ips, and the vascular endothelial cells are mixed in a ratio of 2:1:1 and added to the super-hydrophobic wells.
- the culture medium is basal culture medium + enzymatic casein + hydrocortisone + L-glutamic acid + epidermal growth factor and other added factors.
- the culture environment is 37°C, 5% CO2 , and the cardiac cell spheres are successfully induced after 4 days ( Figure 18).
- the hepatocytes and stellate cells induced by ips are mixed in a ratio of 3:1 and added to another super-hydrophobic well.
- the culture medium is basal culture medium + enzymatic casein + hydrocortisone + L-glutamic acid and other added factors. After 3 days, the hepatocyte spheres are successfully induced ( Figure 19). Finally, the neural stem cells are added to the remaining super-hydrophobic wells.
- the culture medium is D-MEM/F12 plus B27 supplements + antibiotics-antifungal agents, epidermal growth factor and basic fibroblast growth factor, etc., and the neural cell spheres are successfully induced after 7 days ( Figure 20).
- a porous membrane was placed on top of each hole, and then a PDMS plate with a channel was pressed onto the porous membrane, with the position of the channel aligned with the position of the hole. Then, a culture solution containing doxorubicin was circulated in the channel at a flow rate of 1 ⁇ l/min. Liver cell spheres, heart cell spheres and nerve cell spheres were cultured, and doxorubicin would pass through the porous membrane and interact with the cell spheres in the hole, showing toxicity.
- Example 5 Tissue culture based on PDMS organ chips
- Example 6 Construction of a multi-organ chip based on superhydrophobic pores and its application in cisplatin toxicity evaluation
- a multi-organ chip containing 15 kinds of primary microtissues is constructed, and the cross-sectional view and top view of the chip are shown in Figures 25 and 26.
- Figure 27 is a real photo.
- Each well is 5mm in diameter and 5mm deep, and is made of PMMA.
- the tissues in the 15 wells are brain, eye, ear, tongue, trachea, heart, lung, liver, kidney, pancreas, spleen, fat, bone marrow, muscle, and testicular microtissues.
- the culture medium for each tissue in the well is shown in the following table:
- each tissue The bright field photographs of each tissue are shown in Figure 28. These tissues are loaded into the wells, and the culture medium containing cisplatin is circulated in the microchannel at a flow rate of 1 ⁇ l/min. Cisplatin will gradually diffuse into the droplets and interact with various tissues. After 3 days, the live and dead fluorescence photos of each tissue are shown in Figure 29, and it can be seen that doxorubicin shows differentiated toxicity to various tissues. Therefore, the multi-organ chip based on superhydrophobic pores constructed in this embodiment can be used for the toxicity evaluation of cisplatin.
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Abstract
A super-hydrophobic microplate for three-dimensional cell culture. The super-hydrophobic microplate is provided with one or more culture wells. The bottom surfaces of the culture wells are super-hydrophobic surfaces with micro-nano morphology, and the side surfaces of the culture wells are hydrophobic surfaces or super-hydrophobic surfaces. A multi-organ micro-fluidic chip, comprising the super-hydrophobic microplate, a porous membrane, and an upper-layer substrate. The upper-layer substrate caps the super-hydrophobic microplate, and the porous membrane is located between the super-hydrophobic microplate and the upper-layer substrate; the upper-layer substrate is provided with a micro-channel, the micro-channel is in communication with the culture wells in the super-hydrophobic microplate by means of the porous membrane, and the upper-layer substrate is provided with an inlet and an outlet which are in communication with the micro-channel. The super-hydrophobic microplate for three-dimensional cell culture features the micro-nano super-hydrophobic surface that does not adhere to extracellular matrix and proteins, facilitating easy cleaning of deposited substances. This feature allows for repeated and long-term use, and can further be utilized to construct a new platform for integrated cell analysis and drug screening.
Description
本发明涉及细胞三维培养和器官芯片技术领域，具体涉及一种用于细胞三维培养的超疏水孔板，多器官微流控芯片及其应用。The invention relates to the technical field of three-dimensional cell culture and organ chip, and in particular to a super-hydrophobic well plate for three-dimensional cell culture, a multi-organ microfluidic chip and applications thereof.
细胞三维培养比传统的二维贴壁培养更仿生，已日渐成为生物学研究中细胞培养的主流模式。细胞三维培养可以分为无支架三维培养和有支架三维培养两大类。其中无支架培养方式对于细胞的操控更为灵活。而无支架三维培养又依赖于Hanging drop microplate,Micropatterned surface microplate和Ultra-low adhesion microplate(低粘附孔板)三种模式，其中低粘附孔板是使用较普遍和方便的。Three-dimensional cell culture is more bionic than traditional two-dimensional adherent culture and has gradually become the mainstream mode of cell culture in biological research. Three-dimensional cell culture can be divided into two categories: three-dimensional culture without scaffold and three-dimensional culture with scaffold. Among them, the scaffold-free culture method is more flexible for cell manipulation. The three-dimensional culture without scaffold relies on three modes: Hanging drop microplate, Micropatterned surface microplate and Ultra-low adhesion microplate (low adhesion plate), among which the low adhesion plate is more commonly used and convenient.
传统低粘附孔板的原理是在孔的表面修饰化学涂层。这一层化学涂层可以阻碍细胞粘附在孔底，从而可以使细胞悬浮在培养液中自发聚团生长，形成三维的细胞球、原代组织块或者类器官。但传统低粘附孔板的问题在于，原始的细胞、原代组织或者最终聚合的细胞球和类器官会不停的向培养液中分泌细胞外基质和蛋白，化学涂层对这些细胞外基质和蛋白是不抗拒的，这些细胞外基质和蛋白便会沉降并粘附在化学涂层上，使化学涂层失效，从而细胞、原代组织块或者生成的细胞球和类器官便会贴附在孔底、从而影响到三维培养。The principle of traditional low-adhesion well plates is to modify the surface of the wells with a chemical coating. This layer of chemical coating can prevent cells from adhering to the bottom of the wells, so that the cells can be suspended in the culture medium and grow spontaneously in clusters to form three-dimensional cell spheres, primary tissue blocks or organoids. However, the problem with traditional low-adhesion well plates is that the original cells, primary tissues or the final aggregated cell spheres and organoids will continuously secrete extracellular matrix and proteins into the culture medium, and the chemical coating is not resistant to these extracellular matrix and proteins. These extracellular matrix and proteins will settle and adhere to the chemical coating, making the chemical coating ineffective, so that the cells, primary tissue blocks or generated cell spheres and organoids will adhere to the bottom of the wells, thus affecting the three-dimensional culture.
传统低粘附孔板的上述问题导致了三个后果：第一，它并不普适于所有细胞的三维培养，特别是那些细胞外基质和蛋白分泌旺盛的细胞(譬如肾细胞、神经细胞)；第二，对于大多数普通的细胞，传统低粘附孔板一般也很难实现长时间三维培养；第三，传统低粘附孔板一般很难多次使用。此外，传统的低粘附孔板中孔的尺寸、形状、大小是固定的，很难随具体实验的需求而调整；而且，它不能和微流控芯片等下游分析平台集成，实现自动化的细胞分析。The above problems of traditional low-adhesion well plates lead to three consequences: first, it is not universally suitable for the three-dimensional culture of all cells, especially those cells that secrete a lot of extracellular matrix and protein (such as kidney cells and nerve cells); second, for most common cells, traditional low-adhesion well plates are generally difficult to achieve long-term three-dimensional culture; third, traditional low-adhesion well plates are generally difficult to use multiple times. In addition, the size, shape, and size of the holes in traditional low-adhesion well plates are fixed, and it is difficult to adjust them according to the needs of specific experiments; moreover, it cannot be integrated with downstream analysis platforms such as microfluidic chips to achieve automated cell analysis.
上述缺点限制了传统低粘附孔板的应用范围和潜力。
The above shortcomings limit the application scope and potential of traditional low-adhesion orifice plates.
发明内容Summary of the invention
本发明要解决的技术问题是提供一种用于细胞三维培养的超疏水低粘附孔板，该超疏水低粘附孔板解决了传统低粘附孔板的上述问题。The technical problem to be solved by the present invention is to provide a super hydrophobic low adhesion well plate for three-dimensional cell culture, which solves the above-mentioned problems of traditional low adhesion well plates.
为了解决上述技术问题，本发明提供了如下的技术方案：In order to solve the above technical problems, the present invention provides the following technical solutions:
本发明提供了一种用于细胞三维培养的超疏水孔板，所述超疏水孔板具有一个或多个培养孔，所述培养孔的底面为具有微纳米形貌的超疏水表面，其与水相溶液的接触角大于120°；所述培养孔的侧面为疏水表面或者超疏水表面，其与水相溶液的接触角大于90°。The present invention provides a super-hydrophobic well plate for three-dimensional cell culture, wherein the super-hydrophobic well plate has one or more culture wells, wherein the bottom surface of the culture well is a super-hydrophobic surface with a micro-nano morphology, and the contact angle between the bottom surface of the culture well and the aqueous solution is greater than 120°; and the side surface of the culture well is a hydrophobic surface or a super-hydrophobic surface, and the contact angle between the bottom surface of the culture well and the aqueous solution is greater than 90°.
本发明中，利用具有自清洁功能的微纳米超疏水表面，制造低粘附孔板。将水相溶液(如细胞培养液)注入含有超疏水微纳米结构的培养孔后，溶液不能完全覆盖超疏水底面，仅能部分接触甚至不接触，从而以一种半悬空甚至悬空的状态存在，因此，减少了细胞和培养孔底面的接触面积。另外，由于这种微纳米超疏水表面并不粘附细胞外基质和蛋白，因此沉积在上面的这些物质(譬如蛋白，细胞外基质等)很容易清洗掉，从而可以实现超疏水孔板的反复使用和长期使用。In the present invention, the micro-nano super-hydrophobic surface with self-cleaning function is utilized to manufacture low-adhesion orifice plate. After aqueous phase solution (such as cell culture fluid) is injected into the culture well containing super-hydrophobic micro-nano structure, the solution can not completely cover the super-hydrophobic bottom surface, can only partially contact or even not contact, thereby exists in a semi-suspended or even suspended state, therefore, reduces the contact area between cell and culture well bottom surface. In addition, because this micro-nano super-hydrophobic surface does not adhere to extracellular matrix and protein, therefore these materials (such as protein, extracellular matrix etc.) deposited thereon are easy to clean, thereby can realize repeated use and long-term use of super-hydrophobic orifice plate.
进一步地，制作所述超疏水孔板的材料既可以为高分子聚合物材料，例如聚甲基丙烯酸甲酯(PMMA)、聚二甲基硅氧烷(PDMS)、聚苯乙烯(PS)、聚碳酸酯(PC)等；也可以为金属、陶瓷、玻璃、石英、硅等材料；或者上述一种或多种材料的组合。Furthermore, the material for making the super-hydrophobic orifice plate can be a polymer material, such as polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polystyrene (PS), polycarbonate (PC), etc.; it can also be a material such as metal, ceramic, glass, quartz, silicon, etc.; or a combination of one or more of the above materials.
本发明中，对于超疏水孔板上培养孔的数目不限，例如可以为1-2000个。对于培养孔的底面形状和平整度也不做限制，例如可以为圆形、正方形、长方形、三角形或者其他不规则形状，也可以为凹凸不平的形状。同样的，对于培养孔的大小和深度也不做限制，可根据需要进行设置。例如，培养孔的深度可以为0.5-20cm，直径可以为0.5-20cm。In the present invention, the number of culture holes on the super-hydrophobic orifice plate is unlimited, for example, it can be 1-2000. The bottom surface shape and flatness of the culture hole are also not limited, for example, it can be circular, square, rectangular, triangular or other irregular shapes, or it can be an uneven shape. Similarly, the size and depth of the culture hole are also not limited, and can be set as needed. For example, the depth of the culture hole can be 0.5-20cm, and the diameter can be 0.5-20cm.
本发明中，制作所述超疏水孔板的方法包括方法一和方法二：In the present invention, the method for making the super-hydrophobic orifice plate includes method 1 and method 2:
所述方法一包括以下步骤：The method 1 comprises the following steps:
S1.制备超疏水底板；S1. Preparation of super hydrophobic substrate;
S2.制备带通孔的疏水板；S2. Prepare a hydrophobic plate with through holes;
S3.将上述超疏水底板和疏水板贴合封接在一起，即得到所述用于细胞三维培养的超疏
水孔板；S3. The super-hydrophobic bottom plate and the hydrophobic plate are bonded and sealed together to obtain the super-hydrophobic substrate for three-dimensional cell culture. Water hole plate;
所述方法二包括以下步骤：The second method comprises the following steps:
S4.在板材上通过一次成型得到培养孔；S4. Obtaining culture wells on the plate by one-step molding;
S5.对上述培养孔的内表面进行超疏水修饰，即得到所述用于细胞三维培养的超疏水孔板；S5. The inner surface of the culture hole is super-hydrophobic modified to obtain the super-hydrophobic well plate for three-dimensional cell culture;
其中，形成超疏水表面的方法包括表面刻蚀、MEMS加工、表面富集二氧化硅微纳米颗粒、表面喷射超疏水涂料和二次翻模。Among them, the methods for forming a super-hydrophobic surface include surface etching, MEMS processing, surface enrichment of silica micro-nanoparticles, surface spraying of super-hydrophobic coatings and secondary molding.
在一种实施方式中，表面富集二氧化硅微纳米颗粒的方法为：In one embodiment, the method for surface enrichment of silica micro-nanoparticles is:
将纳米二氧化硅颗粒、正己烷和氯仿混合后进行超声处理，使纳米二氧化硅颗粒分散于混合溶液中；接着，将PMMA板材浸入所述混合溶液中，取出晾干，即得到了具有微纳米结构的超疏水表面。Nano-silicon dioxide particles, n-hexane and chloroform are mixed and then ultrasonically treated to disperse the nano-silicon dioxide particles in the mixed solution; then, a PMMA plate is immersed in the mixed solution, taken out and dried, thereby obtaining a super-hydrophobic surface with a micro-nano structure.
优选地，上述表面富集二氧化硅微纳米颗粒的方法具体为：称取0.25-2g直径为2-200nm的疏水性纳米二氧化硅颗粒，量取20-500ml正己烷和0.5-50ml氯仿。将上述材料混合后进行超声处理，使纳米二氧化硅颗粒完全分散在混合溶液中。将PMMA板材浸入上述溶液中约5-300秒，取出晾干，便得到了具有表面超疏水微纳米结构的PMMA表面。Preferably, the method for enriching the silicon dioxide micro-nano particles on the surface is as follows: weigh 0.25-2g of hydrophobic nano-silicon dioxide particles with a diameter of 2-200nm, measure 20-500ml of n-hexane and 0.5-50ml of chloroform. Mix the above materials and perform ultrasonic treatment to completely disperse the nano-silicon dioxide particles in the mixed solution. Immerse the PMMA plate in the above solution for about 5-300 seconds, take it out and dry it, and a PMMA surface with a surface super-hydrophobic micro-nano structure is obtained.
在另一种实施方式中，表面刻蚀的方法为：In another embodiment, the surface etching method is:
将PDMS板材浸入原硅酸四乙酯中使其溶胀，然后取出溶胀的PDMS板材，置于乙二胺水溶液中；接着取出PDMS板材，冲洗后进行热处理，即得到具有微纳米结构的超疏水表面。The PDMS sheet is immersed in tetraethyl orthosilicate to make it swell, and then the swollen PDMS sheet is taken out and placed in an ethylenediamine aqueous solution; then the PDMS sheet is taken out, rinsed and heat-treated to obtain a super-hydrophobic surface with a micro-nano structure.
优选地，上述表面富集二氧化硅微纳米颗粒的方法具体为：将PDMS板材浸入30-70℃的原硅酸四乙酯中10-200分钟，然后，将溶胀的PDMS板材从原硅酸四乙酯溶液中取出，立即漂浮在5％-40％的乙二胺水溶液中。3-24小时后，取出并用去离子水冲洗3-5次，烘箱中热处理0.5-5小时，得到具有表面超疏水微纳米结构的PDMS板材。Preferably, the method for enriching the surface of silicon dioxide micro-nano particles is specifically as follows: immersing the PDMS sheet in tetraethyl orthosilicate at 30-70° C. for 10-200 minutes, then taking out the swollen PDMS sheet from the tetraethyl orthosilicate solution and immediately floating it in a 5%-40% ethylenediamine aqueous solution. After 3-24 hours, taking it out and rinsing it with deionized water for 3-5 times, and heat-treating it in an oven for 0.5-5 hours to obtain a PDMS sheet with a surface super-hydrophobic micro-nano structure.
进一步地，所述二次翻模的方法为：Furthermore, the secondary mold remodeling method is:
a.将弹性树脂的预聚液(譬如PDMS预聚液)浇注在具有表面超疏水微纳米结构的模板上，使弹性树脂预聚液聚合成第一弹性固体，再将第一弹性固体从模板上剥离；
a. pouring a prepolymer of an elastic resin (eg, a PDMS prepolymer) onto a template having a surface super-hydrophobic micro-nanostructure, polymerizing the elastic resin prepolymer into a first elastic solid, and then peeling the first elastic solid from the template;
b.对第一弹性固体的表面进行硅烷化修饰，并以硅烷化修饰的第一弹性固体为模板，再浇注弹性树脂的预聚液，使弹性树脂预聚液聚合成第二弹性固体；b. silanization modification of the surface of the first elastic solid, and using the silanization modification of the first elastic solid as a template, and then pouring a prepolymer of an elastic resin to polymerize the elastic resin prepolymer into a second elastic solid;
c.将第二弹性固体从硅烷化修饰的第一弹性固体模板上剥离，此时，第二弹性固体的表面便形成了表面超疏水微纳米结构。c. Peeling the second elastic solid from the silanized first elastic solid template, at which point a surface super-hydrophobic micro-nano structure is formed on the surface of the second elastic solid.
进一步地，所述具有表面超疏水微纳米结构的模板为天然材料(譬如蝉翼、荷叶等)或通过人工方法制备，所述人工方法包括表面刻蚀、MEMS加工、表面富集二氧化硅微纳米颗粒、表面喷射超疏水涂料。Furthermore, the template with the surface super-hydrophobic micro-nano structure is a natural material (such as cicada wings, lotus leaves, etc.) or is prepared by artificial methods, and the artificial methods include surface etching, MEMS processing, surface enrichment of silica micro-nano particles, and surface spraying of super-hydrophobic coatings.
本发明提供的上述超疏水孔板，水相溶液注入培养孔后，溶液不能完全覆盖超疏水底面，仅能部分接触甚至不接触，从而以一种半悬空甚至悬空的状态存在。超疏水底面是自清洁的，对蛋白，核酸，细胞等的吸附率极低，且表面并不粘附细胞外基质和蛋白，可以有效的防止了细胞的爬出。因此，该超疏水孔板既可以用于细胞球、原代组织块、类器官等细胞的三维培养，也可以用于血细胞、免疫细胞等悬浮细胞的培养。此外，其还可以用于药物成药性评价，包括药效，药代和毒性。The above-mentioned super-hydrophobic orifice plate provided by the present invention, after the aqueous phase solution is injected into the culture hole, the solution cannot completely cover the super-hydrophobic bottom surface, and can only partially contact or even not contact, so as to exist in a semi-suspended or even suspended state. The super-hydrophobic bottom surface is self-cleaning, and the adsorption rate of proteins, nucleic acids, cells, etc. is extremely low, and the surface does not adhere to the extracellular matrix and protein, which can effectively prevent the cells from crawling out. Therefore, the super-hydrophobic orifice plate can be used for the three-dimensional culture of cells such as cell spheres, primary tissue blocks, and organoids, and can also be used for the culture of suspended cells such as blood cells and immune cells. In addition, it can also be used for drug formulation evaluation, including efficacy, pharmacokinetic and toxicity.
进一步地，本发明还提供了一种多器官微流控芯片，包括所述的超疏水低粘附孔板、多孔膜和上层基板；所述上层基板盖合于所述超疏水孔板上，所述多孔膜位于所述超疏水孔板与上层基板之间；所述上层基板上设置有微通道，所述微通道通过所述多孔膜与超疏水孔板上的培养孔连通，且所述上层基板上设有与所述微通道连通的进口与出口；Furthermore, the present invention also provides a multi-organ microfluidic chip, comprising the super-hydrophobic low-adhesion well plate, a porous membrane and an upper substrate; the upper substrate is covered on the super-hydrophobic well plate, and the porous membrane is located between the super-hydrophobic well plate and the upper substrate; a microchannel is provided on the upper substrate, the microchannel is connected to the culture well on the super-hydrophobic well plate through the porous membrane, and the upper substrate is provided with an inlet and an outlet connected to the microchannel;
所述培养孔中用于培养悬浮细胞、细胞球、原代组织块或者类器官，所述微通道中用于添加药物或者外源性刺激物；微通道中的药物或者外源性刺激物可通过所述多孔膜进入到下方的培养孔中。The culture wells are used for culturing suspended cells, cell spheres, primary tissue blocks or organoids, and the microchannels are used for adding drugs or exogenous stimulants; the drugs or exogenous stimulants in the microchannels can enter the culture wells below through the porous membrane.
本发明中，所述上层基板的材质可以为高分子聚合物(聚甲基丙烯酸甲酯、聚二甲基硅氧烷、聚苯乙烯、聚碳酸酯等)、金属、陶瓷、玻璃、石英、硅等材料，且其可以采用与低粘附孔板相同或不同的材质。多孔膜的材质包括聚碳酸酯、聚二甲基硅氧烷、聚乙烯膜、PES(聚醚砜)、纤维素及其衍生物、聚氯乙烯、聚偏氟乙烯PVDF、聚砜、聚丙烯腈、聚酰胺、聚砜酰胺、磺化聚砜、交链的聚乙烯醇、改性丙烯酸聚合物、聚四氟乙烯(PTFE)多孔薄膜、多孔聚氨酯薄膜、中空纤维超滤膜、quantifoil铜网多孔膜，quantifoil二氧化硅支持膜，quantifoil碳膜、多孔氧化铝膜或无机陶瓷膜。In the present invention, the material of the upper substrate can be a polymer (polymethyl methacrylate, polydimethylsiloxane, polystyrene, polycarbonate, etc.), metal, ceramic, glass, quartz, silicon and other materials, and it can adopt the same or different material as the low adhesion orifice plate. The material of the porous membrane includes polycarbonate, polydimethylsiloxane, polyethylene film, PES (polyether sulfone), cellulose and its derivatives, polyvinyl chloride, polyvinylidene fluoride PVDF, polysulfone, polyacrylonitrile, polyamide, polysulfoneamide, sulfonated polysulfone, cross-linked polyvinyl alcohol, modified acrylic polymer, polytetrafluoroethylene (PTFE) porous film, porous polyurethane film, hollow fiber ultrafiltration membrane, quantifoil copper mesh porous membrane, quantifoil silica support membrane, quantifoil carbon film, porous alumina membrane or inorganic ceramic membrane.
进一步地，多孔膜上可以培养有血管内皮细胞和/或免疫细胞。
Furthermore, vascular endothelial cells and/or immune cells may be cultured on the porous membrane.
本发明中，培养孔孔顶和微通道连结，因此培养孔内的培养液可以通过微通道中流动的培养液进行实时更新，从而实现微型组织的长期培养，而且多个孔内不同的组织，可以通过上方微通道进行实时通讯，从而为多器官芯片的构建奠定了基础。另外，可以使用微型组织来构建多器官芯片，可以大幅增加多器官芯片集成器官的数目。In the present invention, the top of the culture well is connected to the microchannel, so the culture solution in the culture well can be updated in real time through the culture solution flowing in the microchannel, thereby realizing the long-term culture of micro-tissues, and different tissues in multiple wells can communicate in real time through the upper microchannel, thus laying the foundation for the construction of multi-organ chips. In addition, micro-tissues can be used to construct multi-organ chips, which can greatly increase the number of integrated organs in multi-organ chips.
进一步地，所述培养孔的底板或者侧壁上开设有贯穿孔，所述贯穿孔用于将微型搅拌桨或传感器伸入到培养孔中，从而可以对培养孔中的培养液进行搅拌或对培养液的一些参数进行检测，也可以向培养孔中的培养液输送氧气，从而维持组织的活力。该贯穿孔的孔径优选地在0.5mm-4mm之间。Furthermore, a through hole is provided on the bottom plate or side wall of the culture well, and the through hole is used to extend a micro stirring paddle or a sensor into the culture well, so that the culture liquid in the culture well can be stirred or some parameters of the culture liquid can be detected, and oxygen can also be delivered to the culture liquid in the culture well to maintain the vitality of the tissue. The aperture of the through hole is preferably between 0.5 mm and 4 mm.
本发明的多器官微流控芯片中，微通道内的液体通过多孔膜与培养孔内的液体连通，微通道内流通的药物或者外源性刺激物可以穿过多孔膜与孔内的微型组织、悬浮细胞、类器官或者细胞球相互作用，从而可以评价该药物的成药性，包括药效，药代和毒性评价。因此本发明还提供了所述的一种多器官微流控芯片在药物成药性评价中的应用。In the multi-organ microfluidic chip of the present invention, the liquid in the microchannel is connected to the liquid in the culture well through a porous membrane, and the drug or exogenous stimulant flowing in the microchannel can pass through the porous membrane and interact with the micro-tissue, suspended cells, organoids or cell spheres in the well, so that the drugability of the drug can be evaluated, including efficacy, pharmacokinetic and toxicity evaluation. Therefore, the present invention also provides an application of the multi-organ microfluidic chip in drug drugability evaluation.
与现有技术相比，本发明的有益效果在于：Compared with the prior art, the present invention has the following beneficial effects:
1.本发明的超疏水孔板，将水相溶液注入含有超疏水微纳米结构的培养孔后，溶液不能完全覆盖超疏水底面，仅能部分接触甚至不接触，从而以一种半悬空甚至悬空的状态存在，因此，减少了细胞和孔底面的接触面积，从而降低了细胞在底面贴壁的概率。1. In the super-hydrophobic well plate of the present invention, after the aqueous solution is injected into the culture wells containing the super-hydrophobic micro-nano structure, the solution cannot completely cover the super-hydrophobic bottom surface, but can only partially contact or even not contact it, so that it exists in a semi-suspended or even suspended state, thereby reducing the contact area between the cells and the bottom surface of the well, thereby reducing the probability of the cells adhering to the bottom surface.
2.本发明的超疏水孔板，微纳米超疏水表面并不粘附细胞外基质和蛋白，因此沉积在上面的这些物质很容易清洗掉，从而可以实现孔板的反复使用和长期使用。2. The super-hydrophobic orifice plate of the present invention has a micro-nano super-hydrophobic surface to which extracellular matrix and protein do not adhere, so these substances deposited thereon can be easily washed off, thereby enabling repeated and long-term use of the orifice plate.
3.本发明中，微纳米超疏水表面可以在多种材质上实现，譬如玻璃，高聚物，陶瓷等等，其中就包括可以用来加工微流控芯片的材质，因此利用微纳米超疏水表面可以把低粘附的孔和微流控芯片集成起来，构建一体化的细胞分析和药物筛选新平台。3. In the present invention, the micro-nano super-hydrophobic surface can be realized on a variety of materials, such as glass, polymer, ceramic, etc., including materials that can be used to process microfluidic chips. Therefore, the micro-nano super-hydrophobic surface can be used to integrate low-adhesion holes and microfluidic chips to construct an integrated new platform for cell analysis and drug screening.
图1为肾原代组织块在传统低粘附孔板表面的粘附和崩解图；FIG1 is a diagram showing the adhesion and disintegration of primary renal tissue blocks on the surface of a traditional low-adhesion plate;
图2为脑原代组织块在传统低粘附孔板表面的粘附和崩解图；FIG2 is a diagram showing the adhesion and disintegration of primary brain tissue blocks on the surface of a traditional low-adhesion well plate;
图3为聚二甲基硅氧烷通过荷叶两次翻模后，表面的电子显微镜照片图；FIG3 is an electron microscope photograph of the surface of polydimethylsiloxane after it has been molded twice through a lotus leaf;
图4为液滴在超疏水PDMS表面的状态；FIG4 shows the state of a droplet on a super-hydrophobic PDMS surface;
图5为制作带通孔的聚二甲基硅氧烷硅板材的步骤；
FIG5 shows the steps of making a polydimethylsiloxane silicon sheet with through holes;
图6为基于聚二甲基硅氧烷的超疏水低粘附孔板原理示意图；FIG6 is a schematic diagram of the principle of a super hydrophobic low adhesion orifice plate based on polydimethylsiloxane;
图7为PDMS超疏水孔中水相溶液的状态；FIG7 shows the state of the aqueous solution in the super-hydrophobic pores of PDMS;
图8为PDMS超疏水孔中，肿瘤球的明场照片；FIG8 is a bright field photograph of tumor spheres in super-hydrophobic pores of PDMS;
图9为重复使用12次后，超疏水孔接触角的变化；FIG9 shows the change in contact angle of the superhydrophobic pore after repeated use for 12 times;
图10为二氧化硅微泡的聚二甲基硅氧烷片材的表面电镜照片；FIG10 is a surface electron microscope photograph of a polydimethylsiloxane sheet of silicon dioxide microbubbles;
图11为超疏水PMMA表面电镜照片；FIG11 is an electron microscope photograph of a superhydrophobic PMMA surface;
图12为PMMA-PDMS超疏水孔中水相溶液的状态；FIG12 is a diagram showing the state of the aqueous solution in the PMMA-PDMS super-hydrophobic pores;
图13为PMMA-PDMS超疏水孔中，肿瘤球的明场照片；FIG13 is a bright field photograph of tumor spheres in PMMA-PDMS super-hydrophobic pores;
图14为PMMA超疏水孔中液滴的状态；FIG14 shows the state of a droplet in a PMMA super-hydrophobic hole;
图15为PMMA超疏水孔中肿瘤球的明场照片；FIG15 is a bright field photograph of tumor spheres in PMMA superhydrophobic pores;
图16为基于超疏水孔的药物筛选微流控芯片设计图；FIG16 is a design diagram of a drug screening microfluidic chip based on superhydrophobic pores;
图17为芯片实物照片(去掉多孔膜)；FIG17 is a photo of the chip (without the porous membrane);
图18为心脏细胞球的明场照片；FIG18 is a bright field photograph of cardiac cell spheroids;
图19为肝细胞球的明场照片；FIG19 is a bright field photograph of hepatocyte spheroids;
图20为神经细胞球的明场照片；FIG20 is a bright field photograph of a neurosphere;
图21为加阿霉素3个小时后，肝细胞球(A)，神经细胞球(B)和心脏细胞球(C)的活死染色图，红色代表死细胞，绿色代表活细胞；FIG21 is a live-death staining image of liver cell spheroids (A), neural cell spheroids (B) and cardiac cell spheroids (C) 3 hours after adding doxorubicin, where red represents dead cells and green represents live cells;
图22为肾组织芯片设计图；FIG22 is a design diagram of a kidney tissue chip;
图23为传统PDMS芯片内肾微组织崩解过程图；FIG23 is a diagram showing the disintegration process of kidney microtissues in a conventional PDMS chip;
图24为超疏水PDMS芯片内肾脏组织第三天的状态；FIG24 shows the state of kidney tissue in the superhydrophobic PDMS chip on the third day;
图25为多器官芯片剖视图；FIG25 is a cross-sectional view of a multi-organ chip;
图26为多器官芯片俯视图；FIG26 is a top view of a multi-organ chip;
图27为多器官芯片实物图(去掉多孔膜)；FIG27 is a real picture of the multi-organ chip (without the porous membrane);
图28为15种原代组织的明场照片；Figure 28 is a bright field photograph of 15 primary tissues;
图29为第3天时，顺铂对15种原代组织的毒性作用结果(绿色代表活细胞，红色代表
死细胞)。Figure 29 shows the toxic effects of cisplatin on 15 primary tissues on day 3 (green represents living cells, red represents Dead Cells).
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“及/或”包括一个或多个相关的所列项目的任意的和所有的组合。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and/or" used herein includes any and all combinations of one or more related listed items.
下述实施例中所使用的实验方法如无特殊说明，均为常规方法，所用的材料、试剂等，如无特殊说明，均可从商业途径得到。Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.
对比例1：原代肾和脑组织块在传统低粘附孔板底面的粘附和崩解Comparative Example 1: Adhesion and disintegration of primary kidney and brain tissue blocks on the bottom surface of a traditional low-adhesion plate
将老鼠的肾和脑的原代组织块分别放入康宁公司的商品化低粘附孔板中培养，正常情况下，由于孔板底面低粘附，肾和脑的原代组织块处于悬浮培养的状态，其形态可以得到很好的保持，但是实际观察到的是，培养到第5天，肾和脑的原代组织块便贴附在孔板底部，而且细胞爬出，组织块崩解，如图1和2所示。The primary tissue blocks of mouse kidney and brain were cultured in Corning's commercial low-adhesion well plates. Under normal circumstances, due to the low adhesion of the bottom of the well plate, the primary tissue blocks of kidney and brain are in a suspended culture state and their morphology can be well maintained. However, it was actually observed that on the fifth day of culture, the primary tissue blocks of kidney and brain adhered to the bottom of the well plate, and the cells crawled out and the tissue blocks disintegrated, as shown in Figures 1 and 2.
实施例1：以天然材料为模板，制作超疏水孔板Example 1: Using natural materials as templates to make super-hydrophobic orifice plates
1.超疏水孔板的制备方法如下：1. The preparation method of the super hydrophobic orifice plate is as follows:
将荷叶固定在玻璃板上，将液态聚二甲基硅氧烷浇注在其表面上，过夜，使液态聚二甲基硅氧烷聚合成固体，将凝固的聚二甲基硅氧烷片从荷叶上剥离作为下一步的模板。再将聚二甲基硅氧烷模板表面进行硅烷化修饰，再在模板上浇注液态聚二甲基硅氧烷，升温进行聚合，将新凝固的聚二甲基硅氧烷片从硅烷化修饰的聚二甲基硅氧烷片模板上剥离。聚二甲基硅氧烷片表面的电子显微镜图片如图3所示，其具有超疏水的纳米结构，接触角>120°(图4)，超疏水的聚二甲基硅氧烷底板制作完毕。The lotus leaf is fixed on a glass plate, and liquid polydimethylsiloxane is poured on its surface, and overnight, the liquid polydimethylsiloxane is polymerized into a solid, and the solidified polydimethylsiloxane sheet is peeled off from the lotus leaf as the template for the next step. The polydimethylsiloxane template surface is then silanized, and liquid polydimethylsiloxane is poured on the template, and the temperature is raised for polymerization, and the newly solidified polydimethylsiloxane sheet is peeled off from the polydimethylsiloxane sheet template of silanization modification. The electron microscope image on the polydimethylsiloxane sheet surface is as shown in Figure 3, and it has a super-hydrophobic nanostructure, and the contact angle>120 ° (Fig. 4), and the super-hydrophobic polydimethylsiloxane base plate is finished.
利用3D打印工艺制备模板，制作带孔的的聚二甲基硅氧烷板，具体流程如图5所示。The template is prepared by 3D printing technology to make a polydimethylsiloxane plate with holes. The specific process is shown in Figure 5.
将上述超疏水聚二甲基硅氧烷底板与带孔的聚二甲基硅氧烷板利用等离子体清洗仪封接，便得到最终的细胞三维培养低粘附孔板，孔的底面是具有表面超疏水微纳米结构的聚二甲基硅氧烷，侧面是疏水的聚二甲基硅氧烷，其结构示意图如图6所示。
The above-mentioned super-hydrophobic polydimethylsiloxane bottom plate and the polydimethylsiloxane plate with holes are sealed using a plasma cleaner to obtain the final cell three-dimensional culture low-adhesion well plate, the bottom surface of the hole is polydimethylsiloxane with a surface super-hydrophobic micro-nano structure, and the side surface is hydrophobic polydimethylsiloxane. Its structural schematic diagram is shown in Figure 6.
将水注入该孔中，如图7所示，水呈现明显的液滴形态。When water is injected into the hole, as shown in FIG7 , the water presents an obvious droplet shape.
2.超疏水孔板的测试2. Testing of super hydrophobic orifice plate
上述超疏水孔板上的方孔的边长是4mm，深4mm。将该孔板用于肿瘤细胞成球实验，混合培养人胚肺成纤维细胞(MRC-5)和人肺腺癌细胞(NCI-H1792)，培养基为RPMI 1640+10％FBS。成纤维细胞与癌细胞的培养比例为1:1，培养环境为37℃，5％CO2。培养3天后，在超疏水孔板中肿瘤细胞成球成功(图8)，达到与传统低粘附孔板相似的效果。The square holes on the super-hydrophobic plate have a side length of 4 mm and a depth of 4 mm. The plate was used for a tumor cell spheroidization experiment, and human embryonic lung fibroblasts (MRC-5) and human lung adenocarcinoma cells (NCI-H1792) were mixed and cultured in RPMI 1640 + 10% FBS. The culture ratio of fibroblasts to cancer cells was 1:1, and the culture environment was 37°C, 5% CO 2 . After 3 days of culture, tumor cells successfully spheroidized in the super-hydrophobic plate (Figure 8), achieving an effect similar to that of a traditional low-adhesion plate.
测试重复性显示，该孔板加入各种细胞培养基，持续48h，然后倒掉，清洗，晾干，重复上述实验12次，再次测量水溶液接触角，仍大于120°(图9)，说明该孔板至少可以重复使用12次，连续使用时间超过24天。The test repeatability showed that the plate was added with various cell culture media for 48 h, then poured out, cleaned, and dried. The experiment was repeated 12 times, and the contact angle of the aqueous solution was measured again, which was still greater than 120° (Figure 9), indicating that the plate can be reused at least 12 times, with a continuous use time of more than 24 days.
实施例2：利用聚二甲基硅氧烷和聚甲基丙烯酸甲酯材料制作超疏水孔板Example 2: Making a super-hydrophobic orifice plate using polydimethylsiloxane and polymethyl methacrylate materials
1.超疏水孔板的制备方法如下：1. The preparation method of the super hydrophobic orifice plate is as follows:
将一块聚二甲基硅氧烷板浸入50℃的原硅酸四乙酯中20分钟。然后，将原硅酸四乙酯溶胀的聚二甲基硅氧烷板从原硅酸四乙酯溶液中取出，立即漂浮在10％乙二胺的水溶液中，在聚二甲基硅氧烷板表面逐渐形成二氧化硅微泡。10小时后，取出带有二氧化硅微泡的聚二甲基硅氧烷板，用去离子水冲洗3次。最后，将带有二氧化硅微泡的聚二甲基硅氧烷板在烘箱中热处理1小时。制备出的超疏水表面的电镜照片如图10所示。A polydimethylsiloxane plate was immersed in tetraethyl orthosilicate at 50°C for 20 minutes. Then, the polydimethylsiloxane plate swollen with tetraethyl orthosilicate was taken out from the tetraethyl orthosilicate solution and immediately floated in an aqueous solution of 10% ethylenediamine, and silica microbubbles gradually formed on the surface of the polydimethylsiloxane plate. After 10 hours, the polydimethylsiloxane plate with silica microbubbles was taken out and rinsed with deionized water 3 times. Finally, the polydimethylsiloxane plate with silica microbubbles was heat treated in an oven for 1 hour. The electron microscope photo of the prepared superhydrophobic surface is shown in Figure 10.
利用激光雕刻制作带孔的聚甲基丙烯酸甲酯板，其中孔是圆形的，孔的内壁超疏水修饰的方法如下：A method for making a polymethyl methacrylate plate with holes by laser engraving, wherein the holes are circular, and the inner wall of the holes is superhydrophobicly modified as follows:
(1)称取0.25g直径为20nm的疏水性纳米二氧化硅颗粒，量取50ml正己烷和2.5ml氯仿，混合后进行超声处理，使纳米二氧化硅颗粒完全分散在混合溶液中；(2)将带通孔的聚甲基丙烯酸甲酯板浸入上述溶液中约15秒，取出晾干，得到的超疏水PMMA的表面电镜照片如图11所示。(1) Weigh 0.25 g of hydrophobic nano-silica particles with a diameter of 20 nm, measure 50 ml of n-hexane and 2.5 ml of chloroform, mix them and then perform ultrasonic treatment to make the nano-silica particles completely dispersed in the mixed solution; (2) Immerse a polymethyl methacrylate plate with through holes in the above solution for about 15 seconds, take it out and dry it, and the surface electron microscope photograph of the obtained super-hydrophobic PMMA is shown in Figure 11.
将超疏水聚二甲基硅氧烷板与带通孔的超疏水聚甲基丙烯酸甲酯板压合，便得到最终的细胞三维培养低粘附孔板，孔的底面是具有表面超疏水微纳米结构的聚二甲基硅氧烷，侧面是超疏水聚甲基丙烯酸甲酯，水相溶液在孔中的形态如图12所示，呈现明显的液滴形态。The superhydrophobic polydimethylsiloxane plate and the superhydrophobic polymethyl methacrylate plate with through holes are pressed together to obtain the final cell three-dimensional culture low-adhesion well plate. The bottom of the well is polydimethylsiloxane with a surface superhydrophobic micro-nano structure, and the side is superhydrophobic polymethyl methacrylate. The morphology of the aqueous solution in the well is shown in Figure 12, showing an obvious droplet morphology.
2.超疏水孔板的测试2. Testing of super hydrophobic orifice plate
上述超疏水孔板的圆孔的直径是3mm，深4cm。将该孔板用于肿瘤细胞成球实验，实验条件与实施例1一致。培养3天后，在超疏水孔板中肿瘤成球成功(图13)，达到与传统低
粘附孔板相似的效果。The diameter of the circular hole of the super hydrophobic plate is 3 mm and the depth is 4 cm. The plate was used for the tumor cell sphering experiment, and the experimental conditions were the same as those in Example 1. After 3 days of culture, tumor sphering was successful in the super hydrophobic plate (Figure 13), reaching the same level as the traditional low-lying Adhesive well plates have similar effects.
测试重复性显示，该孔板加入细胞培养基(1640培养基+10％胎牛血清)，持续48h，然后倒掉，清洗，晾干，重复上述实验12次，最后测量水溶液接触角为135.8°，仍大于120°，说明该孔板至少可以重复使用12次，连续使用时间超过24天。The test repeatability showed that the well plate was added with cell culture medium (1640 culture medium + 10% fetal bovine serum) for 48 hours, then poured out, washed, dried, and the above experiment was repeated 12 times. Finally, the contact angle of the aqueous solution was measured to be 135.8°, which was still greater than 120°, indicating that the well plate can be reused at least 12 times, and the continuous use time exceeds 24 days.
实施例3：利用聚甲基丙烯酸甲酯材料制作超疏水孔板Example 3: Making a super-hydrophobic orifice plate using polymethyl methacrylate material
1.超疏水孔板的制备方法如下：1. The preparation method of the super hydrophobic orifice plate is as follows:
利用激光雕刻制作带孔的聚甲基丙烯酸甲酯板，其中孔是方形的，孔的内壁超疏水修饰的方法如下：(1)称取0.25g直径为20nm的疏水性纳米二氧化硅颗粒，量取50ml正己烷和2.5ml氯仿，混合后进行超声处理，使纳米二氧化硅颗粒完全分散在混合溶液中；(2)将带通孔的聚甲基丙烯酸甲酯板浸入上述溶液中约15秒，取出晾干。另采用相同的方法修饰一块聚甲基丙烯酸甲酯板。A polymethyl methacrylate plate with holes was made by laser engraving, wherein the holes were square and the inner wall of the holes was super-hydrophobicly modified as follows: (1) 0.25 g of hydrophobic nano-silica particles with a diameter of 20 nm were weighed, 50 ml of n-hexane and 2.5 ml of chloroform were measured, and the mixture was ultrasonically treated to completely disperse the nano-silica particles in the mixed solution; (2) the polymethyl methacrylate plate with through holes was immersed in the above solution for about 15 seconds, and then taken out and dried. Another polymethyl methacrylate plate was modified by the same method.
将超疏水聚甲基丙烯酸甲酯板与带通孔的超疏水聚甲基丙烯酸甲酯板利用螺栓压合，便得到最终的细胞三维培养低粘附孔板，孔的底面是具有表面超疏水微纳米结构的聚甲基丙烯酸甲酯板，侧面也是超疏水聚甲基丙烯酸甲酯，水相溶液在孔中的形态如图14所示，呈现明显的液滴形态。The superhydrophobic polymethyl methacrylate plate and the superhydrophobic polymethyl methacrylate plate with through holes are pressed together by bolts to obtain the final cell three-dimensional culture low-adhesion well plate. The bottom of the well is a polymethyl methacrylate plate with a surface superhydrophobic micro-nano structure, and the side is also a superhydrophobic polymethyl methacrylate. The morphology of the aqueous solution in the well is shown in Figure 14, showing an obvious droplet morphology.
2.超疏水孔板的测试2. Testing of super hydrophobic orifice plate
上述超疏水孔板的方孔的边长是4mm，深4cm。将该孔板用于肿瘤细胞成球实验，实验条件与实施例2一致，培养3天后，在超疏水孔板中肿瘤成球成功(图15)，达到与传统低粘附孔板相似的效果。The square hole of the super hydrophobic plate has a side length of 4 mm and a depth of 4 cm. The plate was used for a tumor cell spheroidization experiment under the same experimental conditions as in Example 2. After 3 days of culture, tumor spheroidization was successful in the super hydrophobic plate (Figure 15), achieving an effect similar to that of a conventional low-adhesion plate.
测试重复性显示，该孔板加入细胞培养基(1640培养基+10％胎牛血清)，持续48h，然后倒掉，清洗，晾干，重复上述实验12次，最后测量水溶液接触角为132.6°，仍大于120°，说明该孔板至少可以重复使用12次，连续使用时间超过24天。The test repeatability showed that the well plate was added with cell culture medium (1640 culture medium + 10% fetal bovine serum) for 48 hours, then poured out, washed, dried, and the above experiment was repeated 12 times. Finally, the contact angle of the aqueous solution was measured to be 132.6°, which was still greater than 120°, indicating that the well plate can be reused at least 12 times, and the continuous use time exceeds 24 days.
实施例4：基于超疏水孔板的药物筛选微流控芯片Example 4: Drug screening microfluidic chip based on super-hydrophobic well plate
细胞球具有很好的仿生性，是非常重要药物筛选细胞模型。微流控芯片具有自动化，智能化的特点，也是药物筛选的很好的载体。许多研究是将细胞球装载在微流控芯片上，进行自动化的药物评价。但是这类研究通常是通过传统商品化低粘附孔板产生细胞球后，再将细胞球转移到微流控芯片上，然后进行药物的评价，操作很繁琐。Cell spheroids have good biomimetic properties and are very important cell models for drug screening. Microfluidic chips are automated and intelligent and are also good carriers for drug screening. Many studies load cell spheroids on microfluidic chips for automated drug evaluation. However, this type of research usually produces cell spheroids through traditional commercial low-adhesion well plates, then transfers the cell spheroids to microfluidic chips, and then evaluates drugs, which is very cumbersome.
本实施例设计了一种集成化的细胞球芯片，芯片设计如图16所示，实物图如图17，下
方是一个三孔的PMMA超疏水低粘附孔板，孔的边长均为4mm，深4mm。将ips诱导的心肌细胞和成纤维细胞，血管内皮细胞按2:1:1的比例混合，加入超疏水孔中，培养基为基础培养基+酶解酪蛋白+氢化可的松+左旋谷氨酸+表皮生长因子等添加因子，培养环境为37℃，5％CO2，4天后成功诱导出心脏细胞球(图18)。将ips诱导的肝实质细胞和星状细胞按3:1的比例混合，加入另一个超疏水孔中，培养基为基础培养基+酶解酪蛋白+氢化可的松+左旋谷氨酸等添加因子，3天后成功诱导出肝细胞球(图19)。最后将神经干细胞加入剩下的一个超疏水孔中，培养基为D-MEM/F12加B27补充剂+抗生素-抗真菌剂、表皮生长因子和碱性成纤维细胞生长因子等，7天后成功诱导出神经细胞球(图20)。This embodiment designs an integrated cell ball chip. The chip design is shown in FIG16 , and the actual picture is shown in FIG17 . The square is a three-hole PMMA super-hydrophobic low-adhesion plate, and the side length of the hole is 4mm and the depth is 4mm. The cardiomyocytes and fibroblasts induced by ips, and the vascular endothelial cells are mixed in a ratio of 2:1:1 and added to the super-hydrophobic wells. The culture medium is basal culture medium + enzymatic casein + hydrocortisone + L-glutamic acid + epidermal growth factor and other added factors. The culture environment is 37°C, 5% CO2 , and the cardiac cell spheres are successfully induced after 4 days (Figure 18). The hepatocytes and stellate cells induced by ips are mixed in a ratio of 3:1 and added to another super-hydrophobic well. The culture medium is basal culture medium + enzymatic casein + hydrocortisone + L-glutamic acid and other added factors. After 3 days, the hepatocyte spheres are successfully induced (Figure 19). Finally, the neural stem cells are added to the remaining super-hydrophobic wells. The culture medium is D-MEM/F12 plus B27 supplements + antibiotics-antifungal agents, epidermal growth factor and basic fibroblast growth factor, etc., and the neural cell spheres are successfully induced after 7 days (Figure 20).
将每个孔的上方盖上一张多孔膜，然后将带有通道的PDMS板压在多孔膜上，通道的位置对准孔的位置，接着在通道中以1μl/min的流速流通包含阿霉素的培养液，对肝细胞球，心脏细胞球和神经细胞球进行培养，阿霉素便会穿过多孔膜与孔内的细胞球作用，显示出毒性。可以看到阿霉素作用三天后，心脏细胞球上的细胞几乎全部凋亡，而肝细胞球和神经细胞球上的细胞大部分还存活(图21)，说明了阿霉素显示出明显的心脏毒性。A porous membrane was placed on top of each hole, and then a PDMS plate with a channel was pressed onto the porous membrane, with the position of the channel aligned with the position of the hole. Then, a culture solution containing doxorubicin was circulated in the channel at a flow rate of 1 μl/min. Liver cell spheres, heart cell spheres and nerve cell spheres were cultured, and doxorubicin would pass through the porous membrane and interact with the cell spheres in the hole, showing toxicity. It can be seen that after three days of doxorubicin treatment, almost all cells on the heart cell spheres were apoptotic, while most of the cells on the liver cell spheres and nerve cell spheres were still alive (Figure 21), indicating that doxorubicin showed obvious cardiotoxicity.
实施例5：基于PDMS器官芯片的组织培养Example 5: Tissue culture based on PDMS organ chips
利用超疏水PDMS和普通的PDMS材料分别制作了两个器官芯片，芯片设计如图22。然后往两个器官芯片中分别加入肾脏微组织，进行培养，模拟肾器官，结果如图23所示。三天之内，在普通PDMS芯片上肾脏组织就发生了崩解，在超疏水PDMS芯片上肾脏组织生长正常(图24)。Two organ chips were made using super-hydrophobic PDMS and ordinary PDMS materials, respectively. The chip design is shown in Figure 22. Then kidney microtissues were added to the two organ chips and cultured to simulate kidney organs. The results are shown in Figure 23. Within three days, the kidney tissue on the ordinary PDMS chip disintegrated, while the kidney tissue on the super-hydrophobic PDMS chip grew normally (Figure 24).
实施例6：基于超疏水孔的多器官芯片的构建及在顺铂毒性评价中的应用Example 6: Construction of a multi-organ chip based on superhydrophobic pores and its application in cisplatin toxicity evaluation
本实施例构建了一个包含15种原代微组织的多器官芯片，芯片剖视图和俯视图如图25和26所示。其中底板上有一个孔，可以伸入了一个微型搅拌器，用于每个液滴的搅拌。图27为实物照片。In this embodiment, a multi-organ chip containing 15 kinds of primary microtissues is constructed, and the cross-sectional view and top view of the chip are shown in Figures 25 and 26. There is a hole on the bottom plate, into which a micro stirrer can be inserted for stirring each droplet. Figure 27 is a real photo.
每个孔直径5mm，深5mm，由PMMA制成，15个孔装的组织分别是脑、眼、耳、舌、气管、心、肺、肝、肾、胰、脾、脂肪、骨髓、肌肉和睾丸微组织。孔内每种组织的培养基如下表所示：
Each well is 5mm in diameter and 5mm deep, and is made of PMMA. The tissues in the 15 wells are brain, eye, ear, tongue, trachea, heart, lung, liver, kidney, pancreas, spleen, fat, bone marrow, muscle, and testicular microtissues. The culture medium for each tissue in the well is shown in the following table:
Each well is 5mm in diameter and 5mm deep, and is made of PMMA. The tissues in the 15 wells are brain, eye, ear, tongue, trachea, heart, lung, liver, kidney, pancreas, spleen, fat, bone marrow, muscle, and testicular microtissues. The culture medium for each tissue in the well is shown in the following table:
每种组织的明场照片如图28所示。将这些组织装入孔中，在微通道中以1μl/min的流速流通包含顺铂的培养液，顺铂会逐渐扩散进入液滴中，与各种组织进行相互作用。3天后，每种组织的活死荧光照片如图29所示，可以看出阿霉素对各种组织显示出差异化的毒性。因此，本实施例构建的基于超疏水孔的多器官芯片可用于顺铂的毒性评价。The bright field photographs of each tissue are shown in Figure 28. These tissues are loaded into the wells, and the culture medium containing cisplatin is circulated in the microchannel at a flow rate of 1 μl/min. Cisplatin will gradually diffuse into the droplets and interact with various tissues. After 3 days, the live and dead fluorescence photos of each tissue are shown in Figure 29, and it can be seen that doxorubicin shows differentiated toxicity to various tissues. Therefore, the multi-organ chip based on superhydrophobic pores constructed in this embodiment can be used for the toxicity evaluation of cisplatin.
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.
Claims (14)
- 一种用于细胞三维培养的超疏水孔板，其特征在于，所述超疏水孔板具有一个或多个培养孔，所述培养孔的底面为具有微纳米形貌的超疏水表面，其与水相溶液的接触角大于120°；所述培养孔的侧面为疏水表面或者超疏水表面，其与水相溶液的接触角大于90°。A super-hydrophobic well plate for three-dimensional cell culture, characterized in that the super-hydrophobic well plate has one or more culture wells, the bottom surface of the culture well is a super-hydrophobic surface with a micro-nano morphology, and the contact angle between the bottom surface and the aqueous solution is greater than 120°; the side surface of the culture well is a hydrophobic surface or a super-hydrophobic surface, and the contact angle between the bottom surface and the aqueous solution is greater than 90°.
- 根据权利要求1所述的一种用于细胞三维培养的超疏水孔板，其特征在于，制作所述超疏水孔板的材料包括高分子聚合物、金属、陶瓷、玻璃、硅中的一种或多种，所述高分子聚合物包括聚甲基丙烯酸甲酯、聚二甲基硅氧烷、聚苯乙烯、聚碳酸酯中的一种或多种。According to claim 1, a super-hydrophobic orifice plate for three-dimensional cell culture is characterized in that the material for making the super-hydrophobic orifice plate includes one or more of polymers, metals, ceramics, glass, and silicon, and the polymer includes one or more of polymethyl methacrylate, polydimethylsiloxane, polystyrene, and polycarbonate.
- 根据权利要求1所述的一种用于细胞三维培养的超疏水孔板，其特征在于，制作所述超疏水孔板的方法包括方法一和方法二：The super-hydrophobic orifice plate for three-dimensional cell culture according to claim 1, characterized in that the method for making the super-hydrophobic orifice plate comprises method one and method two:所述方法一包括以下步骤：The method 1 comprises the following steps:S1.制备超疏水底板；S1. Preparation of super hydrophobic substrate;S2.制备带通孔的疏水板；S2. Prepare a hydrophobic plate with through holes;S3.将上述超疏水底板和疏水板贴合封接在一起，即得到所述用于细胞三维培养的超疏水孔板；S3. The super-hydrophobic bottom plate and the hydrophobic plate are bonded and sealed together to obtain the super-hydrophobic well plate for three-dimensional cell culture;所述方法二包括以下步骤：The second method comprises the following steps:S4.在板材上成型得到培养孔；S4. forming culture wells on the plate;S5.对上述培养孔的内表面进行超疏水修饰，即得到所述用于细胞三维培养的超疏水孔板；S5. The inner surface of the culture hole is super-hydrophobic modified to obtain the super-hydrophobic well plate for three-dimensional cell culture;其中，形成超疏水表面的方法包括表面刻蚀、MEMS加工、表面富集二氧化硅微纳米颗粒、表面喷射超疏水涂料和二次翻模中的至少一种。Among them, the method of forming a super-hydrophobic surface includes at least one of surface etching, MEMS processing, surface enrichment of silicon dioxide micro-nano particles, surface spraying of super-hydrophobic coating and secondary molding.
- 根据权利要求3所述的一种用于细胞三维培养的超疏水孔板，其特征在于，所述表面富集二氧化硅微纳米颗粒的方法为：The super-hydrophobic well plate for three-dimensional cell culture according to claim 3, characterized in that the method for enriching silica micro-nanoparticles on the surface is:将纳米二氧化硅颗粒、正己烷和氯仿混合后进行超声处理，使纳米二氧化硅颗粒分散于混合溶液中；接着，将PMMA板材浸入所述混合溶液中，取出晾干，即得到了具有微纳米结构的超疏水表面。Nano-silicon dioxide particles, n-hexane and chloroform are mixed and then ultrasonically treated to disperse the nano-silicon dioxide particles in the mixed solution; then, a PMMA plate is immersed in the mixed solution, taken out and dried, thereby obtaining a super-hydrophobic surface with a micro-nano structure.
- 根据权利要求3所述的一种用于细胞三维培养的超疏水孔板，其特征在于，所述表面刻蚀的方法为： The super-hydrophobic well plate for three-dimensional cell culture according to claim 3, characterized in that the surface etching method is:将PDMS板材浸入原硅酸四乙酯中使其溶胀，然后取出溶胀的PDMS板材，置于乙二胺水溶液中；接着取出PDMS板材，冲洗后进行热处理，即得到具有微纳米结构的超疏水表面。The PDMS sheet is immersed in tetraethyl orthosilicate to make it swell, and then the swollen PDMS sheet is taken out and placed in an ethylenediamine aqueous solution; then the PDMS sheet is taken out, rinsed and heat-treated to obtain a super-hydrophobic surface with a micro-nano structure.
- 根据权利要求3所述的一种用于细胞三维培养的超疏水孔板，其特征在于，所述二次翻模的方法为：The super-hydrophobic well plate for three-dimensional cell culture according to claim 3, characterized in that the secondary mold turning method is:a.将弹性树脂的预聚液浇注在具有表面超疏水微纳米结构的模板上，使弹性树脂预聚液聚合成第一弹性固体，再将第一弹性固体从模板上剥离；a. pouring a prepolymer of an elastic resin onto a template having a surface super-hydrophobic micro-nano structure, polymerizing the elastic resin prepolymer into a first elastic solid, and then peeling the first elastic solid from the template;b.对第一弹性固体的表面进行硅烷化修饰，并以硅烷化修饰的第一弹性固体为模板，再浇注弹性树脂的预聚液，使弹性树脂预聚液聚合成第二弹性固体；b. silanization modification of the surface of the first elastic solid, and using the silanization modification of the first elastic solid as a template, and then pouring a prepolymer of an elastic resin to polymerize the elastic resin prepolymer into a second elastic solid;c.将第二弹性固体从硅烷化修饰的第一弹性固体模板上剥离，此时，第二弹性固体的表面便形成了表面超疏水微纳米结构。c. Peeling the second elastic solid from the silanized first elastic solid template, at which point a surface super-hydrophobic micro-nano structure is formed on the surface of the second elastic solid.
- 根据权利要求6所述的一种用于细胞三维培养的超疏水孔板，其特征在于，所述具有表面超疏水微纳米结构的模板为天然材料或通过人工方法制备得到，所述人工方法包括表面刻蚀、MEMS加工、表面富集二氧化硅微纳米颗粒、表面喷射超疏水涂料。A super-hydrophobic well plate for three-dimensional cell culture according to claim 6, characterized in that the template having a surface super-hydrophobic micro-nano structure is a natural material or is prepared by an artificial method, and the artificial method includes surface etching, MEMS processing, surface enrichment of silica micro-nano particles, and surface spraying of super-hydrophobic coating.
- 权利要求1-7任一项所述的超疏水孔板在细胞球、原代组织块、类器官三维培养中的应用。Use of the super-hydrophobic well plate according to any one of claims 1 to 7 in three-dimensional culture of cell spheroids, primary tissue blocks, and organoids.
- 权利要求1-7任一项所述的超疏水孔板在悬浮细胞培养中的应用。Use of the super-hydrophobic orifice plate according to any one of claims 1 to 7 in suspension cell culture.
- 一种多器官微流控芯片，其特征在于，包括权利要求1-7任一项所述的超疏水孔板、多孔膜和上层基板；所述上层基板盖合于所述超疏水孔板上，所述多孔膜位于所述超疏水孔板与上层基板之间；所述上层基板上设置有微通道，所述微通道通过所述多孔膜与超疏水孔板上的培养孔连通，且所述上层基板上设有与所述微通道连通的进口与出口；A multi-organ microfluidic chip, characterized in that it comprises the super-hydrophobic well plate, a porous membrane and an upper substrate according to any one of claims 1 to 7; the upper substrate is covered on the super-hydrophobic well plate, and the porous membrane is located between the super-hydrophobic well plate and the upper substrate; a microchannel is provided on the upper substrate, the microchannel is connected to the culture well on the super-hydrophobic well plate through the porous membrane, and the upper substrate is provided with an inlet and an outlet connected to the microchannel;所述培养孔中用于培养悬浮细胞、细胞球、原代组织块或者类器官，所述微通道中用于添加药物或者外源性刺激物；微通道中的药物或者外源性刺激物可通过所述多孔膜进入到下方的培养孔中。The culture wells are used for culturing suspended cells, cell spheres, primary tissue blocks or organoids, and the microchannels are used for adding drugs or exogenous stimulants; the drugs or exogenous stimulants in the microchannels can enter the culture wells below through the porous membrane.
- 根据权利要求10所述的一种多器官微流控芯片，其特征在于，所述培养孔的底板或者侧壁上开设有贯穿孔，所述贯穿孔用于将微型搅拌桨或传感器伸入到培养孔中，或用于连通氧气。A multi-organ microfluidic chip according to claim 10, characterized in that a through hole is provided on the bottom plate or side wall of the culture well, and the through hole is used to extend a micro stirring paddle or a sensor into the culture well, or to connect oxygen.
- 根据权利要求11所述的一种多器官微流控芯片，其特征在于，所述贯穿孔的表面进行了超疏水修饰。 The multi-organ microfluidic chip according to claim 11, characterized in that the surface of the through-hole is super-hydrophobic modified.
- 根据权利要求10所述的一种多器官微流控芯片，其特征在于，所述多孔膜上可以培养有细胞，包括血管内皮细胞和/或免疫细胞。The multi-organ microfluidic chip according to claim 10 is characterized in that cells can be cultured on the porous membrane, including vascular endothelial cells and/or immune cells.
- 权利要求10-13任一项所述的一种多器官微流控芯片在药物成药性评价中的应用。 Use of a multi-organ microfluidic chip as described in any one of claims 10 to 13 in drug formulation evaluation.
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