CN115895884A

CN115895884A - Super-hydrophobic pore plate for three-dimensional cell culture, multi-organ microfluidic chip and application of super-hydrophobic pore plate

Info

Publication number: CN115895884A
Application number: CN202211528748.XA
Authority: CN
Inventors: 张秀莉; 曲玥阳; 杨瑒; 王雅坤; 徐曙辉; 季珍妮; 罗勇
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2022-11-30
Filing date: 2022-11-30
Publication date: 2023-04-04
Also published as: WO2024113486A1

Abstract

The invention discloses a super-hydrophobic pore plate for three-dimensional cell culture, which is provided with one or more culture pores, wherein the bottom surfaces of the culture pores are super-hydrophobic surfaces with micro-nano morphology, and the side surfaces of the culture pores are hydrophobic surfaces or super-hydrophobic surfaces. The invention also discloses a multi-organ microfluidic chip, which comprises a super-hydrophobic pore plate, a porous membrane and an upper substrate; the upper substrate covers the super-hydrophobic pore plate, and the porous membrane is positioned between the super-hydrophobic pore plate and the upper substrate; the upper substrate is provided with a micro-channel which is communicated with the culture hole on the super-hydrophobic pore plate through the porous membrane, and the upper substrate is provided with an inlet and an outlet which are communicated with the micro-channel. According to the super-hydrophobic pore plate for three-dimensional cell culture, the extracellular matrix and the protein are not adhered to the micro-nano super-hydrophobic surface, so that deposited substances are easy to clean, repeated and long-term use can be realized, and the super-hydrophobic pore plate can be further used for constructing an integrated new platform for cell analysis and drug screening.

Description

Super-hydrophobic pore plate for three-dimensional cell culture, multi-organ microfluidic chip and application of super-hydrophobic pore plate

Technical Field

The invention relates to the technical field of three-dimensional cell culture and organ chips, in particular to a super-hydrophobic pore plate for three-dimensional cell culture, a multi-organ microfluidic chip and application thereof.

Background

Three-dimensional cell culture is more biomimetic than traditional two-dimensional adherent culture, and has gradually become the mainstream mode of cell culture in biological research. Three-dimensional culture of cells can be divided into two main categories, namely non-scaffold three-dimensional culture and scaffold three-dimensional culture. Wherein the bracket-free culture mode is more flexible to the control of the cells. The three-dimensional culture without the scaffold depends on three modes of Hanging drop microplate, micropattered surface microplate and Ultra-low adhesion microplate, wherein the low adhesion microplate is more universal and convenient to use.

The principle of conventional low-adhesion orifice plates is to modify the surface of the orifice with a chemical coating. The chemical coating can prevent cells from adhering to the bottom of the hole, so that the cells can be suspended in a culture solution to grow in an autonomous clustering way to form a three-dimensional cell ball, a primary tissue block or an organoid. However, the conventional low-adhesion well plate has problems in that the original cells, the primary tissue or the finally polymerized cell balls and organoids continuously secrete extracellular matrix and protein into the culture solution, the chemical coating is not resistant to the extracellular matrix and the protein, the extracellular matrix and the protein are settled and adhered to the chemical coating, the chemical coating is ineffective, and the cells, the primary tissue blocks or the generated cell balls and organoids are attached to the bottom of the well, thereby affecting the three-dimensional culture.

The above-described problems of conventional low adhesion orifice plates result in three consequences: first, it is not universally applicable to three-dimensional culture of all cells, particularly those with vigorous extracellular matrix and protein secretion (e.g., kidney cells, nerve cells); second, conventional low-adhesion well plates are also generally difficult to achieve for long periods of three-dimensional culture for most common cells; third, conventional low adhesion orifice plates are generally difficult to use multiple times. In addition, the size, shape and size of the holes in the traditional low-adhesion hole plate are fixed and are difficult to adjust according to the requirements of specific experiments; moreover, it cannot be integrated with downstream analysis platforms such as microfluidic chips, etc., to achieve automated cell analysis.

The above disadvantages limit the range of applications and potential of conventional low adhesion well plates.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a super-hydrophobic low-adhesion pore plate for three-dimensional cell culture, which solves the above problems of the conventional low-adhesion pore plate.

In order to solve the technical problems, the invention provides the following technical scheme:

the invention provides a super-hydrophobic pore plate for three-dimensional cell culture, which is provided with one or more culture pores, wherein the bottom surfaces of the culture pores are super-hydrophobic surfaces with micro-nano shapes, and the contact angle of the super-hydrophobic surfaces with a water phase solution is more than 120 degrees; the side surface of the culture hole is a hydrophobic surface or a super-hydrophobic surface, and the contact angle of the culture hole and the aqueous phase solution is more than 90 degrees.

In the invention, the micro-nano super-hydrophobic surface with the self-cleaning function is utilized to manufacture the low-adhesion pore plate. After an aqueous phase solution (such as a cell culture solution) is injected into a culture hole containing a super-hydrophobic micro-nano structure, the solution can not completely cover the super-hydrophobic bottom surface and only can be partially contacted or even not contacted, so that the solution exists in a semi-suspended or even suspended state, and therefore, the contact area between cells and the bottom surface of the culture hole is reduced. In addition, since the micro-nano super-hydrophobic surface is not adhered with extracellular matrix and protein, substances (such as protein, extracellular matrix and the like) deposited on the micro-nano super-hydrophobic surface can be easily cleaned, so that the repeated use and long-term use of the super-hydrophobic pore plate can be realized.

Furthermore, the material for manufacturing the superhydrophobic pore plate can be a high molecular polymer material, such as polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polystyrene (PS), polycarbonate (PC), and the like; the material can also be metal, ceramic, glass, quartz, silicon and the like; or combinations of one or more of the above.

In the present invention, the number of culture wells in the superhydrophobic well plate is not limited, and may be, for example, 1 to 2000. The shape and flatness of the bottom surface of the culture well are not limited, and may be, for example, circular, square, rectangular, triangular or other irregular shapes, or may be uneven shapes. Similarly, the size and depth of the culture well are not limited and can be set as required. For example, the culture wells may have a depth of 0.5 to 20cm and a diameter of 0.5 to 20cm.

In the invention, the method for manufacturing the super-hydrophobic pore plate comprises a first method and a second method:

the first method comprises the following steps:

s1, preparing a super-hydrophobic bottom plate;

s2, preparing a hydrophobic plate with a through hole;

s3, attaching and sealing the super-hydrophobic bottom plate and the hydrophobic plate together to obtain the super-hydrophobic pore plate for three-dimensional cell culture;

the second method comprises the following steps:

s4, obtaining culture holes on the plate through one-step forming;

s5, performing super-hydrophobic modification on the inner surface of the culture hole to obtain the super-hydrophobic hole plate for three-dimensional cell culture;

the method for forming the super-hydrophobic surface comprises surface etching, MEMS processing, surface enrichment of silicon dioxide micro-nano particles, surface spraying of super-hydrophobic coating and secondary die turnover.

In one embodiment, the method for enriching the silica micro-nano particles on the surface comprises the following steps:

mixing nano silicon dioxide particles, normal hexane and chloroform, and then carrying out ultrasonic treatment to disperse the nano silicon dioxide particles in the mixed solution; and then, soaking the PMMA board into the mixed solution, taking out and airing to obtain the super-hydrophobic surface with the micro-nano structure.

Preferably, the method for enriching the silica micro-nano particles on the surface specifically comprises the following steps: weighing 0.25-2g of hydrophobic nano-silica particles with the diameter of 2-200nm, and weighing 20-500ml of n-hexane and 0.5-50ml of chloroform. The materials are mixed and then subjected to ultrasonic treatment, so that the nano silicon dioxide particles are completely dispersed in the mixed solution. And (3) immersing the PMMA board into the solution for about 5-300 seconds, taking out and airing to obtain the PMMA surface with the surface super-hydrophobic micro-nano structure.

In another embodiment, the method for surface etching comprises:

soaking the PDMS plate into tetraethyl orthosilicate to swell the tetraethyl orthosilicate, taking out the swelled PDMS plate, and placing the swelled PDMS plate in an ethylene diamine aqueous solution; and taking out the PDMS plate, washing and then carrying out heat treatment to obtain the super-hydrophobic surface with the micro-nano structure.

Preferably, the method for enriching the silica micro-nano particles on the surface specifically comprises the following steps: the PDMS plates were immersed in tetraethyl orthosilicate at 30-70 ℃ for 10-200 minutes, and then the swollen PDMS plates were removed from the tetraethyl orthosilicate solution and immediately floated in a 5% -40% aqueous solution of ethylenediamine. And after 3-24 hours, taking out and washing with deionized water for 3-5 times, and carrying out heat treatment in an oven for 0.5-5 hours to obtain the PDMS plate with the surface super-hydrophobic micro-nano structure.

Further, the secondary turnover method comprises the following steps:

a. pouring a pre-polymerization liquid (such as PDMS pre-polymerization liquid) of elastic resin on a template with a surface super-hydrophobic micro-nano structure, polymerizing the elastic resin pre-polymerization liquid into a first elastic solid, and stripping the first elastic solid from the template;

b. performing silanization modification on the surface of the first elastic solid, pouring the elastic resin pre-polymerization liquid by taking the silanized and modified first elastic solid as a template, and polymerizing the elastic resin pre-polymerization liquid into a second elastic solid;

c. and (3) stripping the second elastic solid from the silanized and modified first elastic solid template, wherein the surface of the second elastic solid forms a surface super-hydrophobic micro-nano structure.

Further, the template with the surface super-hydrophobic micro-nano structure is made of natural materials (such as cicada wing, lotus leaf and the like) or is prepared through an artificial method, and the artificial method comprises surface etching, MEMS processing, surface enrichment of silicon dioxide micro-nano particles and surface spraying of super-hydrophobic coating.

According to the super-hydrophobic pore plate provided by the invention, after the aqueous phase solution is injected into the culture pores, the solution can not completely cover the super-hydrophobic bottom surface, and only can be partially contacted or even not contacted, so that the super-hydrophobic pore plate exists in a semi-suspended or suspended state. The super-hydrophobic bottom surface is self-cleaning, has extremely low adsorption rate to protein, nucleic acid, cells and the like, does not adhere to extracellular matrix and protein on the surface, and can effectively prevent the cells from creeping out. Therefore, the super-hydrophobic pore plate can be used for three-dimensional culture of cells such as cell balls, primary tissue blocks, organoids and the like, and can also be used for culture of suspension cells such as blood cells, immune cells and the like. In addition, it can be used for evaluating the medicine property of medicine, including drug effect, pharmacokinetics and toxicity.

Furthermore, the invention also provides a multi-organ microfluidic chip, which comprises the super-hydrophobic low-adhesion pore plate, a porous membrane and an upper substrate; the upper substrate covers the super-hydrophobic pore plate, and the porous membrane is positioned between the super-hydrophobic pore plate and the upper substrate; the upper substrate is provided with a micro-channel which is communicated with a culture hole on the super-hydrophobic pore plate through the porous membrane, and the upper substrate is provided with an inlet and an outlet which are communicated with the micro-channel;

the culture hole is used for culturing suspension cells, cell balls, primary tissue blocks or organoids, and the micro-channel is used for adding drugs or exogenous stimuli; the drug or exogenous stimulus in the microchannel can pass through the porous membrane into the culture well below.

In the present invention, the upper substrate may be made of a polymer (e.g., polymethyl methacrylate, polydimethylsiloxane, polystyrene, polycarbonate, etc.), metal, ceramic, glass, quartz, silicon, etc., and may be made of the same material as or different from the low adhesion hole plate. The porous membrane may be made of polycarbonate, polydimethylsiloxane, polyethylene membrane, PES (polyethersulfone), cellulose or its derivative, polyvinyl chloride, PVDF, polysulfone, polyacrylonitrile, polyamide, polysulfone amide, sulfonated polysulfone, cross-linked polyvinyl alcohol, modified acrylic polymer, polytetrafluoroethylene (PTFE) porous membrane, porous polyurethane membrane, hollow fiber ultrafiltration membrane, quantifoil copper mesh porous membrane, quantifoil silica support membrane, quantifoil carbon membrane, porous alumina membrane, or inorganic ceramic membrane.

Further, the porous membrane may be cultured with vascular endothelial cells and/or immune cells.

In the invention, the culture Kong Kongding is connected with the microchannel, so that the culture solution in the culture hole can be updated in real time through the culture solution flowing in the microchannel, thereby realizing the long-term culture of the micro tissue, and different tissues in a plurality of holes can be communicated in real time through the upper microchannel, thereby laying a foundation for the construction of the multi-organ chip. In addition, the multiple organ chips can be constructed using micro-tissues, which can greatly increase the number of organs integrated by the multiple organ chips.

Furthermore, a through hole is formed in the bottom plate or the side wall of the culture hole and used for enabling the micro stirring paddle or the sensor to extend into the culture hole, so that the culture solution in the culture hole can be stirred or some parameters of the culture solution can be detected, and oxygen can be conveyed to the culture solution in the culture hole, and therefore the vitality of the tissue can be maintained. The diameter of the through-going hole is preferably between 0.5mm and 4mm.

In the multi-organ microfluidic chip, liquid in the micro-channel is communicated with liquid in the culture hole through the porous membrane, and a medicine or an exogenous stimulant circulating in the micro-channel can pass through the porous membrane to interact with a micro-tissue, a suspension cell, an organoid or a cytosphere in the hole, so that the druggability of the medicine can be evaluated, including the evaluation of the medicine effect, the pharmacokinetics and the toxicity. Therefore, the invention also provides the application of the multi-organ microfluidic chip in drug pharmacy evaluation.

Compared with the prior art, the invention has the beneficial effects that:

1. according to the super-hydrophobic pore plate, after the aqueous phase solution is injected into the culture hole containing the super-hydrophobic micro-nano structure, the solution can not completely cover the super-hydrophobic bottom surface and only can be partially contacted or even not contacted, so that the super-hydrophobic pore plate exists in a semi-suspended or suspended state, the contact area between cells and the bottom surface of the pore is reduced, and the probability of adherence of the cells on the bottom surface is reduced.

2. According to the super-hydrophobic pore plate, the extracellular matrix and the protein are not adhered to the micro-nano super-hydrophobic surface, so that substances deposited on the surface are easy to clean, and the pore plate can be repeatedly used and used for a long time.

3. In the invention, the micro-nano super-hydrophobic surface can be realized on various materials, such as glass, high polymer, ceramics and the like, wherein the materials comprise materials which can be used for processing a micro-fluidic chip, so that the micro-nano super-hydrophobic surface can be used for integrating low-adhesion holes and the micro-fluidic chip to construct a new integrated platform for cell analysis and drug screening.

Drawings

FIG. 1 is a graph of adhesion and disintegration of a kidney primary tissue mass on a conventional low adhesion plaque surface;

FIG. 2 is a graph of the adhesion and disintegration of a brain primary tissue mass on the surface of a conventional low adhesion well plate;

FIG. 3 is an electron micrograph of the surface of PDMS after it has been overmolded twice with lotus leaves;

FIG. 4 is a state of a droplet on a superhydrophobic PDMS surface;

FIG. 5 shows a step of manufacturing a PDMS sheet with through holes;

FIG. 6 is a schematic diagram of a principle of a polydimethyl siloxane based ultra-hydrophobic low-adhesion orifice plate;

FIG. 7 shows the state of aqueous phase solution in PDMS super-hydrophobic pore;

FIG. 8 is a bright field photograph of tumor spheres in PDMS superhydrophobic holes;

FIG. 9 shows the contact angle change of the superhydrophobic hole after 12 times of repeated use;

FIG. 10 is a surface electron micrograph of a polydimethylsiloxane sheet of silica microbubbles;

FIG. 11 is an electron microscope photograph of a superhydrophobic PMMA surface;

FIG. 12 shows the state of aqueous phase solution in PMMA-PDMS super-hydrophobic pores;

FIG. 13 is a bright field photograph of tumor spheres in PMMA-PDMS superhydrophobic holes;

FIG. 14 shows the state of the liquid droplet in the PMMA superhydrophobic hole;

FIG. 15 is a bright field photograph of tumor spheres in PMMA superhydrophobic holes;

FIG. 16 is a design drawing of a drug screening microfluidic chip based on superhydrophobic holes;

FIG. 17 is a photograph of a real object of a chip (with the porous film removed);

FIG. 18 is a brightfield photograph of heart cell spheres;

FIG. 19 is a brightfield photograph of hepatocyte spheroids;

FIG. 20 is a brightfield photograph of a nerve cell sphere;

FIG. 21 is a graph showing the staining of hepatocytes (A), neurocytes (B) and cardiac cytocytes (C) after doxorubicin addition for 3 hours, with red for dead cells and green for live cells;

FIG. 22 shows a kidney tissue chip design;

FIG. 23 is a diagram of the disintegration process of renal micro-tissue in a traditional PDMS chip;

FIG. 24 shows the state of kidney tissue in a superhydrophobic PDMS chip on the third day;

FIG. 25 is a cross-sectional view of a multi-organ-chip;

FIG. 26 is a top view of a multi-organ-chip;

FIG. 27 is a diagram of a multi-organ chip (porous membrane removed);

FIG. 28 is a brightfield photograph of 15 primary tissues;

FIG. 29 is the result of the toxic effect of cisplatin on 15 primary tissues at day 3 (green for live cells and red for dead cells).

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The experimental methods used in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used therein are commercially available without otherwise specified.

Comparative example 1: adhesion and disintegration of Primary Kidney and brain tissue blocks on the bottom surface of conventional Low-adhesion well plates

The primary tissue blocks of the kidney and brain of the mouse were cultured in commercially available low-adhesion well plates of corning corporation, respectively, and under normal conditions, the primary tissue blocks of the kidney and brain were in a suspension culture state due to low adhesion of the bottom surface of the well plates, and the morphology thereof was well maintained, but it was actually observed that the primary tissue blocks of the kidney and brain were attached to the bottom of the well plates and the cells were crawled out, and the tissue blocks were disintegrated by the culture day 5, as shown in fig. 1 and 2.

Example 1: method for manufacturing super-hydrophobic pore plate by using natural material as template

1. The preparation method of the super-hydrophobic pore plate comprises the following steps:

fixing lotus leaves on a glass plate, pouring liquid polydimethylsiloxane on the surface of the lotus leaves, standing overnight to polymerize the liquid polydimethylsiloxane into a solid, and stripping the solidified polydimethylsiloxane sheets from the lotus leaves to be used as a template of the next step. And performing silanization modification on the surface of the polydimethylsiloxane template, pouring liquid polydimethylsiloxane on the template, heating to polymerize, and stripping the newly solidified polydimethylsiloxane sheet from the silanization modified polydimethylsiloxane sheet template. The electron microscope image of the surface of the polydimethylsiloxane sheet is shown in fig. 3, which has a superhydrophobic nanostructure with a contact angle >120 ° (fig. 4), and the fabrication of the superhydrophobic polydimethylsiloxane substrate is completed.

And preparing a template by using a 3D printing process to prepare the polydimethylsiloxane plate with the hole, wherein the specific flow is shown in figure 5.

And sealing the superhydrophobic polydimethylsiloxane bottom plate and the polydimethylsiloxane plate with the holes by using a plasma cleaning instrument to obtain the final cell three-dimensional culture low-adhesion hole plate, wherein the bottom surfaces of the holes are made of polydimethylsiloxane with a superhydrophobic micro-nano structure on the surface, the side surfaces of the holes are made of hydrophobic polydimethylsiloxane, and the structural schematic diagram of the hole plate is shown in fig. 6.

Water was injected into the pores and, as shown in fig. 7, the water exhibited a distinct droplet morphology.

2. Testing of superhydrophobic orifice plates

The side length of the square hole on the super-hydrophobic hole plate is 4mm, and the depth of the square hole is 4mm. The well plate was used for tumor cell spheronization experiments, and human embryonic lung fibroblasts (MRC-5) and human lung adenocarcinoma cells (NCI-H1792) were mixed cultured in RPMI 1640+10% FBS. The culture ratio of the fibroblast to the cancer cell is 1:1, the culture environment is 37 ℃ and 5 percent CO ₂ . After 3 days of culture, tumor cells were successfully pelleted in superhydrophobic well plates (fig. 8), achieving similar effect to conventional low-adhesion well plates.

The repeatability of the test showed that the well plate was filled with various cell culture media for 48h, then poured off, washed, air dried, and the above experiment was repeated 12 times, and the contact angle of the aqueous solution was again measured, still greater than 120 ° (fig. 9), indicating that the well plate could be reused at least 12 times for more than 24 days.

Example 2: method for manufacturing super-hydrophobic pore plate by using polydimethylsiloxane and polymethyl methacrylate materials

a polydimethylsiloxane plate was immersed in tetraethyl orthosilicate at 50 ℃ for 20 minutes. Then, the tetraethyl orthosilicate swollen polydimethylsiloxane plate was taken out of the tetraethyl orthosilicate solution, immediately floated in a 10% ethylenediamine aqueous solution, and silica microbubbles were gradually formed on the surface of the polydimethylsiloxane plate. After 10 hours, the polydimethylsiloxane plate with the silica microbubbles was removed and rinsed 3 times with deionized water. Finally, the polydimethylsiloxane plates with the silicon dioxide microbubbles were heat treated in an oven for 1 hour. An electron micrograph of the prepared superhydrophobic surface is shown in fig. 10.

The polymethyl methacrylate plate with holes is manufactured by utilizing laser engraving, wherein the holes are circular, and the inner wall of each hole is subjected to super-hydrophobic modification by the following method:

(1) Weighing 0.25g of hydrophobic nano silicon dioxide particles with the diameter of 20nm, weighing 50ml of n-hexane and 2.5ml of chloroform, mixing, and performing ultrasonic treatment to completely disperse the nano silicon dioxide particles in the mixed solution; (2) The polymethyl methacrylate plate with the through holes is immersed in the solution for about 15 seconds, taken out and dried, and the surface electron micrograph of the superhydrophobic PMMA obtained is shown in fig. 11.

And pressing the super-hydrophobic polydimethylsiloxane plate and the super-hydrophobic polymethyl methacrylate plate with the through holes to obtain the final cell three-dimensional culture low-adhesion pore plate, wherein the bottom surface of the pore is polydimethylsiloxane with a surface super-hydrophobic micro-nano structure, the side surface of the pore is super-hydrophobic polymethyl methacrylate, and the shape of the aqueous phase solution in the pore is shown in figure 12 and is obvious in droplet shape.

2. Testing of superhydrophobic orifice plates

The diameter of the round hole of the super-hydrophobic pore plate is 3mm, and the depth is 4cm. The well plate was used for tumor cell spheronization experiments under the same conditions as in example 1. After 3 days of culture, tumor balling was successful in superhydrophobic well plates (fig. 13), achieving similar effect to traditional low adhesion well plates.

The test repeatability shows that the pore plate is added with cell culture medium (1640 culture medium +10% fetal bovine serum) for 48 hours, then poured off, cleaned and dried, the experiment is repeated for 12 times, and finally the contact angle of the water solution is measured to be 135.8 degrees and still larger than 120 degrees, which indicates that the pore plate can be repeatedly used for at least 12 times, and the continuous use time is more than 24 days.

Example 3: super-hydrophobic pore plate made of polymethyl methacrylate material

the polymethyl methacrylate plate with holes is manufactured by utilizing laser engraving, wherein the holes are square, and the inner wall of each hole is subjected to super-hydrophobic modification by the following method: (1) Weighing 0.25g of hydrophobic nano silicon dioxide particles with the diameter of 20nm, weighing 50ml of n-hexane and 2.5ml of chloroform, mixing, and performing ultrasonic treatment to completely disperse the nano silicon dioxide particles in the mixed solution; (2) The polymethyl methacrylate plate with the through holes was immersed in the above solution for about 15 seconds, taken out and dried. A polymethylmethacrylate plate was modified in the same manner.

The super-hydrophobic polymethyl methacrylate plate and the super-hydrophobic polymethyl methacrylate plate with the through holes are pressed together by using bolts, so that a final cell three-dimensional culture low-adhesion pore plate is obtained, the bottom surface of the pore is the polymethyl methacrylate plate with the surface super-hydrophobic micro-nano structure, the side surface of the pore is also the super-hydrophobic polymethyl methacrylate, and the shape of the aqueous phase solution in the pore is shown in figure 14, so that the aqueous phase solution is in an obvious liquid drop shape.

2. Testing of superhydrophobic orifice plates

The side length of the square hole of the super-hydrophobic hole plate is 4mm, and the depth of the square hole is 4cm. The well plate is used for a tumor cell balling experiment, the experimental conditions are consistent with those of example 2, and after 3 days of culture, the tumor balling is successful in the super-hydrophobic well plate (figure 15), so that the effect similar to that of the traditional low-adhesion well plate is achieved.

The test repeatability shows that the pore plate is added with a cell culture medium (1640 culture medium +10% fetal bovine serum) for 48 hours, then poured out, cleaned and dried, the experiment is repeated for 12 times, and finally the contact angle of the water solution is measured to be 132.6 degrees and still larger than 120 degrees, which indicates that the pore plate can be repeatedly used for at least 12 times, and the continuous use time is more than 24 days.

Example 4: drug screening micro-fluidic chip based on super-hydrophobic pore plate

The cell balls have good bionic property and are very important cell models for drug screening. The micro-fluidic chip has the characteristics of automation and intellectualization, and is also a good carrier for drug screening. Many studies have carried cell balls on microfluidic chips for automated drug evaluation. However, such studies usually involve the steps of producing cell beads through a conventional commercially available low-adhesion well plate, transferring the cell beads onto a microfluidic chip, and then evaluating the drug, which is cumbersome.

The embodiment designs an integrated cell ball chip, the chip design is shown in fig. 16, the object diagram is shown in fig. 17, a three-hole PMMA super-hydrophobic low-adhesion pore plate is arranged below the chip, and the side length of each hole is 4mm and the depth of each hole is 4mm. Mixing ips-induced cardiomyocytes, fibroblasts and vascular endothelial cells according to a ratio of 2 ₂ After 4 days, heart cell balls were successfully induced (fig. 18). The ips-induced hepatocyte and stellate cell were mixed according to the ratio of 3:1, and added into another super-hydrophobic well, the culture medium was basal medium + enzymatic casein + hydrocortisone + L-glutamic acid and other additive factors, and 3 days later, hepatocyte spheroids were successfully induced (fig. 19). Finally, the neural stem cells were added to the remaining one of the superhydrophobic wells in a medium of D-MEM/F12 plus B27 supplement plus antibiotic-antifungal agent, epidermal growth factor, basic fibroblast growth factor, etc., and the neurospheres were successfully induced 7 days later (fig. 20).

The liver cell pellet, heart cell pellet and nerve cell pellet were cultured by covering the top of each well with a porous membrane, pressing a PDMS plate with channels against the porous membrane, aligning the channels with the wells, and then flowing a culture solution containing adriamycin in the channels at a flow rate of 1 μ l/min, whereby adriamycin penetrated the porous membrane to interact with the cell pellet in the wells, showing toxicity. It can be seen that three days after doxorubicin exposure, cells on the heart cell sphere were almost all apoptotic, while cells on the hepatocyte and neurocyte spheres were mostly still viable (fig. 21), suggesting that doxorubicin shows significant cardiotoxicity.

Example 5: tissue culture based on PDMS organ chip

Two organ chips were fabricated from superhydrophobic PDMS and common PDMS materials, respectively, and the chip design is shown in fig. 22. Then, kidney microtissue was added to each of the two organ chips, and the kidney organs were simulated by culturing, and the results are shown in fig. 23. Within three days, the kidney tissue disintegrated on the ordinary PDMS chip and the kidney tissue grew normally on the superhydrophobic PDMS chip (fig. 24).

Example 6: construction of multi-organ chip based on super-hydrophobic pores and application of multi-organ chip in cisplatin toxicity evaluation

This example constructed a multi-organ-chip containing 15 primary micro-tissues, and the cross-sectional and top views of the chip are shown in FIGS. 25 and 26. Wherein the bottom plate has a hole through which a micro-stirrer can be inserted for stirring each droplet. Fig. 27 is a photograph of a real object.

Each hole is 5mm in diameter and 5mm deep and is made of PMMA, and the tissues filled in the 15 holes are brain, eyes, ears, tongue, trachea, heart, lung, liver, kidney, pancreas, spleen, fat, bone marrow, muscle and testis microtissue respectively. The media for each tissue in the wells are shown in the following table:

a bright field photograph of each tissue is shown in fig. 28. These tissues were loaded into wells and culture broth containing cisplatin was circulated in microchannels at a flow rate of 1 μ l/min, which gradually diffused into the droplets to interact with various tissues. After 3 days, live and dead fluorescence photographs of each tissue are shown in fig. 29, and it can be seen that doxorubicin shows differential toxicity to various tissues. Therefore, the superhydrophobic-pore-based multi-organ chip constructed in the embodiment can be used for toxicity evaluation of cisplatin.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitutions or changes made by the person skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the invention is subject to the claims.

Claims

1. The superhydrophobic pore plate for three-dimensional cell culture is characterized by comprising one or more culture holes, wherein the bottom surfaces of the culture holes are superhydrophobic surfaces with micro-nano shapes, and the contact angle of the superhydrophobic pore plate with a water-phase solution is more than 120 degrees; the side surface of the culture hole is a hydrophobic surface or a super-hydrophobic surface, and the contact angle of the culture hole and the aqueous phase solution is larger than 90 degrees.

2. The superhydrophobic well plate for three-dimensional cell culture according to claim 1, wherein the superhydrophobic well plate is made of a material comprising one or more of high molecular polymers, metal, ceramic, glass and silicon, and the high molecular polymers comprise one or more of polymethyl methacrylate, polydimethylsiloxane, polystyrene and polycarbonate.

3. The superhydrophobic well plate for three-dimensional culture of cells according to claim 1, wherein the method for manufacturing the superhydrophobic well plate comprises a first method and a second method:

the first method comprises the following steps:

s1, preparing a super-hydrophobic bottom plate;

s2, preparing a hydrophobic plate with a through hole;

the second method comprises the following steps:

s4, forming on the plate to obtain a culture hole;

the method for forming the super-hydrophobic surface comprises at least one of surface etching, MEMS processing, surface enrichment of silicon dioxide micro-nano particles, surface spraying of super-hydrophobic coating and secondary mold turnover.

4. The superhydrophobic pore plate for three-dimensional cell culture according to claim 3, wherein the method for enriching the silica micro-nano particles on the surface comprises the following steps:

5. The superhydrophobic pore plate for three-dimensional cell culture according to claim 3, wherein the surface etching method comprises:

6. The superhydrophobic pore plate for three-dimensional cell culture according to claim 3, wherein the method for double rollover is as follows:

a. pouring the pre-polymerization liquid of the elastic resin on a template with a surface super-hydrophobic micro-nano structure, polymerizing the pre-polymerization liquid of the elastic resin into a first elastic solid, and then stripping the first elastic solid from the template;

b. silanizing and modifying the surface of the first elastic solid, pouring the elastic resin prepolymer liquid by taking the silanized and modified first elastic solid as a template, and polymerizing the elastic resin prepolymer liquid into a second elastic solid;

c. and stripping the second elastic solid from the silanized and modified first elastic solid template, wherein the surface of the second elastic solid forms a surface super-hydrophobic micro-nano structure.

7. The superhydrophobic pore plate for three-dimensional cell culture according to claim 6, wherein the template with the surface superhydrophobic micro-nano structure is made of natural materials or prepared by an artificial method, and the artificial method comprises surface etching, MEMS processing, surface silica-enriched micro-nano particles and surface spraying of superhydrophobic coating.

8. Use of the superhydrophobic well plate of any of claims 1-7 in cell spheres, primary tissue pieces, organoid three-dimensional culture.

9. Use of a superhydrophobic well plate of any of claims 1-7 in suspension cell culture.

10. A multi-organ microfluidic chip comprising the superhydrophobic well plate of any one of claims 1-7, a porous membrane, and an upper substrate; the upper substrate covers the super-hydrophobic pore plate, and the porous membrane is positioned between the super-hydrophobic pore plate and the upper substrate; the upper substrate is provided with a micro-channel which is communicated with a culture hole on the super-hydrophobic pore plate through the porous membrane, and the upper substrate is provided with an inlet and an outlet which are communicated with the micro-channel;

the culture hole is used for culturing suspension cells, cell balls, primary tissue blocks or organoids, and the micro-channel is used for adding drugs or exogenous stimuli; drugs or exogenous stimuli in the microchannels can pass through the porous membrane into the culture wells below.

11. The multi-organ microfluidic chip according to claim 10, wherein a through hole is formed on a bottom plate or a side wall of the culture well, and the through hole is used for inserting a micro paddle or a sensor into the culture well or communicating oxygen.

12. The multi-organ microfluidic chip according to claim 11, wherein the surface of said through-hole is super-hydrophobic modified.

13. The multi-organ microfluidic chip according to claim 10, wherein said porous membrane is cultured with cells, including vascular endothelial cells and/or immune cells.

14. Use of a multi-organ microfluidic chip according to any one of claims 10 to 13 for the evaluation of the pharmaceutical properties of a drug.