WO2023103322A1

WO2023103322A1 - Cell microsheet preparation device, and preparation method therefor and use thereof

Info

Publication number: WO2023103322A1
Application number: PCT/CN2022/098968
Authority: WO
Inventors: 张智勇; 王文浩; 宋李治
Original assignee: 广东瑞程医学科技有限公司
Priority date: 2021-12-07
Filing date: 2022-06-15
Publication date: 2023-06-15
Also published as: CN115011477A

Abstract

A cell microsheet preparation device, and a preparation method therefor and the use thereof, wherein the cell microsheet preparation device comprises a base material for culturing cells, the base material is modified with a temperature-sensitive polymer, the temperature-sensitive polymer is modified with an anti-cell-adhesion agent as an isolation strip in a patterned manner, and the surface of the base material modified with the temperature-sensitive polymer is set to be divided into a number of mutually independent areas.

Description

A cell micromembrane preparation device and its preparation method and application

This application claims the priority of the Chinese patent application submitted to the China Patent Office on December 07, 2021, with the application number 202111487278.2, and the title of the invention is "a cell micromembrane preparation device and its preparation method and application", the entire content of which Incorporated in the application by reference.

technical field

The present application relates to the technical field of biomaterials, in particular to a cell micromembrane preparation device and its preparation method and application.

Background technique

Cells have been extensively studied for their applications in tissue repair and regeneration due to their self-renewal and multilineage differentiation potential. According to the clinical trial registration system (Clinicaltrials.gov) managed by the National Institutes of Health in the United States, as of April 14, 2021, there are a total of 5,822 stem cell clinical research projects registered globally, of which 2,451 have completed clinical trial research. In the past three years (2018-2020), the National Center for Drug Evaluation (CDE) has accepted an average of 6 stem cell drugs per year, most of which are stem cell injections. When stem cell injection harvests cells, it is necessary to use trypsin or trypsin substitutes to digest cells, and the cell digestion process will also destroy cell connections and extracellular matrix (extracellular matrix, ECM), which contains collagen, layers Fibronectin, fibronectin, and elastin are key proteins that play a huge regulatory role in cell morphology, adhesion, migration, and differentiation, which not only makes the quality of cells continue to decline, but also leads to a large number of cells withered after injection and transplantation. In addition, the cell loss after single cell suspension injection is serious, and only a very small number of cells can stay in the target host tissue.

Cell sheet technology (CST) is a scaffold-free cell transplantation method that can obtain cells non-invasively, avoiding the damage of cell biological functions by enzymatic digestion, and not only retaining the complete extracellular matrix (extracellular matrix). , ECM), while also retaining important ion channels and growth factor receptors, etc., which can promote the interaction between cells and between cells and extracellular matrix. Regarding the preparation of cell sheet, Okano (Matsuura, K.; Haraguchi, Y.; Shimizu, T.; Okano, T., Cell sheet transplantation for heart tissue repair. Journal of Controlled Release, 2013, 169(3): 336- 340.) discloses the modification of a layer of temperature-sensitive polymer poly(N-isopropylacrylamide) PNIPAM on the surface of tissue culture dishes. When the temperature is 20°C, the surface of tissue culture dishes modified by PNIPAM is hydrophilic , not suitable for cell adhesion; when the temperature is 37°C, the surface is hydrophobic, suitable for cell adhesion and proliferation. Therefore, when the cells are inoculated and cultured to confluence, there is no need to go through the cell digestion step, and a complete cell membrane can be obtained only by changing the culture temperature. The size of the cell membrane used is as high as 4cm, and four membranes are required to be superimposed for use. The transplantation process requires a thoracotomy. The overly complicated transplantation process greatly limits its clinical application scenarios. Hsing-Wen Sung（Chen, CH; Chang, Y.; Wang, CC; Huang, CH; Huang, CC; Yeh, YC; Hwang, SM; Sung, HW, Construction and characterization of fragmented mesenchymal-stem-cell sheets for intramuscular injection. Biomaterials , 2007, 28, 4643-4651.) made further improvements to the cell membrane technology, and proposed a method of using methylcellulose hydrogel to prepare fragmented mesenchymal stem cell membranes (fragmented mesenchymal-stem -cell sheets) technology, first spread a layer of methylcellulose solution on the cell culture dish (it is temperature-sensitive, it is solid at 37°C, and turns into a solution state at 20°C or lower), Then place it in a 37°C incubator for one hour to make it solidify into a gel, and then coat the gel with a type 1 collagen coating to make it suitable for cell adhesion and proliferation. Inoculate mesenchymal stem cells, cut the gel system with a special barbed wire after the cells are full and turn into a cell membrane, and then use cold PBS solution (to make methylcellulose change from a gel state to a solution state) After washing and removing other substances such as methylcellulose, the broken mesenchymal stem cell membrane can be obtained. The broken cell membrane prepared by this method can be injected and transplanted through a syringe because of its small volume and area. However, although the broken stem cell membrane prepared by it can be injected and transplanted, because it needs to use animal-derived type I collagen to coat the methylcellulose hydrogel, there are hidden dangers of viruses and a high probability of rejection. problem; in addition, it also needs to use a special barbed wire to cut the complete cell membrane, and this process will also cause physical damage to the cells, thereby affecting their activity. Therefore, there is an urgent need to provide a technology that does not need to be coated with animal-derived collagen or physically cut with barbed wire to ensure the safety and biological activity of the micro-membrane while preparing a smaller cell membrane. It can be transplanted completely by microinjection technique.

technical problem

The main purpose of this application is to propose a cell micro-membrane preparation device, which aims to solve the technical problems that the cell micro-membrane needs to be coated with animal-derived collagen and needs to be physically cut with barbed wire.

technical solution

In order to achieve the above purpose, the cell micromembrane preparation device proposed by this application includes a substrate for culturing cells, the substrate is modified with a temperature-sensitive polymer, and the temperature-sensitive polymer is patterned and modified with an anti-cell adhesion agent. The isolation zone is set to separate the surface of the substrate modified with the temperature-sensitive polymer into several mutually independent regions.

In one embodiment, the side length or diameter of the region of the unmodified anti-cell adhesion agent is 10 μm-999 μm; the size of the separation region can be regulated by patterned modification, by limiting the cells within a certain region After culturing, cell micromembranes of the expected size can be obtained subsequently.

In one embodiment, the thermosensitive polymer includes but not limited to poly(N-isopropylacrylamide), poly(ethylene glycol) methacrylate, polymethacrylate N,N-dimethyl At least one of aminoethyl ester, poly(2-carboxyisopropylacrylamide), polyethylene oxide, or poly(N,N-diethylacrylamide), theoretically at a temperature suitable for cell culture Thermosensitive polymers that are hydrophobic, suitable for cell adhesion and proliferation, and have a hydrophilic surface at low temperatures, that are not suitable for cell adhesion should be within the protection scope of this application.

In one embodiment, the anti-cell adhesion agent includes but is not limited to at least one of sodium heparin, polyethylene glycol, polyacrylic acid, sodium carboxymethylcellulose or sodium alginate, which can theoretically be used for patterning Modified preparations with anti-cell adhesion effects should be within the protection scope of this application.

In one embodiment, the base material is selected from glass sheet, glass plate, polystyrene culture dish, cell culture bottle, cell culture roller bottle, polyamide, polyethylene terephthalate, polyethylene terephthalate Butyl ester, Delrin, polycarbonate, polyphenylene oxide, polytetrafluoroethylene, polyurethane, polyethylene oxide, polypropylene, polylactic acid, polyvinyl chloride, polymethacrylate, styrene-propylene At least one of nitrile copolymers, acrylonitrile-butadiene-styrene copolymers, and ethylene-vinyl acetate copolymers.

The present application also discloses a method for preparing a cell micro-membrane device. The method for preparing a cell micro-membrane device includes the following steps:

S1. Modifying a layer of thermosensitive polymer on the substrate surface of cultured cells; and

S2. On the substrate modified with temperature-sensitive polymer in step S1, the anti-cell adhesion agent is patterned and modified as an isolation zone, and the surface of the substrate modified with temperature-sensitive polymer is separated into several pieces by patterning modification. The independent area is the cell micromembrane to prepare the device.

In one embodiment, before modifying the temperature-sensitive polymer, the substrate may be cleaned with a cleaning solution selected from deionized water, ethanol, monochloromethane, dichloromethane, chloroform, At least one of acetone, isopropanol, piranha lotion, petroleum ether, trichloroethylene, and tetrachloroethylene.

In one embodiment, the method for modifying the temperature-sensitive polymer in step S1 includes electron beam irradiation, plasma gas phase polymerization, ultraviolet irradiation, solvent casting, spin coating or initiated chemical vapor deposition.

In one embodiment, the patterning modification in step S2 adopts microcontact printing technology; for example, transferring patterns by PDMS stamps.

In one embodiment, when the anti-cell adhesion agent cannot be directly modified on the temperature-sensitive polymer, a layer of intermediate linker can be modified first, and then the anti-cell adhesion agent can be modified by the intermediate linker; for example, when the pattern When modifying heparin sodium, a layer of polydopamine is patterned and modified on the surface of the temperature-sensitive polymer layer, and then the modified polydopamine substrate is further reacted with heparin sodium to obtain a cell micromembrane device modified with heparin sodium . Because heparin sodium is negatively charged, cells will not adhere and proliferate on its surface. After the cells are cultured to confluence, the cells grow on the surface of the substrate in the shape of a micro-membrane separated by areas modified by heparin sodium.

The application also discloses the application of a cell micro-membrane preparation device in preparing the cell micro-membrane.

In one embodiment, after the cell micromembrane preparation device is sterilized, the seed cells are inoculated, and after the cell expansion is completed, the temperature is lowered, and the flaky cell micromembrane will fall off from the cell micromembrane preparation device. Cell micromembranes are thus harvested. The preparation method of the cell micromembrane avoids the use of trypsin or trypsin substitutes when the cells are harvested, protects the integrity of the cell connection and the extracellular matrix to the greatest extent, is conducive to improving the activity and survival rate of stem cells, and increases Its retention at the targeted site ultimately enhances the efficacy of stem cell therapy.

In one embodiment, the seed cells include but not limited to any one of adipose stem cells, airway basal layer cells or umbilical cord mesenchymal stem cells.

Beneficial effect

The cell micromembrane preparation device of the present application is set to independently separate the surface of the substrate into several mutually independent regions by patterning and modifying the anti-cell adhesion agent on the surface of the substrate modified with a temperature-sensitive polymer as an isolation zone , so that the cells cultured on the surface of the substrate can be separated into pieces of independent micro-membrane shapes. The membrane does not need to be coated with animal-derived collagen, nor does it need to be physically cut with barbed wire, which ensures the safety and biological activity of the micro-membrane; at the same time, the shape of the modified area, The advantages of size design, adjusting the size of the cell growth area, can be used for large-scale production of cell micromembranes with a size of tens to hundreds of microns, so that it can be completely transplanted by microinjection technology, which greatly broadens its scope. clinical application scenarios. Using the cell micromembrane preparation device of this application to prepare micromembranes avoids the use of trypsin or trypsin substitutes when harvesting cells, protects the integrity of cell connections and extracellular matrix to the greatest extent, and is conducive to improving stem cell activity and survival rate, increase its retention at the target site, and ultimately improve the efficacy of stem cell therapy.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to the structures shown in these drawings without creative effort.

Figure 1 is a schematic diagram of the structure of a PDMS stamp. The length is 5mm, the width is 5mm, and the height is 1cm. The side length of the hole is 0.25mm, the hole spacing is 0.25mm, and the hole depth is 1mm.

Figure 2 is the XPS pattern analysis. (a) Gla, (b) Gla-PNIPAM, (c) Gla-PNIPAM-PD-Hep.

Figure 3 is a micropatch of human umbilical cord mesenchymal stem cells (hUCMSC). (a) Live/dead staining of the micromembrane before desorption, (b) after desorption.

Figure 4 is a comparison between the human umbilical cord mesenchymal stem cell micropatch and the single cell suspension. (a) Single cell suspension, (6) Micromembrane; It can be seen that there is an abundant extracellular matrix in the micromembrane compared to the single cell suspension.

Figure 5 is a comparison of the inoculation effect of human umbilical cord mesenchymal stem cell micropatch and single cell suspension. (a) Photo of single cell suspension (DCs) 0-4h after inoculation, (b) Photo of micropatch 0-4h after inoculation, (c) F-actin staining photo of single cell suspension during this process, (d) F-actin staining photos of micropatch during this process; it can be seen that compared with single cell suspension, micropatch shows better adhesion and proliferation ability.

Figure 6 is an evaluation of wound healing activity in vivo. (a) Photos of full-thickness skin defects treated with pure Pfs, DC and MTs at 0, 3, 10 and 16 days after operation; (b) Wounds after various treatments at 3 days, 10 days and 16 days after operation Healing rate; (c) Representative H&E staining images of specimens in each group 16 days after operation, scale bar 500 μm; (d) Representative Masson's trichrome staining images of specimens in each group 16 days after surgery, scale bar, 500 μm. *Significant difference, P<0.05; **Extremely significant difference, P<0.01; ***Highly significant difference, P<0.001.

Figure 7 is a micro-membrane of airway basal layer cells.

Fig. 8 is a micromembrane sheet of adipose-derived mesenchymal stem cells.

Embodiments of the present invention

The present application will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in this application are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1 Preparation of device Gla-PNIPAM-PD-Hep for large-scale preparation of cell micromembrane

1. Modified thermosensitive polymer poly(N-isopropylacrylamide) PNIPAM:

(1) Clean the glass sheet with piranha lotion to remove organic impurities on the surface;

Prepare 200mL piranha lotion with hydrogen peroxide and concentrated sulfuric acid at a volume ratio of 3:7, immerse the cover slip in it and place it at 100°C for one hour, take out the cover slip, and wash it several times with deionized water. Blow-dry in nitrogen atmosphere, denoted as Gla.

(2) Modification of thermosensitive polymer poly(N-isopropylacrylamide) PNIPAM by spin coating method;

Prepare 3 wt.% PNIPAM and 10 wt.% 3-aminopropyltriethoxysilane (APTES) solution with ethanol, then take 5mL of PNIPAM solution and 1.5mL of APTES solution and mix evenly to obtain a premix;

Place the cleaned coverslip on a homogenizer, drop 50 microliters of the premix solution in its center, rotate at a speed of 2000rpm/min for 30s, and then place it in a vacuum oven at 160°C (vacuum degree below 100 mTorr ) for 3 days to obtain Gla-PNIPAM.

2. Use microcontact printing technology to pattern and modify a layer of polydopamine on the surface of the PNIPAM-modified cover glass;

Prepare a 2 mg/mL dopamine solution with a Tris-HCl solution with a pH value of 8.5, and place the PDMS stamp (length 5mm, width 5mm, height 1cm, the side length of the hole is 0.25mm, and the hole spacing is 0.25mm) as shown in Figure 1. mm, hole depth 1mm) were immersed in the dopamine solution for 30min, dried under N2 flow, placed on the surface of the PNIPAM-modified coverslip, and reacted at 37°C for 4h after pressing a heavy object. The PDMS stamp was removed, and the coverslip was washed 3 times with deionized water to obtain Gla-PNIPAM-PD.

3. Further modification of heparin sodium

Prepare 5 mg/mL heparin sodium solution with PBS solution. The above-mentioned patterned polydopamine-modified coverslip was immersed in a sodium heparin solution and reacted at 4° C. for 24 h, the coverslip was taken out, and washed 3 times with deionized water to obtain Gla-PNIPAM-PD-Hep.

X-ray photoelectron spectroscopy (XPS; Fig. 2) confirmed the formation of a PNIPAM/APTES film on the surface of the glass substrate. Compared to the XPS spectrum of Gla (Fig. 2a), the spectrum of Gla-PNIPAM (Fig. 2b) has N The 1s signal confirms the formation of Gla-PNIPAM; in the spectrum of Gla-PNIPAM-PD-Hep (Fig. 2c), the appearance of S 2p signal at about 168 eV BE indicates the successful introduction of negatively charged heparin on the surface of Gla-PNIPAM Molecules, that is, the large-scale preparation of micromembrane device Gla-PNIPAM-PD-Hep was successfully prepared.

Example 2 Preparation of device Gla-PNIPAM-PD-Hep for large-scale preparation of cell micromembrane

It is basically the same as Example 1, the only difference is that the preparation of Gla-PNIPAM adopts the following steps: Gla is subjected to oxygen plasma treatment, so that its surface has a large amount of -OH, and then mixed with silane coupling agent 3-aminopropyl three Ethoxysilane (APTES) was reacted to modify -NH2, further reacted with 2-bromoisobutyryl bromide (BIBB), and then triggered ATRP reaction to modify PNIPAM on the Gla surface.

Example 3 Preparation of device Gla-PNIPAM-PD-Hep for large-scale preparation of cell micromembrane

Basically the same as Example 1, the only difference is that the preparation of Gla-PNIPAM adopts the following steps: Gla is subjected to oxygen plasma treatment, so that its surface has a large amount of -OH, and then mixed with silane coupling agent 3-glycidyl ether oxygen Base propyl methyl diethoxysilane reaction, make it take the epoxy ring, then carry out ring-opening reaction to the epoxy ring with the PNIPAM of belt-NH2, prepare Gla-PNIPAM.

Example 4 Preparation of device Gla-PNIPAM-PD-PEG for large-scale preparation of cell micromembrane

1. Modified thermosensitive polymer poly(N-isopropylacrylamide) PNIPAM:

(1) Clean the glass sheet with acetone and absolute ethanol to remove organic impurities on the surface;

The coverslips were submerged in acetone and absolute ethanol and ultrasonically washed for half an hour, respectively, and the coverslips were taken out, washed with deionized water several times, and dried in a nitrogen environment, which was recorded as Gla.

Prepare a 2 mg/mL dopamine solution with a Tris-HCl solution with a pH value of 8.5, immerse the PDMS stamp shown in Figure 1 in the dopamine solution for 30 min, dry it under N2 air flow, and place it on the PNIPAM-modified cover The surface of the glass slide was pressed on a heavy object and reacted at 37°C for 4h. The PDMS stamp was removed, and the coverslip was washed 3 times with deionized water to obtain Gla-PNIPAM-PD.

3. Further modification of polyethylene glycol PEG

Prepare a 5 mg/mL polyethylene glycol solution with Tris-HCl at pH 8.5. The above-mentioned patterned polydopamine-modified coverslip was immersed in a polyethylene glycol solution and reacted at 45° C. for 48 hours, the coverslip was taken out, and washed 3 times with deionized water to obtain Gla-PNIPAM-PD-PEG.

Example 5

The temperature-sensitive polymer poly(N-isopropylacrylamide) PNIPAM in Example 1 was changed to poly(N,N-dimethylaminoethyl methacrylate) PDMAEMA, and the remaining conditions remained unchanged, and the cell microparticles were prepared. The device Gla-PDMAEMA-PD-Hep was fabricated on a large-scale scale.

Example 6

In Example 4, the 5 mg/mL polyethylene glycol solution was changed to 10 mg/mL polyacrylic acid (PAA) solution, and the rest remained unchanged, and the cell micromembrane large-scale preparation device Gla-PNIPAM-PD-PAA was prepared. .

Application Example 1 Preparation of Human Umbilical Cord Mesenchymal Stem Cell (hUCMSC) Micropatch

Put the modified glass sheet in Example 1 into a culture dish with a diameter of 3.5 cm after being sterilized by irradiation, and inoculate human umbilical cord mesenchymal stem cells hUCMSC at a density of 1.5×104/cm2. Since heparin sodium is negatively charged, Therefore, stem cells will not adhere and proliferate on the surface. After the cells are cultured to confluence, it is observed that the stem cells grow on the coverslip as a micro-membrane shape separated by areas modified by sodium heparin (as shown in Figure 3a As shown), the size of the micro-membrane is about 250 μm * 250 μm; put it in a refrigerator at 4°C for 20 minutes, and the micro-membrane will automatically fall off from the cover glass. Because the micro-membrane is smaller, it will fall off faster. Since no sharp objects such as barbed wire or cell scrapers were used during the harvesting of the micropatch, live/dead staining showed that the micropatch had a very high cell survival rate (as shown in Figure 3b).

The prepared human umbilical cord mesenchymal stem cell micro-membrane was further characterized. Since the whole process did not use proteolytic enzymes to digest the cells, the micro-membrane retained a large amount of extracellular matrix, as shown in Figure 4. Compared with the single cell suspension (Fig. 4a), the micromembrane (Fig. 4b) contained a large amount of extracellular matrix components, such as Fibronectin, Laminin and Collagen I. Figure 5a and Figure 5b are the photos of single cell suspension (DCs) (Figure 5a) and micromembrane (MTs) (Figure 5b) after inoculation 0-4h, respectively, and Figure 5c and Figure 5d are the single cell suspension (Figure 5b). 5c) and the F-actin staining photos of the micropatch (Fig. 5d) during this process, it can be seen that the micropatch shows better adhesion and proliferation ability than the single cell suspension.

The feasibility of the prepared human umbilical cord mesenchymal stem cell micropatch to enhance soft tissue repair in vivo was further evaluated, and full-thickness skin defects in rats were used as a model system for soft tissue defects. Compared with the traditional full-thickness skin defect with a diameter of 10 mm, a wider full-thickness skin defect model with a diameter of 25 mm was used in this study. And using porcine fibrin adhesive (Pfs) as the adhesive, the MTs The effect of (MTs-Pfs) on wound healing was compared with pure Pfs and DCs (DCs-Pfs). As shown in Figure 6a, a reduction in wound size was observed in the MTs-Pfs treatment group at day 3 after surgery compared with other groups. On day 10, wounds treated with MTs-Pfs showed a significant difference in healing rate compared to wounds in other groups. After 16 days, the wounds treated with MTs-Pfs were completely closed and the epidermis was smooth, while the wounds treated by other treatments were not completely healed. Quantification of the percentage of wound closure at different time points (days 3, 10, and 16) confirmed that MTs-Pfs-treated wounds healed significantly faster than other treatments (Fig. 6b). In addition, hematoxylin and eosin (H&E) staining was performed to observe tissue regeneration during wound healing. As shown in Fig. 6c, after 16 days, only in the MTs-Pfs group, we observed complete re-epithelialization and more regenerated hair follicles. Histological analysis of Masson's trichrome-stained sections showed that wounds treated with MTs-Pfs showed increased collagen deposition and collagen fibers showed a regular wavy shape compared with other treatment groups (Fig. 6d). These results confirmed that the MTs-Pfs group exhibited the best soft tissue repair in vivo compared with the DCs-Pfs group.

Application Example 2 Preparation of airway basal cells (BCs) micropatch

The human umbilical cord mesenchymal stem cells in Application Example 1 were replaced with airway basal cells to prepare airway basal cell micropatch, and live/dead staining also showed that the airway basal cell micropatch had a very high Cell viability (as shown in Figure 7).

Application example 3 Preparation of adipose-derived mesenchymal stem cells (ADSCs) micromembranes

The human umbilical cord mesenchymal stem cells in Application Example 1 were replaced with adipose-derived mesenchymal stem cells to prepare adipose-derived mesenchymal stem cell micromembranes, and live/dead staining also showed that the adipose-derived mesenchymal stem cell micromembranes had a very high The cell survival rate (as shown in Figure 8).

The above results show that the large-scale preparation technology of the micro-membrane of the present application is universal and applicable to the preparation of various stem cell micro-membranes, such as human umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, airway basal layer cells, Etc., avoiding the use of trypsin or trypsin substitutes when harvesting cells, neither needing to use animal-derived collagen for coating, nor needing to physically cut with barbed wire, ensuring the safety and Biological activity protects the integrity of cell connections and extracellular matrix to the greatest extent, which is conducive to improving the activity and survival rate of stem cells, increasing their retention at the target site, and ultimately improving the efficacy of stem cell therapy. At the same time, because its size is controlled between tens to hundreds of microns, it can be completely transplanted by microinjection technology, which greatly broadens its clinical application scenarios.

Claims

A cell micromembrane preparation device, including a substrate for culturing cells, the substrate is modified with a temperature-sensitive polymer, and the temperature-sensitive polymer is patterned and modified with an anti-cell adhesion agent as an isolation zone, which is set to modify the The surface of the substrate with the thermosensitive polymer is divided into several independent regions.
According to the cell micromembrane preparation device according to claim 1, the side length or diameter of the region of the unmodified anti-cell adhesion agent is 10 μm-999 μm.
According to the device prepared by the cell micromembrane of claim 1, the thermosensitive polymer is poly(N-isopropylacrylamide), poly(ethylene glycol) methacrylate, polymethacrylic acid N,N - at least one of dimethylaminoethyl ester, poly(2-carboxyisopropylacrylamide), polyethylene oxide or poly(N,N-diethylacrylamide).
According to the cell micromembrane preparation device of claim 1, the anti-cell adhesion agent is at least one of heparin sodium, polyethylene glycol, polyacrylic acid, carboxymethylcellulose sodium or sodium alginate.
According to the described cell micromembrane preparation device of claim 1, described base material selects glass sheet, glass plate, polystyrene petri dish, cell culture bottle, cell culture roller bottle, polyamide, polyethylene terephthalate ester, polybutylene terephthalate, polyoxymethylene resin, polycarbonate resin, polyphenylene ether, polytetrafluoroethylene, polyurethane, polyethylene oxide, polypropylene, polylactic acid, polyvinyl chloride, polymethyl At least one of acrylate, styrene-acrylonitrile copolymer, acrylonitrile-butadiene-styrene copolymer, and ethylene-vinyl acetate copolymer.
The preparation method of the cell micromembrane preparation device described in any one of claims 1-5, comprising the steps of:

S1. Modifying a layer of thermosensitive polymer on the substrate surface of cultured cells; and

S2. On the substrate modified with temperature-sensitive polymer in step S1, the anti-cell adhesion agent is patterned and modified as an isolation zone, and the surface of the substrate modified with temperature-sensitive polymer is separated into several pieces by patterning modification. The independent area is the cell micromembrane to prepare the device.
According to the preparation method of the cell micromembrane preparation device according to claim 6, the method for modifying the temperature-sensitive polymer in step S1 includes electron beam irradiation, plasma gas phase polymerization, ultraviolet irradiation, solvent casting, spin coating or starting chemical vapor deposition.
According to the preparation method of the cell micromembrane preparation device described in claim 6, the patterned modification in step S2 adopts microcontact printing technology.
According to the preparation method of the cell micromembrane preparation device described in claim 6, before step S1, the base material is cleaned with a cleaning solution, and the cleaning solution is selected from deionized water, ethanol, monochloromethane, dichloromethane, At least one of chloroform, acetone, isopropanol, piranha lotion, petroleum ether, trichloroethylene, and tetrachloroethylene.
According to the method for preparing a cell micromembrane device according to claim 6, before step S2, a layer of intermediate linker is patterned and modified on the surface of the temperature-sensitive polymer layer, and then the anti-cell adhesion agent is modified by the intermediate linker.
The application of the cell micro-membrane preparation device according to any one of claims 1-5 in the preparation of cell micro-membrane.
According to the application of claim 11, after the cell micro-membrane preparation device is sterilized, the seed cells are inoculated, and after the cell amplification is completed, the temperature is lowered, and the sheet-like cell micro-membrane will be removed from the cell micro-membrane preparation device. detached, thereby harvesting the cell micromembrane.
According to the application of claim 12, the seed cells include any one of adipose stem cells, airway basal layer cells or umbilical cord mesenchymal stem cells.