CN101629162A - Cell sheet engineering and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of tissue engineering, and in particular relates to a tissue engineering cell sheet and a preparation method thereof. The cell sheet is prepared by the following method: first, polyN-isopropylacrylamide hydrogel is synthesized on the surface of the cell culture support by electron accelerator radiation; then the polyN-isopropylacrylamide hydrogel is a hydrophobic phase by controlling the temperature , culture cells on this phase; change the temperature to make the hydrogel a hydrophilic phase, and separate the tissue engineered cell sheet by polymer membrane adsorption or clamp clamping method. The obtained cell sheet is cultured in multi-layers or adhered by protein adhesive fluid to obtain a multi-layered cell sheet. The cell sheet obtained by the present invention can maintain the desmosome structure between cells, ZO-1 tight junction structure and other intercellular junction structures, can retain basement membrane protein and many components related to adhesion such as integrin, etc., and also has A certain intensity.

Description

Tissue engineering cell sheet and preparation method thereof

technical field

The invention belongs to the field of tissue engineering, and in particular relates to a tissue engineering cell sheet cultured by temperature-sensitive material-N isopropylacrylamide hydrogel and a preparation method thereof.

Background technique

Poly-N isopropylacrylamide (poly(N-isopropylacrylamide), PIPAAm) hydrogel is a temperature-responsive intelligent polymer, which has a certain temperature, which is commonly referred to as the lower critical solution temperature (lower critical solution temperature, There is a reversible discontinuous volume phase transition near LCST), and the LCST of PNIPAAm hydrogel is around 32 °C. This material has a very broad application prospect in the fields of machinery, chemistry and biomedicine. In recent years, its application in the field of tissue engineering has attracted more and more attention. Due to the existence of LCST, it has outstanding effects when used in cell culture. This is because, when the ambient temperature is slightly higher than the LCST, the volume of the PIPAAm hydrogel will suddenly shrink sharply. At this time, it is hydrophobic, and the cells can be planted on it, and the cells can adhere and grow; when the ambient temperature drops below the LCST, The hydrogel re-swells, at which point the hydrogel is hydrophilic, and the cells detach from its surface. So we can inoculate the cells when the hydrogel is hydrophobic and then grow them. When the cells proliferate to a certain extent, the surface of the hydrogel can be made hydrophilic by lowering the temperature below the LCST, so that the cells are separated from the hydrogel to obtain the entire cell sheet, avoiding the damage to the cells caused by enzyme digestion. The resulting cell sheet can be used for transplantation in vivo to reconstruct three-dimensional tissue.

The synthesis methods of PNIPAAm hydrogel mainly include chemical polymerization and radiation polymerization. The chemical polymerization method of PIPAAm hydrogel is very mature, and there are many reports in the literature. The temperature-responsive PIPAAm hydrogel prepared by the chemical synthesis method has the following disadvantages that limit its application in the preparation of tissue engineering cell sheets: (1) The chemical synthesis method has many residues and has certain toxicity for subsequent cell culture . (2) The chemical synthesis method cannot graft the PIPAAm hydrogel onto the polystyrene culture vessel, therefore, the cultured cells cannot be attached to the surface of the PIPAAm hydrogel, and cell sheets cannot be obtained. Radiation polymerization is the use of ionizing radiation energy to initiate the polymerization of organic monomers to obtain polymer compounds. The main difference between radiation polymerization and initiator-initiated polymerization (ie, chemical polymerization) lies in the way of initiation. Once the chain reaction starts, the subsequent chain growth and chain termination are no different from ordinary methods. Radiation polymerization can graft PNIPAAm into ordinary polystyrene culture dish or culture plate while carrying out PNIPAAm monomer polymerization, and the culture dish or culture plate can be used for culturing cells. Radiation polymerization is mainly divided into polymerization induced by radionuclide, ultraviolet irradiation and electron accelerator according to the radiation source. Compared with the other two radiation methods, the electron beam of the electron accelerator is directional, and it also has the characteristics of high dose rate, high power, short radiation time, large processing capacity and low electron beam penetration, especially suitable for the production of thin-layer PIPAAm Temperature-responsive hydrogel Petri dishes. However, when the pure electron accelerator polymerizes PIPAAm hydrogel, when the total radiation dose is lower than 240kGy, it is not easy to form a network-like polymer, usually only a linear polymer is formed, and the linear polymer will dissolve after soaking in ionized water (Fig. 1). The Fourier transform infrared spectrum found that the image of PIPAAm produced after electron accelerator radiation polymerization without cross-linking agent was consistent with the image of linear PIPAAm, suggesting that the PIPAAm produced after electron accelerator radiation polymerization without cross-linking agent only showed linear PIPAAm, not A network-like cross-linked PIPAAm hydrogel can be formed (Figure 2). If an electron accelerator with a dose of more than 200kGy is used to cross-link PIPAAm hydrogel, although a network-like polymer can be obtained, high-dose electron acceleration is accompanied by instantaneous high temperature, which usually causes damage to the deformation of polystyrene culture vessels (Fig. 3).

Contents of the invention

In view of the above-mentioned problems in the prior art, the object of the present invention is to provide a tissue engineered cell sheet cultured with PIPAAm hydrogel.

Another object of the present invention is to provide a method for preparing the above-mentioned tissue engineered cell sheet.

Above-mentioned purpose is realized through following technical scheme:

A method for preparing a tissue engineering cell sheet, specifically comprising the following steps:

(1) The surface of the cell culture support is covered with a N-isopropylacrylamide monomer solution containing a mass concentration of 0.1% to 1.5% cross-linking agent N, N'-methylenebisacrylamide; the N-isopropylacrylamide The mass concentration of the propylacrylamide monomer is 40%-60%.

(2) Perform electron accelerator radiation synthesis on the solution obtained in step (1), the total radiation dose is 240kGy-300kGy, three to six consecutive radiation synthesis times, each time the radiation dose is less than 200kGy. A cell culture support covered with poly-N-isopropylacrylamide hydrogel was obtained;

(3) Put the cell culture support obtained in step (2) into deionized water at 4°C, soak for 5-8 days, change the water 4-7 times during the period, dry and sterilize, and seal it for later use;

(4) Control the temperature to make the poly-N-isopropylacrylamide hydrogel a hydrophobic phase, inoculate the cells into the cell culture support obtained in step (3), and cultivate the cells; change the temperature to make the poly-N-isopropyl acrylamide The acrylamide hydrogel is a hydrophilic phase, and the tissue engineered cell sheet is separated by polymer membrane adsorption or clamp clamping method.

The N-isopropylacrylamide monomer described in step (1) is preferably a recrystallized monomer. The recrystallization of the monomer is preferably carried out using benzene and isopropanol in a volume ratio of 1:1.

The N-isopropylacrylamide monomer solution in step (1) preferably uses isopropanol as a solvent. When water is used as the NIPAAm monomer solvent, the solution cannot spread evenly on the surface of the culture vessel, which may be related to the fact that water is a polar solvent and isopropanol is a non-polar solvent. In addition, the cross-linking effect of ionizing radiation carried out with water as the NIPAAm monomer solvent is not uniform, some local effects are good, and some parts cannot be completely cross-linked. Therefore, it is better to use isopropanol as the NIPAAm monomer solvent.

The covering amount of N-isopropylacrylamide monomer on the surface of the cell culture support in step (1) is preferably 3 mg-5 mg/cm ² .

The added amount of the cross-linking agent in step (1) is preferably 10 μg-30 μg/cm ² .

The cell culture support in step (1) is preferably a polystyrene plate or a polytetrafluoroethylene material, such as a polystyrene culture dish (tissue culture polystyrene, TCPS) or a six-hole plate, or a MILLICELL-CM insert culture dish (diameter: 30 mm, pore size: 0.4 μm, polytetrafluoroethylene (Polytetrafluoroethylene, PTFE) membrane material).

Preferably, the vacuum degree of the electron accelerator in step (2) is ≤2.2×10 ^-6 KPa.

The sterilization in step (3) is preferably ethylene oxide sterilization.

In step (4), the phase transition temperature is used to separate the tissue engineered cell sheet, preferably the following scheme:

Inoculate the cells onto the culture vessel grafted with PIPAAm hydrogel; culture in a 5% CO ₂ incubator at 37°C, and when the cultured cells reach 80-90% confluence, transfer the culture vessel to an incubator at 20°C and incubate for 1 hour Finally, the cells float in sheets on the surface of the swollen and thickened hydrogel, and the carrier-free tissue engineering cell sheets are separated by polymer membrane adsorption or clamp clamping.

The inoculation of the above-mentioned cells is suitable for the conventional inoculation method of cell culture, for example: take the cells in the exponential growth phase in the culture, absorb the culture solution, add 0.25% trypsin-0.02% EDTA, it is advisable to submerge the cells, and put them in the incubator (5 %CO ₂ , 37°C) for several minutes, when the adherent cells were found to be round, immediately add serum-containing medium to terminate the digestion. Pipette into a single cell suspension and centrifuge. Remove the supernatant, add the culture medium, pipette into a single cell suspension, and inoculate it on the culture vessel grafted with PIPAAm hydrogel according to the required concentration.

Of course, the present invention also provides the tissue engineering cell sheet obtained by the above method, because the bioengineering cell sheet separated from the surface of the PIPAAm hydrogel retains natural ECM components, and these ECM components have the effect of an adhesive, so it can be conveniently These cell sheets are glued to the tissues and organs that need to be transplanted without sutures.

The inserted culture dish grafted with PIPAAm hydrogel can also be used to culture cell sheets in the same way as above. In addition, feeder cells can be cultured in the bottom culture dish, and the inserted culture dish of PIPAAm hydrogel can be used to construct cell sheets. The cell sheet with better viability can be obtained by the method; the insert culture dish of PIPAAm hydrogel can also obtain the epithelial cell sheet with stratified cells through the air-liquid culture technique.

The present invention also provides a multi-layered cell sheet, which is obtained by multi-layered culture of the above-mentioned tissue engineering cells according to a conventional method, or obtained by vertically integrating the adhesive protein solution;

The adhesive protein liquid includes at least one of the following proteins:

10μg～100μg/ml laminin, 5μg～50μg/ml fibronectin, 100μg～1mg/ml peptide arginyl-glycyl-aspartyl-serine tetrapeptide (Arg-Gly-Asp-Ser, RGDS ), 100mg～300mg/ml fibrinogen, 10μg～100μg/mL type I collagen, 10μg～100μg/ml type IV collagen;

The collagen is diluted with 0.006-0.1mol/L acetic acid, and other proteins are diluted with NaHCO ₃ solution at pH 8.3;

After the fibrinogen is added, an equal amount of thrombin 200-1000 U/ml and an equal amount of 10% calcium chloride solution need to be added.

Compared with the prior art, the present invention has the following beneficial effects:

1. The synthesis of PNIPAm hydrogel adopts the method of combining chemical cross-linking and electron accelerator radiation, which overcomes the shortcomings of the two methods used alone. It not only obtains the network-like PIPAAm hydrogel polymer and the effect of grafting with polystyrene culture vessels, but also does not cause deformation and damage to the culture vessels. After adding a very small amount of cross-linking agent, it is synthesized into a polymer and passed After immersion treatment, there is little or no residue, and it is basically non-toxic to cell growth.

2. The cell sheet made of temperature-responsive PIPAAm hydrogel can maintain the desmosome structure between cells, ZO-1 tight junction structure and other intercellular junction structures, and can retain basement membrane proteins and many components related to adhesion, such as Integrin, etc., the complete tissue engineering cell sheet also has a certain strength.

3. At present, multi-layer cell sheets are placed by overlapping multiple single-cell sheets, relying on the adhesion protein of the cell sheet itself to bond and integrate into a multi-layer cell sheet. However, in some cases, this overlapping and integrated multi-layer The cell sheet may not be cohesive enough and is easy to loosen, and the application of adhesive protein solution can make up for this deficiency.

Description of drawings

Figure 1 shows that when the electron accelerator polymerizes PIPAAm hydrogel, when the total radiation dose is lower than 240kGy, it is not easy to form a network polymer, usually only a linear polymer is formed, and the linear polymer will dissolve after soaking in ionized water.

Figure 2 is the Fourier transform infrared spectrum of PIPAAm hydrogel. It is found that the image of PIPAAm (lower line) produced after electron accelerator radiation polymerization without cross-linking agent is consistent with the image of linear PIPAAm (upper line).

Figure 3 shows that when the electron accelerator polymerizes PIPAAm hydrogel, a dose of more than 200kGy of electrons is accelerated at a time, accompanied by instantaneous high temperature, which usually causes deformation and damage to the polystyrene culture vessel, and the NIPAAm monomer does not polymerize, turning into popcorn shape.

Figure 4 shows the morphology of the culture vessel grafted with PIPAAm hydrogel at a temperature higher than the LCST.

Figure 5 shows the morphology of culture dishes grafted with PIPAAm hydrogel at temperatures below the LCST.

Fig. 6 is the Fourier transform infrared spectrum of PIPAAm produced after adding cross-linking agent electron accelerator radiation polymerization.

Fig. 7 is the Fourier transform infrared spectrum of the PIPAAm hydrogel synthesized by chemical method.

Figure 8 is the Fourier transform infrared spectrum of PIPAAm hydrogel: wherein, the upper curve represents the PIPAAm hydrogel produced by the electron accelerator radiation polymerization of NIPAAm monomer; the lower curve represents the PIPAAm hydrogel produced by chemical crosslinking.

Figure 9 is a 2D image of PIPAAm hydrogel atomic force microscope.

Figure 10 is a 3D image of PIPAAm hydrogel atomic force microscope. Atomic force microscopy revealed that the surface morphology of PIPAAm hydrogel was slightly rough.

Figure 11 is a scanning electron microscope image of the PIPAAm hydrogel. The surface of the PIPAAm hydrogel is dense and smooth, without obvious wrinkles, and the rough edges of the hydrogel are more obvious.

Figure 12 shows the popcorn-like morphology formed when the NIPAAm monomer coverage is greater than 5 mg/cm ² .

Fig. 13 is the culture vessel of the grafted PIPAAm hydrogel ready for use after being sterilized in an ethylene oxide sterilizer.

Fig. 14 is an image under an inverted microscope on day 5 of the corneal epithelial cells cultured by enzymatically digesting the tissue block for cell isolation, and the tissue block remains.

Fig. 15 is an image under an inverted microscope of corneal epithelial cells cultured for 9 days, and the fused corneal epithelial cells and their edge structures can be seen.

Figure 16 is oral mucosal epithelial cells growing adherently on the surface of PIPAAm hydrogel.

Figure 17 shows the corneal stromal cells growing adherently on the surface of PIPAAm hydrogel.

Figure 18 shows the corneal stromal cells growing adherently on the edge of the surface of the PIPAAm hydrogel, showing the orderly edge structure of the corneal stromal cell sheet.

Figure 19 is an H&E stained histological section image of a single corneal stromal cell sheet containing stratified cells without a carrier.

Figure 20 is a scanning electron microscope image of a single corneal stromal cell sheet, showing a densely packed surface structure.

Figure 21 is a scanning electron microscope image of a single corneal stromal cell sheet showing the edge structure of the corneal stromal cell sheet.

Figure 22 shows 3T3 cells growing adherently on the surface of PIPAAm hydrogel.

Figure 23 is an H&E stained histological section image of two composite keratocyte stromal cell sheets without carrier.

Figure 24 is a scanning electron microscope image of multiple composite corneal stromal cell sheets, showing a densely packed surface structure.

Figure 25 is an H&E-stained tissue section image of multiple composite corneal stromal cell sheets without a carrier in the early stage of formation, and the adhesion between each cell sheet is not tight enough.

Figure 26 is an image of H&E stained tissue sections formed by multiple composite corneal stromal cell sheets without a carrier, and each cell sheet is tightly bonded to form a complete 7-10-layer corneal stromal cell sheet.

Detailed ways

The present invention will be described in further detail below in conjunction with the embodiments, but the implementation of the present invention is not limited thereto.

Embodiment 1 Construction of corneal epithelial cell sheet

1. Preparation of PNIPAm hydrogel grafted culture dish

(1) Recrystallized monomer

The purchased N-isopropylacrylamide monomer (N-isopropylacrylamide, NIPAAm) (Shanghai Wujing Technology Co., Ltd.) was recrystallized using benzene and isopropanol at a volume ratio of 1:1, and the recrystallized The NIPAAm monomer was dried for use.

(2) Preparation of monomer solution

Prepare a NIPAAm monomer solution with a mass concentration of 40%, containing a mass concentration of 0.1% cross-linking agent N, N'-methylene bisacrylamide (N, N-methylene bisacrylamide, MBA), all of which are passed through the solution isopropyl Alcohol preparation, shake and mix well before use.

(3) Radiation Synthesis of Monomer Solution

Take the above-prepared NIPAAm monomer solution, add it to a 35mm tissue culture polystyrene (TCPS) culture dish, and spread it evenly on the surface of the culture vessel through the tension of the monomer solution itself. The covering amount of the monomer on the surface of the culture vessel is 4 mg/cm ² . Put it on the trolley of electron accelerator (Dinamic accelerator, manufactured by Shanghai Institute of Nuclear Research, Chinese Academy of Sciences) and carry out electron radiation synthesis. The data during electron accelerator radiation synthesis is: the radiation dose is 240kgy. The accelerator vacuum is 2.2×10 ^-6 KPa. After electron accelerator radiation, the NIPAAm monomer is polymerized into PIPAAm hydrogel, and at the same time, the NIPAAm monomer is grafted and polymerized with the culture vessel.

The PIPAAm hydrogel grafted in this embodiment has temperature response characteristics: when the temperature is higher than the LCST, the volume shrinks and becomes a milky white thin layer (Figure 4), and when the temperature is lower than the LCST, the volume swells and becomes transparent water gel (Figure 5). The Fourier transform infrared spectrum of PIPAAm hydrogel found that the band appearing near 1644cm-1 is produced by the carboxyl stretching vibration of amide, that is, the amide I band; while the band at 1538cm-1 is the amide II band, which is resulting from the combined absorption of NH bending vibrations and CH stretching vibrations in the amide group. In addition, there are absorption peaks around 1386cm-1 and 1367cm-1, which is the characteristic spectrum of symmetrical CH vibration in isopropyl-CH(CH ₃ ) ₂ of NIPAAm. The Fourier transform infrared spectrum image of PIPAAm hydrogel after adding a small amount of cross-linking agent is consistent with the main component image of chemical synthesis, suggesting that the PIPAAm hydrogel component produced by the electron accelerator radiation polymerization of NIPAAm monomer is the same as the PIPAAm produced by chemical crosslinking. The main components of the hydrogels are similar (Fig. 6, Fig. 7, Fig. 8). Autoprobe CP Research Atomic Force Microscope (Thermo Company, USA) found that the surface morphology of PIPAAm hydrogel was slightly rough (Fig. 9, Fig. 10). The JEOLT-300SEM scanning electron microscope (Japan Electronics Co., Ltd.) further confirmed that the surface of the PIPAAm hydrogel was dense and smooth, with no obvious wrinkles, and the rough edges of the hydrogel were obvious (Figure 11). The coverage of the monomer on the surface of the culture vessel is 3mg-5mg/cm ² , when the coverage is less than 3mg/cm ² , it cannot be radiated and polymerized into sheet-like PIPAAm hydrogel; when the coverage is greater than 5mg/cm ² , it is easy A popcorn-like appearance was formed, and a thin layer of PIPAAm hydrogel was not formed (FIG. 12). In addition, it is difficult for cultured cells to attach thereto. The growth of cultured cells attached to the surface of PIPAAm hydrogel is related to the adsorption of cell adhesion proteins (such as fibronectin) to the hydrogel. If the monomer coverage is too large, a thick layer of PIPAAm hydrogel is formed, which is difficult to Adsorbs components such as fibronectin. The amount of MBA added is generally 10μg-30μg/cm ² , too little MBA has little crosslinking or only linear crosslinking, and loosens after absorbing water; too much MBA has too much crosslinking degree and loses water absorption characteristics.

When water is used as the NIPAAm monomer solvent, the solution cannot spread evenly on the surface of the culture vessel, which may be related to the fact that water is a polar solvent and isopropanol is a non-polar solvent. In addition, the cross-linking effect of ionizing radiation carried out with water as the NIPAAm monomer solvent is not uniform, some local effects are good, and some parts cannot be completely cross-linked. Therefore, it is better to use isopropanol as the NIPAAm monomer solvent.

(4) Processing of PIPAAm hydrogel after radiation synthesis

Put the temperature-sensitive hydrogel synthesized by radiation into deionized water at 4°C, soak for 5 days, change the water 4 times during this period, and put it in an oven at 50°C to dry it. The culture vessel grafted with PIPAAm hydrogel is sealed with paper-plastic packaging by a sealing machine, and a piece of ethylene oxide sterilization indicator paper is placed before sealing, and it is put into the HM-23 ethylene oxide sterilizer (Shenyang Delong Electronics Co., Ltd.), when the test paper changes from red to blue, it means that the sterilization is up to the standard, take it out for use, and then unpack it for cell culture (Figure 13).

2. Cell culture and transplantation on hydrogel

(1) culture medium

Culture medium: A. Supplemental hormonal epithelial medium (SHEM): DMEM/F12 (3:1), 10 μg/L epidermal growth factor, 1000 U/L insulin, 0.1 mg/L cholera toxin, 0.18 mM/L adenine, 0.5mg/L hydrocortisone, 1.2g/L NaHCO3, 10% fetal bovine serum (FBS), 100,000 U/L penicillin and 100mg/L streptomycin. B. Low-calcium keratinocyte medium (keratinocyte serum freemedium, KSFM), the concentration of Ca ²⁺ is 0.07mM-0.09mM. Unless otherwise specified, the culture reagents were the products of Gibco, USA. Unless otherwise specified, other chemical reagents used the products of Sigma Company in the United States.

(3) Take materials

As much as possible, use sterilized instruments under sterile conditions for related operations. Extraction of isolated eyeballs: Separate the limbal ring tissue with a width of about 2 mm, and cut it into 8 pieces on average. Autologous living eyeball material: The superficial separation of the limbus is about 2×3×1mm ³ .

(4) Enzymatic digestion of tissue blocks for cell separation

Put the above small pieces of limbal tissue in the corneal epithelial cell culture medium without serum containing 1.2U/ml neutral protease and 100mM sorbitol at 4°C for about 10 hours. Under a dissecting microscope, separate the loose epithelial layer with micro tweezers . Place in a 15ml centrifuge tube, add about 5ml trypsin solution (0.05% trypsin and 0.02% EDTA) into the centrifuge tube, digest at 37°C for 30 minutes, and centrifuge at 1000rpm/min for 5 minutes after the digestion is terminated. After centrifugation, the supernatant was sucked away, 10% fetal bovine serum was added, and the DMEM/F12 culture medium was pipetted to make a cell suspension, which was inoculated at a cell number of 2×10 ⁴ /ml for primary cell culture. Under the DM/L inverted biological microscope (Leica, Germany), it was found that the corneal epithelium and the corneal epithelial cells fused into sheets were digested with enzymes for cell isolation and cultured (Fig. 14, Fig. 15). Inoculate the culture vessel at a cell density of 1×10 ⁵ /ml for subculture, and use the subculture within 5 passages.

(5) Corneal epithelial cell sheets of stratified cells

The cultured limbal epithelial cells were seeded on the surface of the culture dish obtained in step 1 at a cell density of 2×10 ⁴ /ml for single cell culture.

(6) Separation and acquisition of corneal epithelial cell sheets

Adhesive cell sheets were isolated directly using nitrocellulose membranes.

(7) Corneal epithelial cell sheet transplantation

Transplanted objects: (1) Experimental animals: For New Zealand white rabbits or other animals, use curved scissors to remove about 0.1-1 mm of corneal epithelium and conjunctival epithelium from a place 4 mm away from the limbus to make an animal model of ocular surface stem cell deficiency. Fluorescein ocular surface staining confirmed complete removal of corneal epithelial cells. After 4 weeks, the animal's ocular surface was covered by the remaining conjunctival epithelium, and corneal surface turbidity and corneal neovascularization appeared. (2) Clinical patients: patients with persistent corneal epithelial defect in the acute stage of chemical injury, patients with corneal neovascularization and corneal opacity caused by scar tissue invasion in the chronic stage of chemical injury, ocular surface diseases with limbal stem cell deficiency, and limbal dysfunction Keratopathy, ocular pemphigoid and other patients. The transplanted corneal epithelial cell sheets without any biomaterials can be used for the treatment of various corneal diseases, which is expected to solve various postoperative reactions due to the degradation of biomaterials or the interaction between tissues and biomaterials in the body. Autologous corneal epithelial cell sheets can be obtained by culturing autologous healthy corneal epithelial cells, and their transplantation can solve the contradiction of insufficient corneal donors and remove the postoperative rejection of allogeneic corneal epithelial cell sheet transplantation.

Surgical method: The entire superficial layer of the diseased cornea is separated and resected, the turbidity, conjunctival epithelialization and neovascularization of the corneal surface tissue are removed, the bleeding is fully stopped and cleaned, the transparent corneal stroma is exposed, and a recipient implant bed is made. The above-mentioned functionally active artificial corneal epithelium sheet adhered to the nitrocellulose membrane is contacted and attached to the implantation bed, and the cell sheet transplantation is achieved through the tissue adhesion between the cell sheet adhesion protein and the corneal stroma, and the nitrocellulose membrane is removed after transplantation. If necessary, a bandage-type contact lens can be used to protect the transplanted cell sheet. After the operation, antibiotic eye drops and eye ointment were used locally, 2 times a day. After the corneal epithelial cell sheet is transplanted into the body, fluorescein staining is used to check whether the transplanted corneal epithelial cell sheet has a barrier function, and to confirm the transplantation effect of the epithelial cell sheet.

In addition to limbal epithelial cells, oral mucosal epithelial cells ( FIG. 16 ) and other cells can also be used as seed cells for the above-mentioned construction of the corneal epithelial cell sheet and transplantation. When it is difficult to obtain healthy autologous corneal limbal epithelial cells, autologous oral mucosal epithelial cells and other cells can be used as substitute seed cells, which can also solve the contradiction of insufficient corneal donors and reduce postoperative rejection of allogeneic corneal epithelial cell sheet transplantation Advantages of the response.

Embodiment 2 Construction of corneal epithelial cell sheet

1. Preparation of PNIPAm hydrogel grafted culture dish

(1) Recrystallized monomer

With embodiment 1.

(2) Preparation of monomer solution

Prepare a NIPAAm monomer solution with a mass concentration of 60%, containing a mass concentration of 0.4% cross-linking agent N, N'-methylene bisacrylamide (N, N-methylene bisacrylamide, MBA), all of which are passed through the solution isopropyl Alcohol preparation, shake and mix well before use.

(3) Radiation Synthesis of Monomer Solution

Take the above prepared NIPAAm monomer solution and add it to the surface of the MILLICELL-CM insert culture dish [diameter: 30mm, pore size: 0.4μm, polytetrafluoroethylene (PTFE) (Millipore Company, USA) membrane material] On the surface of the culture vessel, it is spread evenly on the surface of the culture vessel by the tension of the monomer solution itself. The coverage of the monomer on the surface of the culture vessel was 4.5 mg/cm ² . Put it on the trolley of the electron accelerator for electron radiation synthesis. The data of the electron accelerator radiation synthesis is: the radiation dose is 300kgy, and the trolley is used for six consecutive radiation synthesis, and the vacuum degree of the electron accelerator is 2.2×10 ^-6 KPa. After electron accelerator radiation, the NIPAAm monomer is polymerized into PIPAAm hydrogel, and at the same time, the NIPAAm monomer is grafted and polymerized with the culture vessel.

The obtained grafted PIPAAm hydrogel has similar characteristics to the hydrogel obtained in the examples.

(4) Processing of PIPAAm hydrogel after radiation synthesis

The temperature-sensitive hydrogel synthesized by radiation was placed in deionized water at 4°C, soaked for 8 days, and the water was changed 7 times during the period, and dried in an oven at 50°C. The subsequent processing is the same as in Example 1.

2. Cell culture and transplantation on hydrogel

(1) culture medium

With embodiment 1.

(3) Take materials

With embodiment 1.

(4) Enzymatic digestion of tissue blocks for cell separation

Place the above-mentioned small pieces of limbal tissue in corneal epithelial cell culture medium containing 2.4 U/ml neutral protease and 100 mM sorbitol without serum at 4°C for about 18 hours. Under a dissecting microscope, separate the loose epithelial layer with micro tweezers . Place in a 15ml centrifuge tube, add about 5ml trypsin solution (0.05% trypsin and 0.02% EDTA) into the centrifuge tube, digest at 37°C for 30 minutes, and centrifuge at 1000rpm/min for 5 minutes after the digestion is terminated. After centrifugation, the supernatant was sucked away, 10% fetal bovine serum was added, and the DMEM/F12 culture medium was pipetted to make a cell suspension, which was inoculated at a cell number of 1×10 ⁶ /ml for primary cell culture. Inoculate the culture vessel at a cell density of 1×10 ⁶ /ml for subculture, and use the subculture within 5 passages.

(5) Corneal epithelial cell sheets of stratified cells

The cultured limbal epithelial cells were inoculated on the surface of the insert culture dish obtained in step 1 at a cell density of 1×10 ⁶ /ml for cell co-culture: pre-cultured fused 3T3 fibroblasts or human embryonic fibroblasts with Incubate with 4 μg/mL mitomycin C for 2 hours, wash twice with PBS, digest with trypsin, and inoculate the culture dish or culture plate at 1×10 ⁴ /cm ² . Only then are the inserts containing limbal epithelial cells placed within this dish or plate. Put it in a 5% _CO2 incubator at 37°C, 97% humidity, change the medium once every 2-3 days, after 7-10 days of conventional culture, carry out gas-liquid culture, that is, reduce the amount of culture medium added, add culture medium The plane only reaches the surface of the inserted culture dish, so that the surface of the cultured epithelial cells is more in contact with the air, feeder cells that have been treated with mitomycin are added every week, and stratified corneal epithelial cell sheets are obtained after air-liquid culture for 7-10 days.

(6) Separation and acquisition of corneal epithelial cell sheets

The method for obtaining the stratified corneal epithelial sheet is as follows: after the above-mentioned stratified corneal epithelial cell sheet is cultured, reduce the culture medium in the insert culture dish, cover the cultured cells with a nitrocellulose membrane, and put them in an incubator at 20°C for 30 minutes. At a temperature below 20°C, the nitrocellulose membrane that adhered to the entire cell sheet was peeled off from the hydrophilic PIPAAm with a loose coil structure.

(7) Corneal epithelial cell sheet transplantation

Cell sheets can be transplanted in the same manner as in Examples.

Example 3 Corneal endothelial cell sheet

The seed cell culture was carried out according to the conventional culture method of corneal endothelial cells, and the corneal endothelial cell sheet was constructed by a method similar to the construction of the corneal epithelial cell sheet described in Example 1 or 2. Corneal endothelial cell sheet transplantation can treat patients with corneal endothelial cell lesions and endothelial cell function decompensation: remove the diseased corneal posterior lamellar and corneal endothelial layer, through similar penetrating keratoplasty, deep lamellar corneal endothelial Corneal endothelial cell sheet transplantation is performed by surgical methods such as elastic membrane stripping and corneal endothelial transplantation. Corneal endothelial cell sheet transplantation usually does not require sutures, and the tissue adhesion between the cell sheet adhesion protein and the posterior corneal lamina is used to achieve cell sheet transplantation. Or use at least one of the above adhesive protein solutions to adhere the cell sheet to the posterior corneal lamina. Slit lamp observation of corneal transparency, check whether there is corneal edema, and confirm the effect of corneal endothelial cell sheet transplantation. In addition to using corneal endothelial cells as the seed cells for the above-mentioned construction of the corneal endothelial cell sheet and transplantation, other cells can also be used.

Example 4 Corneal stromal cell sheet

Carry out seed cell culture according to the conventional culture method of corneal stromal cells, and use the above-mentioned corneal epithelial cell sheet to construct a similar method to construct corneal stromal cell sheet (Figure 17, Figure 18)), and reduce the temperature separation by the above method to obtain carrier-free corneal stromal cells piece. The histological examination of the H&E stained tissue section of the corneal stromal cell sheet under an optical microscope revealed that the cells in the carrier-free single corneal stromal cell sheet were tightly arranged and contained 2-3 layers of stratified cell structure (Figure 19). Examination under the scanning electron microscope revealed that the surface structure (Figure 20) and the edge structure (Figure 21) of the monolithic corneal stromal cell sheet cells were tightly arranged. Corneal stromal cell sheet transplantation can treat patients with keratopathy: remove the corneal stromal layer lesion tissue, and perform corneal stromal cell sheet transplantation through a method similar to lamellar keratoplasty. Corneal stromal cell sheet transplantation usually does not require sutures, and cell sheet adhesion is used The tissue adhesion between the protein and the cornea achieves cell sheet transplantation. Or use at least one of the above adhesive protein solutions to adhere the cell sheet to the corneal tissue. Slit lamp observation of the cornea to confirm the effect of corneal stromal cell sheet transplantation. In addition to corneal stromal cells, other cells can also be used as the seed cells for the construction and transplantation of the corneal stromal cell sheet ( FIG. 22 ).

Example 5 Preparation of Multiple Composite Corneal Stromal Cell Sheets

The seed cell culture was carried out according to the conventional culture method of corneal stromal cells, and a single corneal stromal cell sheet was constructed by a method similar to the construction of the above corneal epithelial cell sheet, and multiple single corneal stromal cell sheets were obtained by the above method of separating and obtaining the cell sheet. Obtain multiple composite keratocyte stromal cell sheets according to the following method of stacking single sheets and integrating multiple sheets of culture: after constructing keratocyte stromal cell sheets using a method similar to the above-mentioned keratocyte stromal cell sheet construction, reduce the culture medium of the culture vessel to the bottom of the culture vessel, and place The above-mentioned multiple single corneal stromal cell sheets are stacked vertically one by one and integrated into a multi-layered cell sheet to prevent the cell sheet from curling and detachment. After standing for several minutes to several hours, add enough culture medium and perform routine culture for several hours to several days. . Using the above cell sheet separation and acquisition method to obtain a multi-sheet composite keratocyte stromal cell sheet, and make two (Fig. 23) or multiple composite keratoma stromal cell sheets (Fig. 24) that can be transplanted. In the early stage of multi-composite corneal stromal cell sheets, the adhesion between each cell sheet is not tight enough (Figure 25). 10-layer corneal stromal cell sheet (Fig. 26). Since the single piece of corneal stromal cell sheet isolated from the surface of PIPAAm hydrogel retains the natural ECM protein components, these ECM proteins have the role of adhesives, therefore, the cell structure can be obtained by the method of stacking and integrating multiple pieces of culture. multi-composite corneal stromal cell sheets. The transplantation method of multiple composite corneal stromal cell sheets is the same as that in Example 4.

Example 6 Multiple Composite Keratocyte Sheets Vertically Integrated with Various Cell Sheets

At least two of the above three corneal cell sheets (corneal epithelial cell sheet, corneal endothelial cell sheet, and corneal stromal cell sheet) are obtained by the following three methods to obtain multiple composite keratocyte sheets that are vertically integrated with various cell sheets:

The first method is single-sheet overlapping and integrated multi-sheet culture method: the single-sheet overlapping and integrated multi-sheet culture method prepared by the multi-sheet composite corneal stromal cell sheet in Example 5 is used, and the natural ECM protein adhesion between cell sheets is used to obtain Multiple composite keratocyte sheets vertically integrated with various cell sheets.

The second method is the adhesive protein solution adhesion method: After constructing the three kinds of corneal cell sheets using the method similar to the above cell sheet construction, reduce the culture medium of the culture vessel to the bottom of the culture vessel, and use the above adhesive protein solution, one or more of the adhesive protein solutions For this kind of use, the above-mentioned three kinds of corneal cell sheets are adhered vertically one by one to prevent the cell sheets from curling and detachment. After standing for several minutes to several hours, enough culture medium is added, and the conventional culture is carried out for several hours to several days. A multi-piece composite keratocyte sheet in which various cell sheets are vertically integrated is obtained by adopting the method for separating and obtaining the above cell sheets.

The third method is the mixed application of the above two methods: the multi-sheet culture method relies on the adhesion protein of the cell sheet itself to adhere and integrate into multiple composite keratocyte sheets. However, in some cases, the overlapping placement The integrated multi-piece composite cell sheet may not be well bonded and easy to loosen, but the mixed application of adhesive protein solution can make up for this deficiency.

The multiple composite corneal cell sheets of the above-mentioned multiple cell sheets vertically integrated include: corneal epithelial cell sheet (single sheet) and corneal stromal cell sheet (multiple sheets) can be used for anterior lamellar keratoplasty after vertical integration; corneal endothelial cell sheet (single sheet) Posterior lamellar keratoplasty can be performed after longitudinal integration of corneal stromal cell sheets (multiple sheets) and corneal stromal cell sheets (multiple sheets); corneal epithelial cell sheets (single sheet) are vertically integrated with corneal stromal cell sheets (multiple sheets) Full-thickness corneal transplantation can be performed. The above-mentioned corneal transplantation can treat related lesions in different parts of the cornea.

Example 7 retinal pigment epithelial cell sheet

Isolation and primary culture of retinal pigment epithelial cells by enzymatic digestion or tissue block attachment method, conventional culture of retinal pigment epithelial cells, construction of retinal pigment epithelial cell sheets according to the method similar to the above corneal epithelial cell sheet construction, the retinal pigment epithelial cell sheet Transplanted in the subretinal space, it can treat fundus diseases caused by metabolic disorders of retinal pigment epithelial cells. RPE cell sheet transplantation is superior to RPE cell suspension injection transplantation in that the former can maintain the normal polarity of RPE cells in physiological state after transplantation, and the correct polarity distribution is important for maintaining the function of RPE cells. is of great significance. In addition to retinal pigment epithelial cells and iris epithelial cells, other pigment epithelial cells can also be used as the seed cells for the above-mentioned construction of the retinal pigment epithelial cell sheet and transplantation.

Example 8 retinal neurosensory layer cell sheet

Seed cell culture was carried out according to the conventional culture method of retinal neurosensory layer cells (photoreceptor cells, interneurons, ganglion cells), and the optic nerve cell sheet was constructed by a method similar to the above-mentioned corneal epithelial cell sheet construction. Transplantation of neurosensory retinal cell sheets can treat patients with retinopathy. In addition to using retinal precursor cells (retinal progenitor cells), other cells can also be used for the construction of the retinal neurosensory layer cell sheet and the seed cells for transplantation.

Example 9 retinal cell sheet

The retinal pigment epithelial cell sheet and the retinal nerve sensory layer cell sheet are longitudinally integrated by overlapping single cell sheets or adhered to a composite retinal cell sheet by adhesive protein solution. One or more adhesive protein solutions are used for retinal cell sheet transplantation. It can treat patients with various retinal diseases.

Embodiment 10 skin cell sheet

Similar to the construction of corneal cell sheets, epidermal epithelial cell sheets and dermal cell sheets can be constructed separately for transplantation treatment, and epidermal epithelial cell sheets and dermal cell sheets can be vertically integrated into multiple composite skin cell sheets through overlapping integration or adhesion. Transplantation is then performed to treat skin lesions.

Example 11 Periodontal tissue cell sheet

Isolate periodontal ligament fibroblasts by enzymatically digesting tissue blocks and carry out routine cell culture, construct periodontal ligament fibroblast sheets according to the above method, and transplant them in periodontium. Gingival regeneration can be observed after the operation, and the regenerated gingiva and tooth adhesion Adhering tightly, it can treat receding gums or other gum diseases.

Embodiment 12 cardiomyocyte sheet

According to the method above to construct a cardiomyocyte sheet, multiple cardiomyocyte sheets can be stacked and vertically integrated into multiple composite cardiomyocyte sheets, or can be vertically integrated into multiple composite cell sheets through adhesion. The pulsation and electrophysiological activities of multiple composite cell sheets can be observed, and multiple composite cell sheets can be transplanted to myocardial defect sites.

According to the above, cell sheets such as liver tissue, urinary system, and respiratory system can also be constructed and transplanted.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (10)

1. A method for preparing a tissue engineering cell sheet, characterized in that it specifically comprises the following steps: (1) covering the surface of the cell culture support with an N-isopropylacrylamide monomer solution containing a cross-linking agent N,N'-methylenebisacrylamide at a mass concentration of 0.1% to 1.5%; The mass concentration of the N-isopropylacrylamide monomer is 40%-60%. (2) Carry out electron accelerator radiation synthesis to the solution obtained in step (1), the total radiation dose is 240kGy～300kGy, and it is continuously divided into three to six times of radiation synthesis, each radiation dose is lower than 200kGy, and the poly N-isopropyl group is covered. Acrylamide hydrogel cell culture support; (3) Put the cell culture support obtained in step (2) into deionized water at 4° C., soak for 5 to 8 days, change the water 4 to 7 times during the period, dry and sterilize, and seal it for later use; (4) Control the temperature to make the poly-N-isopropylacrylamide hydrogel a hydrophobic phase, inoculate the cells into the cell culture support obtained in step (3), and cultivate the cells; change the temperature to make the poly-N-isopropyl acrylamide The acrylamide hydrogel is a hydrophilic phase, and the tissue engineered cell sheet is separated by polymer membrane adsorption or clamp clamping method. 2. The preparation method of tissue engineering cell sheet according to claim 1, characterized in that: the N-isopropylacrylamide monomer in step (1) is recrystallized. 3. The method for preparing tissue engineered cell sheets according to claim 2, characterized in that: the recrystallization is carried out using benzene and isopropanol at a volume ratio of 1:1. 4. The preparation method of tissue engineered cell sheet according to claim 1, characterized in that: the covering amount of N-isopropylacrylamide monomer on the surface of the cell culture support in step (1) is 3 mg-5 mg/cm ^2. The added amount of the cross-linking agent is 10 μg-30 μg/cm ² . 5. The preparation method of tissue engineered cell sheet according to claim 1, characterized in that: the cell culture support in step (1) is made of polystyrene or polytetrafluoroethylene. 6. The preparation method of tissue engineered cell sheet according to claim 1, characterized in that: the vacuum degree of the electron accelerator in step (2) is ≤2.2×10 ^-6 KPa. 7. The method for preparing tissue engineered cell sheets according to claim 1, characterized in that the sterilization in step (3) is ethylene oxide sterilization. 8. The preparation method of tissue engineering cell sheet according to claim 1, characterized in that: in step (4), the following method is used to separate and obtain said tissue engineering cell sheet: Inoculate the cells onto the culture vessel grafted with poly-N isopropylacrylamide hydrogel; culture in a 5% CO ₂ incubator at 37°C, and when the cultured cells reach 80-90% confluence, transfer the culture vessel to 20°C The incubator was incubated for 1 hour, and the cells floated in sheets on the surface of the swollen and thickened hydrogel, and the carrier-free tissue engineering cell sheet was separated by polymer membrane adsorption or clamp clamping method. 9. A tissue engineered cell sheet obtained by the method according to any one of claims 1-8. 10. A multilayered tissue engineered cell sheet, characterized in that: the tissue engineered cell sheet according to claim 9 is stacked vertically and obtained by multilayered culture; or obtained by vertically integrating the adhesive protein solution; The adhesive protein liquid includes at least one of the following proteins: 10μg～100μg/ml laminin, 5μg～50μg/ml fibronectin, 100μg～1mg/ml peptide arginyl-glycyl-aspartyl-serine tetrapeptide, 100mg～300mg/ml fibrinogen, 10μg～100μg/mL type I collagen, 10μg～100μg/ml type IV collagen; The collagen is diluted with 0.006-0.1mol/L acetic acid, and other proteins are diluted with NaHCO ₃ solution at pH 8.3; After the fibrinogen is added, an equal amount of 200-1000 U/ml thrombin and an equal amount of 10% calcium chloride solution need to be added.

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