CN114887118B

CN114887118B - Method for culturing keratinocyte and constructing double-layer tissue engineering epidermis by taking autologous KC as seed cells

Info

Publication number: CN114887118B
Application number: CN202210426459.2A
Authority: CN
Inventors: 李志清; 吴炳义; 吴晗; 何珊; 陈琼
Original assignee: Guangzhou Xiangxue Nanfang Precision Medical Technology Co ltd
Current assignee: Guangzhou Xiangxue Nanfang Precision Medical Technology Co ltd
Priority date: 2022-04-21
Filing date: 2022-04-21
Publication date: 2023-03-24
Anticipated expiration: 2042-04-21
Also published as: CN114887118A

Abstract

The application discloses a method for culturing keratinocytes and constructing a double-layer tissue engineering epidermis by taking autologous KC as seed cells, (1) adding Rock inhibitor Y-27632 into a serum-free culture medium, inoculating primary keratinocytes from autologous skin, and performing amplification and subculture; separating to obtain seed cells; (2) And (3) re-suspending the obtained seed cells by a serum-free culture medium, and then inoculating the seed cells into a double-layer three-dimensional collagen sponge scaffold material to construct a double-layer tissue engineering epidermis. The upper layer of the double-layer three-dimensional collagen sponge support material is a medical silicon rubber semipermeable membrane, the lower layer of the double-layer three-dimensional collagen sponge support material is a three-dimensional porous collagen sponge support, and the seed cells are inoculated to the three-dimensional porous collagen sponge support layer. The method solves the problems of limited rapid amplification and culture of autologous seed cells in the construction of the epidermis in tissue engineering, degradation of a scaffold material, histocompatibility and the like.

Description

Method for culturing keratinocyte and constructing double-layer tissue engineering epidermis by taking autologous KC as seed cells

Technical Field

The application relates to the technical field of tissue engineering skin, in particular to a method for constructing a double-layer tissue engineering epidermis by culturing autologous keratinocytes and the constructed double-layer tissue engineering epidermis using the autologous keratinocytes as seed cells.

Background

The Tissue Engineering Epidermis (TEE), also called epidermis substitute, is an epidermal cell membrane formed by separating epidermal cells from skin tissues and culturing and amplifying in vitro by using a tissue engineering technology. Seed cells, scaffold materials and growth factors are 3 basic elements of the culture medium. At present, the epidermis substitutes for experiment and clinical test and application include autologous epidermis substitutes, variant epidermis substitutes, autologous/variant epidermal cell mixed membranes, autologous epidermal cell membranes inoculated in carriers, epidermal stem cell membranes and the like. If the seed cells are non-receptor autologous sources, the seed cells can be rejected after transplantation, cannot survive for a long time and only can temporarily cover the wound surface; however, if the receptor autologous cells are used as seed cells, the activity of the cells can be maintained, rejection can be avoided, and the cells can survive permanently to repair the wound surface.

Tissue engineering technology has been developed rapidly, but at present, tissue engineering epidermis still faces many problems: for example, autologous keratinocytes are difficult to obtain and rapidly expand in a short time on a large scale as seed cells; the traditional epidermal cell membrane cells are thin and easy to fall off, are difficult to operate, have low transplanting survival rate, are easy to lose epidermal cells after being transplanted, and have poor wear resistance, easy burst, contracture and the like after wound surfaces are healed; whether the scaffold material used as the epidermal cell membrane scaffold has a good microporous structure, a proper degradation period, histocompatibility and the like is favorable for the adhesion, migration, proliferation and the like of KC, so that the application of the tissue engineering epidermis is limited. Therefore, no tissue engineering epidermis commercial product using the source of the autologous epidermal cells as seed cells is available in China.

Disclosure of Invention

The application provides a scheme for promoting the rapid adherence and growth of Keratinocyte (KC), which solves the problem of limited rapid expansion culture of autologous seed cells; the application also provides a construction method of the double-layer tissue engineering epidermis taking the keratinocyte from the self as the seed cell, the bracket material has good micropore structure, proper degradation period, histocompatibility and the like, is beneficial to the adhesion, migration and proliferation of KC, and the upper medical silicon rubber semipermeable membrane has the functions of barrier protection and mechanical support.

In the traditional cell culture method, the requirement of the in vitro culture and amplification technical condition of epidermal cells is high, the time consumption is long, and at least 3-4 weeks are needed from material taking to complete membrane culture; the amplification passage number is limited, and the clinical requirements are difficult to meet in a short time; in the process of culturing epidermal cells in vitro, if a culture medium containing xenogenous animal serum is adopted and the culture medium is mixed with xenogenous cells (such as rat cells and 3T3 cells), exogenous pathogenic factors and risks caused by immunological rejection can be brought; if the seed cells are non-receptor autologous sources, the seed cells are easy to reject after transplantation, cannot survive for a long time and only can temporarily cover the wound surface.

The application finds in experimental studies:

on one hand, the application adopts an animal serum-free and xeno-free cell culture system, can avoid the risk caused by exogenous pathogenic factors and immunological rejection caused by adding animal serum, xeno-cells and the like, and is beneficial to obtaining safer and more effective cell products. The addition of ROCK inhibitor (Y-27632) into KC culture medium can promote KC adherence, inhibit its apoptosis and aging, increase its proliferation ability, and increase the number of passage times. The final culture time of the culture method is 10-14 days.

On the other hand, the medical sponge stent adopts a double-layer three-dimensional collagen sponge stent material, and the upper layer is a medical silicon rubber semipermeable membrane, so that the medical sponge stent has the functions of barrier protection and mechanical support; the lower layer is a three-dimensional porous collagen sponge scaffold, namely the collagen-chondroitin sulfate composite material, has a good microporous structure (aperture, porosity, pore shape, connectivity and the like), a proper degradation period and histocompatibility, and is beneficial to adhesion, migration and enrichment of KC. The transplantation survival rate is higher than that of the prior epidermal cell membrane, and the loss is not easy to occur; the skin after the repair has improved wear resistance and is not easy to break, contracture and the like.

The double-layer three-dimensional collagen sponge scaffold material is a product which can be directly purchased and commercialized. For example, shenzhen zikang medical device GmbH, a double-layer dermal repair material model BAS-0505, or PELNAC (Pi Naike) dermal repair material produced by GUNZE corporation of Japan.

Research based on this application finds that:

on the one hand, the application provides the application of double-deck three-dimensional collagen sponge scaffold material in constructing the tissue engineering epidermis, the upper strata of double-deck three-dimensional scaffold material is medical silicon rubber pellicle, the lower floor is three-dimensional porous collagen sponge scaffold. The material has a good micropore structure, a proper degradation period, histocompatibility and the like, so that the material is beneficial to the adhesion, migration, proliferation and the like of KC.

In another aspect, the present application also provides a method for constructing a double-layer tissue-engineered epidermis using autologous keratinocytes as seed cells, comprising:

(1) Adding Rock inhibitor Y-27632 into serum-free culture medium, inoculating primary keratinocyte derived from autologous skin, and performing amplification and subculture; separating to obtain seed cells;

(2) The obtained seed cells are re-suspended by a serum-free culture medium and then inoculated to a double-layer three-dimensional collagen sponge scaffold material, and the double-layer tissue engineering epidermis enriched with the seed cells in the three-dimensional porous collagen sponge scaffold is obtained through culture; the upper layer of the double-layer three-dimensional collagen sponge scaffold material is a medical silicon rubber semipermeable membrane, the lower layer of the double-layer three-dimensional collagen sponge scaffold material is a three-dimensional porous collagen sponge scaffold, and the seed cells are inoculated to the three-dimensional porous collagen sponge scaffold layer.

Optionally, in the step (1), the concentration of the ROCK inhibitor Y-27632 in a serum-free culture medium is 3-8 mu M; more preferably 4 to 6. Mu.M; most preferably 5. Mu.M.

Optionally, the serum-free culture medium in the step (1) is replaced by a human mesenchymal stem cell conditioned culture solution, and the human mesenchymal stem cell conditioned culture solution contains Rock inhibitor Y-27632.

Optionally, the concentration of the ROCK inhibitor Y-27632 in the human mesenchymal stem cell conditioned medium is 3-8 μ M; more preferably 4 to 6. Mu.M; most preferably 5. Mu.M.

Optionally, in step (1): and (3) changing the culture medium for the first time after 70-75 hours of inoculation, wherein the frequency of changing the culture medium in the early stage of culture is 2-3 days/time, and the frequency of changing the culture medium is increased to 1 day/time when the cell fusion degree in the culture medium is increased to 30-40%.

Optionally, in step (2): adding a ROCK inhibitor to a serum-free medium in which the seed cells are resuspended; in this step, the concentration of the ROCK inhibitor in the serum-free medium is 3 to 8. Mu.M.

Optionally, the conditions for culturing the seed cells, culturing the human mesenchymal stem cells and inoculating the seed cells on the scaffold are as follows: 36-38 deg.C, 3-6% of CO ₂ 85-95% humidity; further 37 ℃,5% CO ₂ And 90% humidity. The seeding density of the cells is 15,000-20,000/cm when the seed cells are cultured, the human mesenchymal stem cells are cultured and the seed cells are seeded on the bracket ² 。

Optionally, the ROCK inhibitor is Y-27632; the serum-free culture medium is a KSFM culture medium; the human mesenchymal stem cells are umbilical cord mesenchymal stem cells.

Alternatively, the primary keratinocytes may be digested from the patient's skin ex vivo.

Optionally, the digestion method comprises:

(1) Cutting skin of patient 2-3cm ² Washing with 1% double antibody (penicillin and streptomycin sulfate) PBS buffer solution for 3-5 times;

(2) Transferring the skin to a 60mm petri dish and cutting to 1 × 1cm ² Adding Dispase II with the final concentration of 0.25wt% diluted by KSFM culture medium, and standing overnight at 4 ℃;

(3) After overnight digestion, the skin was transferred to a large dish and washed 2-3 times with PBS buffer;

(4) Cutting skin with scissors, adding 0.25% pancreatin digestive juice containing EDTA, incubating at 37 deg.C for about 10min, and adding isovolumetric 0.5% pancreatin Inhibitor (Trypsin Inhibitor) to stop digestion;

(5) The digestion solution was collected using 100 μm and 70 μm cell filters in this order, centrifuged at 1000rpm/min for 10min to collect cells, and washed.

The application also provides the double-layer tissue engineering epidermis which is constructed by the construction method and takes the autologous KC as seed cells.

A double-layer tissue engineering epidermis comprises a double-layer three-dimensional collagen sponge scaffold material and seed cells growing in the double-layer three-dimensional collagen sponge scaffold material, wherein the upper layer of the double-layer three-dimensional collagen sponge scaffold material is a medical silicon rubber semipermeable membrane, the lower layer of the double-layer three-dimensional collagen sponge scaffold material is a three-dimensional porous collagen sponge scaffold, and the seed cells grow in the three-dimensional porous collagen sponge scaffold; the seed cells are autologous keratinocytes;

compared with the prior art, the application has at least one of the following beneficial effects:

(1) The application discovers for the first time that the publicly reported three-dimensional collagen sponge dermal material can be used for constructing the tissue engineering epidermis and successfully solves the technical problem of constructing the tissue engineering epidermis.

(3) The constructed double-layer three-dimensional collagen sponge scaffold has good histocompatibility with KC, and cells can not only be adhered to the surface of the material, but also migrate and proliferate to the interior of the material along the structure of the collagen scaffold to form a double-layer tissue engineering epidermis enriched with seed cells.

(4) The double-layer tissue engineering epidermis can obviously promote the healing of the wound of animals and reduce the healing time of the wound, has higher abrasion resistance, and the bracket is easy to biodegrade.

(5) When the culture fusion degree of primary Keratinocyte (KC) is about 30-40%, the liquid is changed every day, the method can delay the aging of KC cells in vitro and obtain more KC cells with vigorous activity.

(6) The KC can be promoted to proliferate by adding a ROCK inhibitor (Y-27632) and umbilical cord mesenchymal stem cell conditioned medium into a KC culture medium, and the umbilical cord mesenchymal stem cells in the application have the function of promoting the growth of seed cells in the double-layer three-dimensional scaffold material by using the conditioned medium and do not serve as the seed cells.

(7) The application discovers that the ROCK inhibitor is added in a plurality of times, namely the ROCK inhibitor is added in the processes of culturing stem cells and seed cells and preparing tissue engineering epidermis, so that the proliferation capacity of KC in a double-layer three-dimensional collagen sponge scaffold can be obviously promoted.

Drawings

FIG. 1 is a diagram showing the culture conditions of primary keratinocytes in example 1;

FIG. 2 is a graph showing the growth of primary keratinocytes on a collagen film in example 2;

FIG. 3 is a graph of wound healing in the animal model of example 3;

FIG. 4 is a graph showing the degradation of the bioscaffold in example 3;

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

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 application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

The upper layer of the double-layer three-dimensional collagen sponge scaffold material used in the application is a medical silicon rubber semipermeable membrane, and has the functions of barrier protection and mechanical support; the lower layer is a three-dimensional porous collagen sponge bracket. The double-layer three-dimensional collagen sponge scaffold material is directly purchased. For example, shenzhen zikang medical device Limited, model BAS-0505, a double-layer dermal repair material. However, the material is publicly reported to be used for constructing the tissue engineering dermis.

The application aims to research the tissue engineering epidermis, and finds that: on one hand, in the traditional construction method, the adopted epidermal cell membrane scaffold has imperfect structure and function, and is not as rich in more epidermal cells as a three-dimensional porous collagen sponge scaffold; the scaffold material has proper microporous structure, proper degradation speed and histocompatibility, and is closely related to adhesion, proliferation and migration of epidermal cells. The previous epidermal cell membrane is thin and fragile and is difficult to operate; the survival rate of transplantation is not high, epidermal cells are easy to lose after transplantation, the wear resistance and elasticity of wound surfaces are poor after healing, the wound surfaces are easy to burst and contracture, and the appearance and the function are influenced.

On the other hand, in the traditional cell culture method, the requirement on the in vitro culture amplification technical condition of the epidermal cells is high, the time consumption is long, and at least 3-4 weeks are needed from the material taking to the culture of the epidermal cells into a complete membrane; the amplification passage number is limited, and the clinical requirements are difficult to meet in a short time; in the process of culturing epidermal cells in vitro, if a culture medium containing xenogenous animal serum is adopted and the culture medium is mixed with xenogenous cells (such as rat cells and 3T3 cells), exogenous pathogenic factors and risks caused by immunological rejection can be brought; if the seed cells are non-receptor autologous sources, the seed cells are easy to reject after transplantation, cannot survive for a long time and only can temporarily cover the wound surface.

In the research process of the application, the tissue engineering epidermis is constructed by directly using a commercial double-layer artificial dermis repairing material; however, in the research process, the material is only directly used for constructing the tissue engineering epidermis, and the tissue engineering epidermis cannot be successfully constructed or the effect is poor.

In order to solve the problem, the research of the application discovers that KC which is self-sourced is used as seed cells; and ROCK inhibitor with certain concentration and the like is added in the seed cell culture process, so that the adhesion, migration and proliferation of KC in the double-layer three-dimensional collagen sponge scaffold material can be remarkably promoted.

In order to promote the growth of KC in the double-layer three-dimensional collagen sponge scaffold material, the culture process of seed cells is further improved, namely a serum-free culture medium can be replaced by a human mesenchymal stem cell conditioned culture solution, a ROCK inhibitor is also added into the culture solution, the proliferation of the stem cell conditioned culture solution on the KC cells can be promoted, and the growth of the KC cells in the double-layer three-dimensional scaffold material is further promoted.

Furthermore, the application also finds that the addition of the ROCK inhibitor in the culture process after the seed cells are inoculated on the double-layer three-dimensional scaffold material can further promote the growth of the KC cells in the double-layer three-dimensional scaffold material.

In the application, on the one hand, the animal serum-free culture medium and the xeno-free cell culture system are adopted, so that the risk caused by exogenous pathogenic factors and immunological rejection due to the addition of animal serum, xenogeneic cells and the like can be avoided, and a safer and more effective cell product can be obtained. On the other hand, the addition of the ROCK inhibitor (Y-27632) into a culture medium of the KC cells can promote the KC cells to adhere to the wall, inhibit the KC cells from apoptosis and aging, improve the proliferation capacity of the KC cells and obviously increase the number of passages. The final culture time of the culture method is 10-14 days.

In a word, the technical obstacle of the double-layer collagen sponge three-dimensional scaffold material in the construction of the tissue engineering epidermis is successfully solved through the selection of seed cells and the improvement of a seed cell culture method.

The lower layer of the double-layer collagen sponge three-dimensional scaffold material is a collagen-chondroitin sulfate composite material, has a good microporous structure (aperture, porosity, pore shape, connectivity and the like), a proper degradation period and histocompatibility, and is beneficial to adhesion, migration and enrichment of KC. After the double-layer collagen sponge three-dimensional scaffold material is successfully applied to the construction of the tissue engineering epidermis, the transplantation survival rate of the finally constructed tissue engineering epidermis is higher than that of the prior epidermal cell membrane, and the tissue engineering epidermis is not easy to lose; the skin after the repair has improved wear resistance and is not easy to break, contracture and the like.

Primary Keratinocyte (KC) is separated, amplified and subcultured, and inoculated into a collagen sponge scaffold with a three-dimensional structure to construct a full-layer skin deletion acute wound surface mouse model and examine the performance of tissue engineering epidermis.

The following is illustrated by specific examples:

preparation of culture medium and umbilical cord mesenchymal stem cell conditioned medium used in the following examples:

serum-free culture medium for stem cells: human mesenchymal stem cell culture kit produced by dacco corporation has no phenol red, and the product number is: 6914521;

KSFM medium: complete serum-free medium, product number 10744019, produced by Gibco;

y-27632: dow company, inc. has a Y-27632 powder product number 146986-50-7.

Preparing umbilical cord mesenchymal stem cell conditioned medium A:

inoculating Mesenchymal Stem Cells (MSC) into stem cell serum-free medium at 37 deg.C with 5% CO ₂ And culturing the mesenchymal stem cells under the condition of 90% humidity until the cell fusion degree reaches 80-90%, centrifuging, taking supernatant, and filtering to prepare conditioned medium.

Preparing umbilical cord mesenchymal stem cell conditioned medium B:

inoculating Mesenchymal Stem Cells (MSC) into a stem cell serum-free medium containing 5 μ M Y-27632, and 5% CO at 37 ℃% ₂ Culturing the mesenchymal stem cells MSC under the condition of 90% humidity until the cell fusion degree reaches 80-90%, centrifuging, taking supernatant, and filtering to prepare the mesenchymal stem cells MSCConditioned medium.

Mesenchymal Stem Cells (MSCs) can be isolated and extracted from tissues such as umbilical cord, bone marrow, fat, etc. using extraction methods well established in the art, and MSCs used in the following examples are references (k. Self, d. Troyer, m.l. Weiss. Method to isolate media-like cells from white's jelly of lithium cord. Cell biol.,2008, 86.https://doi.org/10.1016/S0091-679X(08) 00006-X) The tissue block adherent culture method is adopted to separate and extract from the umbilical cord, and when the isolated MSC is cultured to the P4-P5 generation, the stem cell culture supernatant is obtained in the embodiment. In addition to the self-extraction route, commercial MSC cells can also be purchased.

Seed cell culture medium:

(a) KSFM medium;

(b) KSFM medium containing 5. Mu. MY-27632;

(c) Umbilical cord mesenchymal stem cell conditioned medium A;

(d) Umbilical cord mesenchymal stem cell conditioned medium B.

The double-layer three-dimensional collagen sponge scaffold material used in the following examples is purchased from Shenzhen Qikang medical instruments GmbH, model number BAS-0505.

In the following examples, concentrations in percentage terms refer to mass percent concentrations unless otherwise specified.

Example 1

1. KC isolation and culture

(1) Cutting skin of patient 2-3cm ² And shaking and washing the mixture for 3-5 times by adopting PBS buffer solution containing 1% double antibody (penicillin and streptomycin sulfate).

(2) Transferring the skin to a 60mm petri dish and cutting to 1 × 1cm ² Size, dispase II diluted in KSFM medium to a final concentration of 0.25wt% was added overnight at 4 ℃.

(3) After overnight digestion, the skin was transferred to a large dish and washed 2-3 times with PBS buffer.

(4) The skin was minced with scissors, 0.25% Trypsin digest containing EDTA was added after the skin was minced, incubated at 37 ℃ for about 10min, and to ensure adequate digestion, an equal volume of 0.5% Trypsin Inhibitor (Trypsin Inhibitor) was added to stop the digestion.

(6) After cell counting, the cells were counted at a certain density (20,000 cells/cm) ² ) Cells were inoculated into the following media, respectively:

experimental group (a): KSFM;

experimental group (b): KSFM medium (KSFMY or KSFM + Y) containing 5. Mu.MY-27632;

experimental group (c): umbilical cord mesenchymal stem cell conditioned medium A;

experimental group (d): umbilical cord mesenchymal stem cell supernatant B containing 5 mu MY-27632.

37℃，5％CO ₂ And culturing under 90% humidity condition.

(7) The liquid is changed for the first time after 72 hours of inoculation, and the liquid changing speed at the prophase of the cells is 2-3 days/time. When the cell density is increased to 40%, the liquid is changed every day for 4-6 days, and the cells reach 70% density and can be passaged.

The experimental results are shown in fig. 1:

in FIG. 1, A is a comparison graph of keratinocyte growth conditions between the case of changing solution every day for 2-3 days when the cell growth fusion degree reaches about 30% -40%, wherein the case of changing solution every day for 2-3 days is shown on the left, and the case of changing solution every day is shown on the right. From the results shown in A in FIG. 1, it can be seen that when the confluency of cell growth reaches about 30% -40%, the amount of cell expansion increases if the solution is changed for 2-3 days, and the process is irreversible; if the liquid is changed every day, KC maintains the elliptical shape with high nucleus-to-mass ratio. From this, it was found that the solution was changed every day when the degree of fusion of KC was about 30% to 40% after 1 week of culture. The method can delay the aging of KC in vitro and obtain more vigorous KC cells. If the solution is changed once in 2-3 days, the cells can rapidly expand and age after being cultured to the 3 rd generation, which is not beneficial to the large-scale expansion of KC.

In FIG. 1, B is a diagram showing morphological changes in culture of immunofluorescence-identified KC cells (corresponding to experiment group (d)). Cell nucleus DAP1 stained blue (top right), β -Integrin protein stained red (bottom left), CK14 protein stained green (bottom right). As shown in the results B in FIG. 1, the KC cultured by the method has regular morphology and vigorous activity, and the expression levels of the surface markers CK14 and beta-Integrin are significant.

The left table of C in FIG. 1 is a statistical table of adherent cells and total cell counts at different fields of KC culture on day 5 in KSFM medium supplemented with 5. Mu. M Y-27632, and the right panel is a histogram of significance analysis of the data. Therefore, Y-27632 can obviously promote KC adherence; the anchorage rate of cells added with 5 μ M of Y-27632 is improved by more than 6 times than that of KC cultured in KSFM medium alone. In the table of C: a represents the KSFM group (i.e., experimental group (a)), and B represents the KSFM + Y group (i.e., experimental group (B)).

The left table in fig. 1 shows the cell cycle data of KC cells in 6 different culture conditions (where the numbers a, b, c, D and NC1 are the experimental groups (a), (b), (c), (D), and NC2 are the control groups, where IL2 added to the NC1 group is a cytokine that promotes cell growth, proliferation, and differentiation, which is used to illustrate a positive control), and the right drawing shows the growth curve of KC. The result shows that the proliferation capacity and the growth speed of the KC cells are enhanced after the Y-27632 is added, and the proliferation capacity and the growth speed of the KC cells are strongest under the culture condition of the experimental group d. Therefore, the cell proliferation capacity and the growth rate are enhanced after the Y-27632 is added; meanwhile, Y-27632 can promote the function of MSC conditioned medium on KC cell proliferation.

Example 2

(1) According to the size of a wound surface, a double-layer two-dimensional collagen scaffold material (the upper layer is a medical silicon rubber semipermeable membrane, the lower layer is single-layer achilles tendon collagen without a special good microporous structure, 2D-TEES) and a double-layer three-dimensional collagen sponge cell scaffold material (3D-TEES, shenzhen Qikang medical equipment Limited, model number BAS-0505) are respectively cut into a proper size in a biological safety cabinet, the collagen layer is arranged on the upper part, and the silicon rubber membrane layer is arranged on the lower part and is horizontally placed in a culture plate.

(2) The resuspended KC cells in KSFM medium containing 5. Mu. M Y-27632 (group (d) culture in example 1) were collected at 20,000 cells/cm ² The seeding density and volume were 350. Mu.L seeded on two collagen scaffold materials, 2D-TEES and 3D-TEES, respectively.

(3) After the cells are respectively inoculated to the collagen membrane 2D-TEES and the collagen membrane 3D-TEES, the cells are basically adhered completely after static culture for 6 hours, and at the moment, a KSFM culture medium can be supplemented in a culture plate until the cells submerge the membrane. 37 ℃ C., 5% CO ₂ The incubation was continued overnight at 90% humidity.

(4) The following day, the solution was changed and washed twice with PBS buffer to obtain cell patches (3D-TEES cell patch and 2D-TEES cell patch).

The growth of primary keratinocytes on collagen membranes is shown in fig. 2:

FIG. 2A is a schematic diagram of 2D-TEES and 3D-TEES structures. Wherein the pore diameter of the 2D-TEES collagen layer is small. The 3D-TEES three-dimensional collagen sponge bracket has the advantages that the collagen has slightly larger aperture, uniform density and thin film, can be flatly spread in a culture dish and can stably bear liquid drops.

FIG. 2B is a graph showing the culture conditions of KC in 2D-TEES and 3D-TEES. KC was cultured on 2D-TEES, cells adhered mainly to the collagen scaffold surface rather than uniformly distributed throughout the collagen layer; because the pore diameter of the collagen fiber is too small and dense, cells are adhered to the surface of the collagen and cannot migrate to the inside of the collagen layer; when the cell density is too high, KC cells are easily exfoliated from the collagen smooth surface in a sheet. KC was cultured on 3D-TEES, and cells were evenly distributed throughout the patch, and grew inward, filling the entire collagen sponge layer. The gray dots in the figure represent DAPI-stained KC cells.

In FIG. 2, C is KC in the form of droplets seeded on 3D-TEES, and the 3D-TEES seeded with cells was observed under a microscope in a bright field in which cell adhesion and the pore size of a collagen membrane sheet were observed (right panel in C).

FIG. 2D shows DAPI staining of cells in 30 μ M thick collagen scaffold material, which were seeded on 3D-TEES with the same number of KC cells and cultured with KSFM and KSFM supplemented with 5 μ M Y-27632 for 4 days, respectively. It can be seen that the addition of 5. Mu. M Y-27632 resulted in a greater number of KC cells and a stronger adhesion to 3D-TEES.

FIG. 2E is the two-dimensional fluorescent staining of keratinocytes after 4 days of growth on 3D-TEES collagen scaffolds.

In FIG. 2, F is a three-dimensional superimposed image of cellular immunofluorescence on a patch with a thickness of 30 μ M taken by a confocal laser microscope under the E-picture field.

From the results of fig. 2, it can be seen that:

(1) The 3D-TEES membrane is thinner, a culture medium is not needed to be soaked in advance, the membrane is smoother, the cell suspension can be evenly and uniformly dripped above the membrane, the material density is more uniform, and the density and the growth space of the cells at each position in the collagen sponge layer are more uniform.

(2) After Y-27632 was added, the number of KC cells cultured on 3D-TEES was greater, indicating that Y-27632 can promote the proliferation and adhesion of KC in 3D-TEES.

(3) The 3D-TEES membrane has good affinity for the keratinocytes, and the cells can not only adhere to the surface of the material, but also grow towards the interior of the material along the structure of the collagen scaffold, so that the three-dimensional membrane filled with the cells is formed.

Example 3 cellular Membrane on wound repair

(1) Anesthetized mice were induced under sterile surgical conditions using an isoflurane induction chamber (3% at 0.6L/min).

(2) Using an ophthalmic scissors to cut off the whole skin layer of the back at both sides of the spine, and reserving the muscle tissue below to form 2cm ² Left and right square areas, similar to the size of the graft.

(3) After bleeding was controlled with appropriate pressure, the wounds were covered with different cell patches (prepared in example 2) cultured in vitro and sutured to the host's defective skin, where the tissue engineered epidermis of the wounds on both sides of the back were different for control, and the dressing was removed after two weeks.

(4) Animals were sacrificed after anesthesia to obtain experimental area skin for pathological sectioning and immunofluorescent staining analysis, using 1, 2, and 3 weeks as time nodes.

The experimental results of the wound healing situation of the animal model are shown in fig. 3:

in fig. 3, a is an appearance diagram of acute full-thickness wound surfaces at different time nodes of each group: wherein "cell patch/blank control" indicates that the left wound on the back of the mouse in the row is covered with the tissue engineering epidermis, the right side is blank control, and the left cell patch is the 3D-TEES cell patch prepared in example 2; the expression "cell patch/simple patch set" means that the left wound on the back of the mouse in the row is covered with the tissue engineering epidermis, the right side is made of a simple collagen scaffold material, the left tissue engineering epidermis is the 3D-TEES tissue engineering epidermis prepared in example 2, and the right simple patch set is the 2D-TEES cell patch prepared in example 2. The results show that the 3D-TEES tissue engineering prepared in example 2 has the best healing capacity of epidermis.

FIG. 3B is a statistical chart of the unhealed area of the wound on the A-picture;

the data in the table on the left of C in fig. 3 represent the time required for the final healing of acute full-thickness wounds of the same size in each group of wounds, the proportion of the scar area to the initial wound area at the third week, and the statistical analysis of wound shrinkage, respectively. (. P <0.0001 has statistical significance). The right panel is a bar graph corresponding to the table.

The results in fig. 3 show that the 3D-TEES tissue engineering epidermis can significantly promote the healing of the wound of animals and shorten the healing time of the wound.

The degradation of collagen scaffolds in the tissue engineering epidermis is shown in fig. 4:

in FIG. 4, A is 3D-TEES tissue engineering epidermis subcutaneous neonatal basal tissue of wound at 1 st, 3 rd and 6 th weeks to be pathological section (HE), black arrow in the figure is the condition that TEES exists in subcutaneous tissue, and experiment shows that no obvious stent residue is seen at 6 th week.

In FIG. 4B, it can be seen that compared with the blank group, the 3D-TEES tissue engineering epidermal group and the 2D-TEES cell membrane group have a large amount of new blood vessels formed (see arrows in the figure), and the 3D-TEES tissue engineering epidermal group has a thin layer of epidermal cells generated (see square area in the figure), and the structures are complete and coherent.

In FIG. 4C, the left 3D-TEES tissue engineering epidermal group has a complete structure, and the healed skin has a more ordered structure and a complete basal layer compared with the right 2D-TEES cell patch (see the box area in the figure).

From the results of fig. 4, it can be seen that: the 3D-TEES tissue engineering epidermis can realize biodegradation and can serve as a 'cell bank', provides autologous keratinocytes required for wound repair, and promotes wound healing; and can promote the formation of a large amount of new capillaries of the animal wound base which is removed from the whole layer of the skin, so that the newly healed skin structure is more similar to a similar dermal structure, and the elasticity and the wear resistance of the healed wound surface are improved.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for culturing autologous keratinocytes and constructing a double-layer tissue-engineered epidermis using the autologous keratinocytes as seed cells, comprising:

(1) Adding Rock inhibitor Y-27632 into a human mesenchymal stem cell conditioned culture solution, inoculating primary keratinocyte derived from autologous skin, performing amplification and subculture, wherein the concentration of the ROCK inhibitor Y-27632 in the human mesenchymal stem cell conditioned culture solution is 3-8 mu M; changing the culture medium for the first time after 70-75 h inoculation, wherein the culture medium changing frequency is 2-3 days/time in the early stage of culture, and the culture medium changing frequency is increased to 1 day/time when the cell fusion degree in the culture medium is increased to 30-40%; separating to obtain seed cells;

(2) The obtained seed cells are re-suspended by a serum-free culture medium and then inoculated to a double-layer three-dimensional collagen sponge scaffold material, and the double-layer tissue engineering epidermis with rich keratinocyte in the collagen sponge scaffold is obtained through culture; the upper layer of the double-layer three-dimensional collagen sponge scaffold material is a medical silicon rubber semipermeable membrane, the lower layer of the double-layer three-dimensional collagen sponge scaffold material is a three-dimensional porous collagen sponge scaffold, and the seed cells are inoculated to the three-dimensional porous collagen sponge scaffold layer; adding a ROCK inhibitor Y-27632 into a serum-free culture medium for resuspending the seed cells; in this step, the concentration of ROCK inhibitor Y-27632 in serum-free medium is 3-8. Mu.M.

2. The method of claim 1, wherein in step (1): the inoculation density is 15,000-25,000 pieces/cm ² (ii) a The culture conditions were: 36-38 deg.C, 3-6% of CO ₂ 85-95% humidity.

3. A tissue engineered epidermis constructed according to the method of any one of claims 1 to 2.