CN109971703B

CN109971703B - Culture method and culture medium for autologous tissue engineering epidermis

Info

Publication number: CN109971703B
Application number: CN201910269786.XA
Authority: CN
Inventors: 柴勋
Original assignee: SHANGHAI BIOMED-UNION CO LTD
Current assignee: Shanghai Biomed Union Biotechnology Co ltd
Priority date: 2019-04-04
Filing date: 2019-04-04
Publication date: 2021-02-26
Anticipated expiration: 2039-04-04
Also published as: CN109971703A

Abstract

A method of culturing an autologous tissue-engineered epidermis using umbilical cord mesenchymal cells as trophoblast cells. The method has the advantages that umbilical cord mesenchymal cells are used as trophoblast cells and matched with the serum-free culture medium, the umbilical cord mesenchymal cells do not need to be replaced by a differentiation culture medium, and the consumption of manpower, financial resources and time is reduced; the culture plate is prepared by using temperature-sensitive hydrogel in advance, so that secondary digestion of epidermal tissues by enzyme is avoided, damage to cell activity is reduced, and the peeling speed between the epidermal tissues and the culture plate can be accelerated.

Description

Culture method and culture medium for autologous tissue engineering epidermis

Technical Field

The invention relates to the technical field of cell culture, in particular to a culture method of autologous tissue engineering epidermis and a culture medium thereof.

Background

At present, the tissue engineering epidermis is mainly used for a series of skin diseases such as burns, scalds and leucoderma, and has wide application range, so great attention is paid to how to improve the quality and culture speed of the tissue engineering epidermis. The existing culture technology has two technical difficulties:

first, epidermal cells were cultured. In 1975, the method for culturing the cells by taking a mouse embryonic fibroblast line (3T3) as a trophoblast was invented by Rheinwald and Green, and in order to solve the safety problem caused by an animal-derived cell line, a lot of people also use autologous fibroblasts as trophoblasts for culturing; the method mainly has the following four defects: a) safety issues with animal-derived cell trophoblasts; b) the autologous epidermal cells are mainly from the dermis layer and can generate wound surfaces; c) the trophoblast cells need to be prepared in advance, so that the skin of a patient needs to be taken for 2 times, and the injury is increased; d) fibroblasts of different people have activity and function difference, the stability of the trophoblasts is different, and the quality of cultured epidermal cells is difficult to guarantee.

And secondly to the construction of tissue engineering. The tissue engineering construction is that under the traditional trophoblast method and serum condition, cultured epidermal cells can be vertically differentiated into normal physiological structures of epidermis, and can be torn off from a culture vessel after being digested by DISPASE enzyme. However, this method still presents the risk: a) the basal layer is easy to damage after digestion by DISPASE enzyme, cultured epidermal cells are not divided into epidermal structures under the condition of a serum-free culture medium, and the epidermal cells need to be inoculated on a scaffold material to complete the construction of the tissue engineering epidermis: b) the epidermal cells are difficult to distribute uniformly on the scaffold material and cannot have complete epidermal structure and function; c) the scaffold material is mainly chemically synthesized, has high cost and increases clinical risk.

Disclosure of Invention

The invention aims to provide a culture method of autologous tissue engineering epidermis and a culture medium used by the culture method, wherein the culture method is simple to operate, short in culture time and stable in cell quality.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method of culturing an autologous tissue-engineered epidermis using umbilical cord mesenchymal cells as trophoblast cells.

In order to avoid the secondary digestion of enzymes, the umbilical cord mesenchymal cells are pre-seeded on a culture plate coated with a temperature-sensitive hydrogel.

Preferably, the culture method is performed using a serum-free medium throughout the culture.

Preferably, the serum-free medium is replaced every 24 h.

A culture medium for culturing autologous tissue engineering epidermis comprises basal medium, growth component, calcium secretion component, serum substitute and enzyme inhibiting component; the calcium secreting component can stimulate the umbilical cord mesenchymal cells to secrete calcium ions.

Preferably, the calcium secreting component comprises dexamethasone, dexamethasone and beta-glycerophosphate.

Preferably, the growth components include insulin, cholera toxin, hydrocortisone and EGF.

Preferably, the enzyme inhibitor is a ROCK1 inhibitor.

Compared with the prior art, the invention has the advantages that: 1. umbilical cord mesenchymal cells are used as trophoblast cells and are matched with the serum-free culture medium, so that the umbilical cord mesenchymal cells do not need to be replaced by a differentiation culture medium, and the consumption of manpower, financial resources and time is reduced; 2. the culture plate is prepared by using temperature-sensitive hydrogel in advance, so that secondary digestion of epidermal tissues by enzyme is avoided, damage to cell activity is reduced, and the peeling speed between the epidermal tissues and the culture plate can be accelerated; 3. calcium ions are secreted by umbilical cord mesenchymal cells by adding a calcium secreting component into a culture medium, and the maturation and differentiation of the cells are promoted along with the increase of the concentration of culture time; 4. the ratio of melanocytes to keratinocytes of the epidermal tissue cultured by the culture method of the invention is similar to that of normal epidermal tissue, and no melanocytes are required to be added, so that the safety is high.

Drawings

FIG. 1 is a schematic diagram showing cell climbing-out of epidermal tissue cells after 24h of seeding.

FIG. 2 is a schematic diagram of cell fusion of epidermal tissue cells according to an embodiment of the present invention.

FIG. 3 is a schematic representation of DOPA staining of epidermal tissue cells in accordance with an embodiment of the present invention.

FIG. 4 is a schematic diagram of the verification of DOPA staining ratio of epidermal tissue basal layer cells according to the embodiment of the present invention.

FIG. 5 is a schematic diagram of cell stratification of epidermal tissue cells in accordance with an embodiment of the present invention.

FIG. 6 is a schematic diagram showing the peeling of epidermal tissue cells and a culture plate according to an embodiment of the present invention.

FIG. 7 is a schematic diagram of the cell structure of epidermal tissue cells according to an embodiment of the present invention.

FIG. 8 is a schematic diagram showing cell climbing-out of epidermal tissue cells of comparative experiment 1 of the present invention after 24 hours of seeding.

FIG. 9 is a schematic view showing cell fusion of epidermal tissue cells in comparative experiment 1 of the present invention.

FIG. 10 is a schematic diagram showing DOPA staining of epidermal tissue cells in comparative experiment 1 of the present invention.

FIG. 11 is a schematic representation of the cell stratification of epidermal tissue cells of group B of comparative experiment 1 of the present invention.

FIG. 12 shows the case of culturing epidermal tissue of group B of comparative experiment 1 of the present invention on a culture plate.

FIG. 13 is a schematic diagram showing the cell structure of epidermal tissue cells of group B of comparative experiment 1 of the present invention.

FIG. 14 is a schematic diagram showing cell climbing-out of epidermal tissue cells of comparative experiment 2 of the present invention after 24 hours of seeding.

FIG. 15 is a schematic view showing cell fusion of epidermal tissue cells in comparative experiment 2 of the present invention.

FIG. 16 is a schematic representation of DOPA staining of epidermal tissue cells of comparative experiment 2 of the present invention.

FIG. 17 is a schematic diagram showing the cell structure of epidermal tissue cells of group C of comparative experiment 2 of the present invention.

The magnification of the above figures is 40 times.

Detailed Description

Examples

The culture method of the autologous tissue engineering epidermis comprises the following steps:

1. pretreatment of epidermal tissue 1: cleaning normal epidermal skin 2cm x 2cm with DPBS containing antibiotic for several times, cutting into small pieces of about 5mm, adding 10ml of Dispase II separating enzyme, and digesting at 4 deg.C for 17 hr to obtain cultured umbilical cord mesenchymal cells.

2. Plating trophoblast cells: preparing temperature-sensitive hydrogel on a culture plate for 24h, and taking cultured umbilical cord mesenchymal cells as trophoblast cells according to the ratio of 2 x 10⁴/cm²And pre-inoculating the culture plate prepared by the temperature-sensitive hydrogel.

3. Pretreatment of epidermal tissues 2: removing Dispase II separating enzyme from epidermal tissue, cleaning with DPBS for 3 times, peeling epidermal layer with tooth bent forceps, transferring epidermal layer into centrifuge tube, washing with DPBS, cutting, removing DPBS, adding tissue digestive juice, and shaking at 37 deg.C for 10 min.

4. Inoculating epidermal tissue cells: the epidermal tissue cells obtained in step 3 are expressed as 4 x 10⁴/cm²The cell amount of (2) was inoculated on umbilical cord mesenchymal cells, and observed after adding 10ml of serum-free medium to culture for 24 hours, and the results are shown in FIG. 1.

5. Epidermal tissue cell fusion: then replacing serum-free culture medium solution every 24h until epidermal tissue cells are fused, taking out a small amount of cell fluid after the cells are fused, carrying out DOPA staining, and observing the content of melanocytes, wherein the results are shown in figures 2-3.

6. Epidermal tissue cell stratification: after the epidermal histiocyte is fused, the serum-free culture medium solution is continuously replaced every 24h for culture, the epidermal histiocyte is layered after 7 days of culture, the result is shown in figure 5, a small amount of cell basal layer is taken for DOPA staining, the proportion of melanocyte and keratinocyte is verified, and the result is shown in figure 4.

7. Epidermal tissue and plate separation: the plate was transferred to an incubator at 30 ℃ and after 2 hours, the skin pieces were separated from the bottom of the plate and observed, and the results are shown in FIGS. 6 to 8.

Among them, umbilical cord mesenchymal cells have the following advantages: the source is rich; secondly, autologous fibroblasts of the patient do not need to be collected, and secondary or even tertiary damage to the patient is avoided, so that when epidermis is cultured, only the epidermis layer of the patient needs to be taken, no suture is needed, and no scar is left; the stable heredity, the stable heredity function of the umbilical cord mesenchymal stem cells without establishing a line, and the stable quality guarantee of each batch of the produced cells can be ensured; the umbilical cord mesenchymal stem cells have high safety and no immunogenicity, can be directly injected into veins in the treatment of various diseases at present, and have more obvious safety than animal-derived and xenogenous-derived trophoblast cells; umbilical cord mesenchymal cells can promote wound healing, and the tissue engineering epidermis manufactured by taking the umbilical cord mesenchymal cells as a trophoblast has better wound healing effect; and the epidermis cultured by taking the umbilical cord mesenchymal stem cells as a trophoblast contains melanocytes with normal proportion, so that a complete tissue engineering epidermis with pigments can be constructed without additionally adding melanocytes.

The temperature-sensitive hydrogel is a segmented copolymer of poly-N-isopropylacrylamide and polyethylene glycol, is in a liquid state at low temperature, is condensed at high temperature, and the change of the state is reversible along with the temperature. When the cells are cultured, the cells adhered to the wall can be suspended by changing the temperature without enzyme secondary digestion and damaging the interaction among the cells. So that the skin piece can be completely taken down from the bottom of the culture dish.

Comparative experiment 1

1. Pretreatment of epidermal tissue 1: cleaning normal epidermal skin 2cm x 2cm with DPBS containing antibiotic for several times, cutting into small pieces of about 5mm, adding 10ml of Dispase II separating enzyme, and digesting at 4 deg.C for 17 hr to obtain cultured autologous fibroblasts.

2. Plating trophoblast cells: preparing temperature sensitive hydrogel on a culture plate for 24h, and taking cultured autologous fibroblasts as trophoblast cells according to the ratio of 2 x 10⁴/cm²And pre-inoculating the culture plate prepared by the temperature-sensitive hydrogel.

4. Inoculating epidermal tissue cells: the epidermal tissue cells obtained in step 3 are expressed as 4 x 10⁴/cm²The cell amount of (2) was inoculated on autologous fibroblasts, and observed after adding 10ml of serum-free medium to culture for 24 hours, and the results are shown in the figure.

5. Epidermal tissue cell fusion: serum-free medium solution was replaced every 24h thereafter until epidermal tissue cells were fused and the melanocyte content was observed, and the results are shown in the figure.

6. Epidermal tissue cell stratification: dividing the fused epidermal tissues in the step 7 into A, B groups, wherein the epidermal tissues of the group A are continuously cultured by replacing serum-free culture medium solution every 24 hours, observing the layering condition of epidermal tissue cells, and finding that the epidermal tissue cells cannot be layered, thus knowing that the culture fails. And the epidermal tissue of the group B is continuously cultured by replacing the differentiation medium, the differentiation medium solution is replaced every 24 hours, and the epidermal tissue cells are layered after 7 days of culture, and the result is shown in the figure.

7. Epidermal tissue and plate separation: the plate was transferred to an incubator at 30 ℃ and after 2 hours, the separation of the epidermal tissue from the plate was observed, and the results are shown in the figure.

Comparative experiment 2

2. Plating trophoblast cells: 2 x 10 percent of cultured autologous fibroblasts serving as trophoblast cells⁴/cm²And pre-inoculated onto a culture plate.

4. Inoculating epidermal tissue cells: the epidermal tissue cells obtained in step 3 are expressed as 4 x 10⁴/cm²The cell amount of (2) was inoculated on autologous fibroblasts, and the cells were cultured for 24 hours in 10ml of serum-free medium, and the results are shown in FIG. 1.

5. Epidermal tissue cell fusion: after that, the medium solution was changed every 24 hours until the epidermal tissue cells were fused, and the melanocyte content was observed, as shown in FIG. 9.

6. Epidermal tissue cell stratification: and (3) replacing the differentiation medium solution after the epidermal tissue cells are fused, replacing the differentiation medium solution every 24 hours for culture, and layering the epidermal tissue cells after 7 days of culture, wherein the results are shown in the figure.

7. Epidermal tissue and plate separation: the plate obtained in step 6 was transferred to an incubator at 30 ℃ and cultured while dividing the plate into C, D groups, group C was cultured with Dispase II isolation enzyme, group D was cultured without any substance, and the separation of epidermal tissue and the plate was observed after 2h and 24h of culture, respectively, as shown in the figure.

Results and analysis

1. As shown in fig. 1, the cells in the examples adhere well, and the keratinocytes melanocytes are distributed among the trophoblast cells. As shown in fig. 8 and 14, the cell-crawl for comparative experiment 1 and comparative experiment 2, respectively, and the cell-crawl results for comparative experiments 1 and 2 are similar: the cell adherence rate is low, and no black cell adheres to the wall.

2. As shown in FIG. 2, the keratinocytes of the example were fused into pieces, the trophoblasts were apoptotic, and the melanocytes were interspersed among the keratinocytes. As shown in fig. 3, the dendritic melanocytes were seen by DOPA staining with healthy morphology and intact ratio, and it was confirmed from fig. 4 that the ratio of the melanocytes to the keratinocytes was 1:12, which is close to the normal ratio of human epidermis. As shown in fig. 9 and 15, on the other hand, neither of comparative experiment 1 and comparative experiment 2 showed melanocytes after cell fusion. Also, the DOPA staining patterns of FIGS. 10 and 16 demonstrate that epidermal tissues cultured in comparative experiment 1 and comparative experiment 2 do not contain melanocytes.

3. As shown in FIG. 5, the cells of the examples were tightly connected, and the epidermal cells keratinized, thickened and clearly layered. In the group A in the comparative experiment 1, because a differentiation medium solution is not used, epidermal tissue cells cannot be layered and extruded to die; group B uses differentiation medium solution, as shown in FIG. 11, epidermal tissue cells are obviously layered and densely linked; the epidermal cells in comparative experiment 2 are also obviously layered and densely linked. As shown in fig. 6-7, the epidermis was completely separated from the bottom of the dish, the epidermis flap was structurally intact, stratified, and not damaged, and HE staining indicated that the in vitro constructed artificial epidermis flap had a layered structure with intact human normal epidermis. As shown in FIGS. 12-13, the epidermal tissue of group B in comparative experiment 1 could be completely separated from the bottom of the culture dish, and the epidermal cells were clearly layered with a clear basal layer. In comparative experiment 1, the epidermis of group C could not be separated from the culture dish after 2h, and the epidermis of group C could be separated from the culture dish after 24h, and the cells of group C epidermal sheets were remarkably layered and had a remarkable basal layer as shown in FIG. 17, while group D could not be completely separated from the culture dish.

In order to further reduce the influence of the culture method on the cells and improve the culture efficiency, the invention also provides a serum-free culture medium which comprises a basal culture medium, a growth component, a calcium secretion component, a serum substitute and an enzyme inhibition component. The basic culture medium is mainly prepared by mixing L-DMEM and F12, and each component comprises the following factors in concentration in the basic culture medium respectively:

growth components: insulin 5ug/ml, cholera toxin 0.1ng/ml, hydrocortisone 0.4ug/ml, EGF10 ng/ml;

calcium secreting component: dexamethasone 4ug/ml, Vc 50ug/ml, beta-glycerophosphate 0.2 mg/ml;

serum replacement: elite gro 5%;

enzyme inhibiting component: y2763220 ng/ml.

Insulin is used for promoting cell growth, maintaining cell morphology, and preventing rapid aging of epidermal cells; cholera toxin is used for stimulating the attachment and growth of melanocytes; hydrocortisone is used for promoting the growth of epidermal cells; EGF can promote the growth of epidermal cells and black malignant cells.

Vitamin C, dexamethasone and beta-glycerophosphate can stimulate umbilical cord mesenchymal cells to secrete calcium ions, so that the calcium ions in the early stage of culture are in a non-or low-concentration state, and the concentration gradually rises along with the culture time, so that the calcium ions in the later stage of culture are in a high-concentration state, and the mature differentiation of epidermal cells is promoted.

The action of elite gro in place of serum was used to prepare serum-free media.

y27632 is a ROCK1 inhibitor, reduces apoptosis of cells and improves cloning efficiency.

The serum-free culture medium mixed with the various component factors and in reasonable proportion promotes the growth of epidermal tissue cells and solves the safety problem of animal serum. The culture method of the autologous tissue epidermis combines the serum-free culture medium to overcome the defect that the traditional culture method can not differentiate the cells, does not need to replace the differentiation culture medium, and reduces the consumption of manpower, financial resources and time.

The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make several improvements and modifications without departing from the technical principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention.

Claims

1. A culture method of autologous tissue engineering epidermis is characterized in that: the method comprises the following steps: 1) the epidermal tissue cells are expressed by 4 × 10⁴/cm²Inoculation ofCulturing on umbilical cord mesenchymal cells, 2) using a serum-free culture medium, and replacing every 24h until epidermal tissue cells are layered to obtain a tissue engineering epidermis;

the culture method uses umbilical cord mesenchymal cells as trophoblast cells;

the serum-free culture medium comprises a basic culture medium, a growth component, a calcium secretion component, a serum substitute and an enzyme inhibition component;

the calcium secretion component comprises dexamethasone 4 mu g/ml, Vc 50ug/ml and beta-glycerophosphate 0.2mg/ml, and can stimulate umbilical cord mesenchymal cells to secrete calcium ions.

2. The method for culturing the autologous tissue engineering epidermis of claim 1, wherein the culture medium comprises: the method comprises the step 1) of pre-inoculating umbilical cord mesenchymal cells to a culture plate coated with temperature-sensitive hydrogel before the step.

3. The method for culturing the autologous tissue engineering epidermis of claim 1, wherein the culture medium comprises: the serum-free culture medium in the step 2) needs to be replaced every 24 hours.

4. The method for culturing the autologous tissue engineering epidermis of claim 1, wherein the culture medium comprises: the growth components include insulin, cholera toxin, hydrocortisone and EGF.

5. The method for culturing the autologous tissue engineering epidermis of claim 1, wherein the culture medium comprises: the enzyme inhibitor is a ROCK1 inhibitor.