CN117757719A - Micro-tissue for accelerating skin injury repair and application thereof - Google Patents

Abstract

The invention belongs to the field of biological medicine, and relates to a micro-tissue for accelerating skin injury repair and application thereof. In particular, the present invention provides a method for preparing micro tissues (microtissues) for repairing skin lesions, which comprises the steps of loading epidermal stem cells on microcarriers (3D microcarriers,MC) and performing a spin culture. Preferably, the spin culture is performed at a speed of 15 rpm after days 1-3, 4-7, 10 rpm. The micro-tissue has a repairing function on skin injury, and is particularly superior to other treatment methods in repairing the full-thickness defect of the skin.

Description

Micro-tissue for accelerating skin injury repair and application thereof

Technical Field

The invention belongs to the field of biological medicine, and relates to a micro-tissue for accelerating skin injury repair and application thereof.

Background

The skin is used as the organ with the largest area of the human body, has the functions of blocking foreign matters and pathogens from entering, preventing body fluid from losing and the like, and is the first barrier for directly resisting external damage of the body. Severe wounds, burns, diabetic refractory skin ulcers, tumor resection procedures, and the like often result in extensive defects of skin tissue. The most mild wounds are limited to the skin epidermis, with skin and subcutaneous tissue breaks in the slightly heavier individuals, and wounds appear; severe trauma may be a break in muscles, muscle bonds, nerves, and fractures. The mild wounds can be healed through epithelial regeneration, the last two wounds can cause the appearance form and histopathology of normal skin tissues to change to form scars, the scars grow beyond a certain limit, various complications such as the damage of the appearance, dysfunction and the like can occur, and huge physical and mental pains, especially scars left after burns, scalds and severe trauma are brought to patients. Although research and application of tissue engineering skin have been advanced, some skin grafts, skin substitutes, etc. have been used clinically, but none of them meets the needs of patients with severe skin defects well.

Epidermal stem cells (Epidermal stem cells, epSCs) are a type of pluripotent cell that address the formation and differentiation of functional epidermis. As the role of EpSCs in wound healing and tissue regeneration has become of increasing interest to researchers, the number of EpSCs-based therapies is increasing. The improvement of the isolated culture method of EpSCs is of great importance for the clinical application thereof.

Traditional static cell cultures are performed in culture flasks or dishes. Whether cells or tissues are grown in a two-dimensional planar space and contact a glass or plastic surface. Such a manner affects the expression of genes in cells and does not continue to grow and differentiate. A three-dimensional (3D) cell culture model is a system in which a carrier having a 3D structure is co-cultured with different types of cells in vitro to simulate an in vivo microenvironment. This novel cell culture model has been shown to be similar to the natural system in vivo, producing a biological response in the processes of cell attachment, migration, mitosis and apoptosis that is different from that of monolayer cell culture. The rotational culture system (Rotary Cell Culture System, RCCS) is a rotationally dynamic, suspended culture technique for adherent or suspended cell culture in the laboratory. The system provides a reproducible, complex 3D in vitro culture solution for use in conducting research into structural processes and regulatory molecules that control normal tissue differentiation and tumor tissue transformation.

Disclosure of Invention

To provide a more effective method for treating skin lesions, the present invention provides a microstructure (microclasses) formed by self-assembly of epidermal stem cells with 3D Microcarriers (MC) and uses thereof, the self-assembly occurring in a rotating culture system (Rotary Cell Culture System, RCCS).

In a first aspect, the present invention provides a method for preparing micro-tissues (microtissues) for repairing skin lesions, said method comprising the steps of loading epidermal stem cells on microcarriers (3D microcarriers,MC) and performing a spin culture.

In particular, the skin damage refers to a damaged state of skin structure, including closed wounds or open wounds.

Preferably, the skin injury comprises an abrasion, a cut, a tear, a blister or a pressure ulcer.

More specifically, the skin injury is a skin defect.

Preferably, the spin culture is performed at a speed of 15 rpm after days 1-3, 4-7, 10 rpm.

Preferably, the rotational culture lasts for 14 days or more in total.

Specifically, a person skilled in the art can select an appropriate reaction system according to the target yield and the specific condition of the culture apparatus, and each reaction system can load a single microcarrier or load a plurality of microcarriers in one reaction system at the same time.

Preferably, the culture system of the rotary culture is about 5-20mL.

Preferably, the culture system of the rotary culture is about 10mL.

Specifically, the rotation culture is performed in a rotation culture system (microgravity rotation cell culture system, rotary Cell Culture System, RCCS); more specifically, in the synthetic HARVs system.

Preferably, any medium suitable for culturing epidermal stem cells, preferably a serum-free medium, including commercially available serum-free medium and self-contained serum-free medium, may be used in the culture. Specific examples of the medium include: minimal essential medium (MinimumEssential Medium, MEM), dulbecco's Modified Eagle's Medium (DMEM), hami F12 medium (Ham's F medium), basal medium Eagle (Basal Medium Eagle, BME), iscove's Modified Dulbecco's Medium (IMDM), roswell park souvenir college medium (Roswell Park Memorial Institute medium, RPMI), defined keratinocyte serum-free medium (Defined Keratinocyte-SFM, DKFM), and the like, and mixtures thereof.

Most preferably, KGM-gold medium (keratinocyte medium) is used in the specific examples of the invention.

Preferably, the medium is changed every 2-3 days.

Preferably, the microcarrier is a solid.

Preferably, the microcarrier is a tablet.

Preferably, the microcarrier forms a porous structure of 100-300 μm when exposed to liquid (FIG. 3 BC).

Preferably, the microcarrier has an effective growth area>9000cm ² /g

Preferably, the microcarrier porosity is >90%.

Preferably, the microcarriers are present in a dose of 20mg per tablet.

Preferably, the microcarrier is biodegradable.

Preferably, the epidermal stem cells are present in an amount of 2.5X10 ⁶ -5×10 ⁶ A dose of 20mg microcarrier (250-500 tens of thousands/20 mg microcarrier) was loaded on the microcarrier.

Preferably, the epidermal stem cells are present in an amount of 2.5X10 ⁶ A dose of 20mg of microcarrier was loaded on the microcarrier.

Preferably, the microcarrier is a 3D TableTrix.

Preferably, the loading is for about 24 hours.

More specifically, the loading operation steps are: after 2 hours of incubation, the culture was continued with the addition of complete medium, and transferred to a 10mL RCCS culture vessel for 24 hours for rotation.

Preferably, the epidermal stem cells can be isolated from skin samples discarded after circumcision, can be induced to differentiate from stem cells, and can be commercial epidermal stem cell lines.

Preferably, the stem cells are classified into embryonic stem cells and adult stem cells according to the developmental stage in which they are located. Pluripotent stem cells and multipotent stem cells can be classified according to their developmental potential. According to the function of the adult stem cells, the adult stem cells can be classified into one or more of bone marrow mesenchymal stem cells, hematopoietic stem cells, skin stem cells, mesenchymal stem cells, muscle stem cells, liver stem cells and neural stem cells.

The term "epidermal stem cell" as used herein means a cell population which has an unlimited proliferation potential, self-renews by symmetrical or asymmetrical division and continuously generates functional cells to maintain epidermal homeostasis, has morphologically characteristics of undifferentiated cells, has a small cell volume, large nucleus and less cytoplasm, large nuclear plasma ratio, low intracellular RNA content, less organelles and is immature, and is relatively fixed in position in a tissue structure.

In another aspect, the invention provides a microstructure prepared by the above method.

Experimental results in the specific embodiment of the invention show that compared with the direct transplantation of the epidermal stem cells, the transplanted micro tissue can store more cells and form a complete epithelial structure more quickly; that is, the micro-tissue prepared by the method has a significantly better therapeutic effect on repairing skin injury and promoting wound healing than the transplanted epidermal stem cells.

Preferably, the micro-tissue preparation may be preserved in a liquid.

In particular, the liquid comprises a physiologically acceptable sterile aqueous or anhydrous solution; more specifically, phosphate buffered saline (English: phosphate buffered saline, abbreviated as PBS), physiological saline used in clinic, and the like.

In another aspect, the invention provides the use of microcarriers (3D microcarriers,MC), micro tissues (microtissues) obtained by loading epidermal stem cells on microcarriers for repairing skin lesions, promoting wound healing.

Preferably, the skin injury comprises an abrasion, a cut, a tear, a blister or a pressure ulcer.

More specifically, the skin injury is a skin defect.

More preferably, the microstructure is prepared by the aforementioned preparation method.

Preferably, the microtissue is prepared for administration as an injection.

Preferably, the injection comprises, for example, a physiologically acceptable sterile aqueous or anhydrous solution; more specifically, phosphate buffered saline and saline used in clinic.

Preferably, the micro-tissue may be administered to the subject by a route of subcutaneous administration, transdermal administration, intradermal administration, subdermal administration.

Preferably, the micro-tissue is subcutaneously injected at the center or edge of a skin wound (repair target). The skin wound is not limited to skin where it can be applied to various body parts.

Preferably, the microcarrier is a solid.

Preferably, the microcarrier is a tablet.

Preferably, the microcarrier forms a porous structure of 100-300 μm when exposed to liquid.

Preferably, the microcarrier is a 3D TableTrix.

In another aspect, the present invention provides a method of using the micro-tissue prepared by the above method, i.e., a method of repairing and/or preventing skin damage, the method comprising: the aforementioned micro-tissue is applied to a subject with skin damage.

Preferably, the mode of administration is subcutaneous injection at the center or edge of the skin wound. The skin wound is not limited to skin where it can be applied to various body parts.

The term "subject" as used herein refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that will become the recipient of a particular treatment. In general, the terms "subject" and "patient" are used interchangeably herein when referring to a human subject.

Preferably, the subject is a human.

Preferably, the microtissue is administered in a pharmaceutically effective amount. For the purposes of the present invention, a "pharmaceutically effective amount" refers to an amount sufficient to treat a disease in a reasonable benefit/risk ratio for medical treatment, and the effective amount standard may be determined based on factors including the type of disease, the severity of the disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and rate of expulsion, the time of treatment, the concurrent use of the drug, and other factors well known in the medical arts. The micro-tissue according to the present invention may be administered alone as an individual therapeutic agent or concurrently with other therapeutic agents, sequentially or simultaneously with existing therapeutic agents, and may be administered singly or multiply.

The optimal cellular therapeutic agent content can be easily determined by those skilled in the art and can be adjusted according to various factors such as the kind of disease, the degree of disease, the content of other components contained in the composition, the kind of dosage form and the age, weight, general health condition, sex and diet of the patient, the administration time, the administration route and secretion rate of the composition, the treatment period, and the simultaneous use of the drugs. It is important that the elements are taken into consideration entirely, and that the amount in which the maximum effect can be obtained in the smallest amount without side effects is contained.

In particular, the effective amount may be at least 10 ⁵ At least 10 ⁶ At least 10 ⁷ At least 10 ⁸ At least 10 ⁹ Or more cells.

The beneficial effects are that:

the invention utilizes a microgravity bioreactor (RCCS) and a GMP-grade biodegradable microcarrier to carry out 3D suspension amplification culture on epidermal stem cells, and prepares the micro-tissue which has a repairing function on skin injury, and is particularly obviously superior to other treatment methods in repairing the full-thickness defect of the skin.

Drawings

FIG. 1 is a graph showing the results of examination of EpSCs obtained by the preparation.

FIG. 2 is a graph comparing the results of cell activity assays of EpSCs in 3D and 2D planar cultures.

FIG. 3 is a graph showing the results of in vitro static 3D culture.

FIG. 4 is a graph showing the results of in vitro 3D dynamic culture.

FIG. 5 is a graph showing the results of dynamic culture and static culture.

FIG. 6 is a graph at 5X 10 ⁶ A graph of the results of comparison of dynamic and static culture at cell concentration.

FIG. 7 is a graph showing the results of cell migration ability.

FIG. 8 shows the cell morphology of RCCS cultured micro-tissue scanned by a scanning electron microscope.

FIG. 9 shows the detection results of markers in RCCS cultured micro-tissues.

Fig. 10 is a graph of results of verifying skin repair effects in a full-loss skin defect model.

FIG. 11 is a graph of statistical results of the epithelial thickness in each experimental group.

FIG. 12 is a graph showing the results of statistics of the collagen content in each experimental group.

Detailed Description

The present invention will be further described with reference to specific embodiments, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 isolation and planar culture of epidermal Stem cells

Cell treatment method: skin samples discarded after adult circumcision were taken, incubated overnight at 4℃with 0.5% protease II (17105041, gibco), the epidermis pieces were separated from the dermis, and gently digested with 0.25% trypsin at 37℃for 8-10 min to form single cells. Subsequently, the single cell suspension was filtered with a 70 μm filter (15-1070, biologic), centrifuged at 1000rpm for 5 minutes and resuspended in complete KGM-gold medium (00192060, lonza). 5X 10 ⁶ Cells were seeded in T75 flasks coated with 100. Mu.g/mL type IV collagen (C5533, sigma) and adhered at 37℃for 10 minutes. After discarding non-adherent cells, culturing in KGM medium, changing medium every 2 days, culturing at 37deg.C with 5% CO ₂ Is cultured in an incubator of (a). When confluence reaches 70-80%, primary cells are passaged by 0.25% trypsin, and the passaged 1-3 generation cells are subjected to subsequent experiments.

The study has been approved by the ethics committee of the general hospital of the release army and informed consent was obtained from the patients.

Cells were observed daily for growth and morphological changes and photographed under an inverted phase contrast microscope (Nikon, TI2-U, japan) on days 1, 3, 5, 7. The cell image is shown in FIG. 1A, and the cells are small and bright and have the same characteristics as the characteristics of the epidermal stem cells.

Flow cytometry analysis showed positive expression rates of CK19 and integrin- β1 were 97.3% and 87.4%, respectively (fig. 1B), indicating strong expression of the EpSCs-specific markers in EpSCs. Immunofluorescence results showed that almost all isolated cells expressed high levels of CK19, integrin- β1 and p63 proteins (fig. 1C).

The results show that the high-purity EpSCs are prepared and can be used for subsequent experiments.

Example 2, 3D Static Culture (SC)

To determine the optimal cell seeding number, 300. Mu.L of cells were plated in 6-well plates according to 1.25X10 ⁶ ，2.5×10 ⁶ ，5×10 ⁶ ，1×10 ⁷ ，2×10 ⁷ Cell concentration of cells/sheet seeded in 3DMicrocarriers (W01-200, beijing Hua niche Biotechnology Co., ltd., 20 mg/tablet). Subsequently, 10mL of fresh complete medium was added to each well, with no agitation, except for microcarrier cell samples. Half of the medium was changed every 2 days. To prevent evaporation of the medium and to ensure a moist environment, PBS or deionized water was added to the compartment.

No significant difference in cell activity was seen between 3D Static Culture (SC) and 2D planar culture (FIG. 2), indicating 3DThe microcarrier has good compatibility with the epidermal stem cells.

A schematic diagram of in vitro static 3D culture is shown in FIG. 3A, 3DThe microcarriers rapidly expand to a porous shape within a few seconds after encountering the liquid, with a diameter of 100-300 μm (FIGS. 3B and C).

The results on days 1, 3 and 7 of the culture are shown in FIG. 3D. With the increase in cell number, the cell density is high, living cells are easy to aggregate, and the number of dead cells tends to increase. Cells were at 1.25X10 due to cell density rarefaction and lack of cell attachment ⁶ Almost no growth was possible at the cell concentration. With the cultivation, 2.5X10 ⁶ Cells and 5X 10 ⁶ Cells proliferate gradually, 1X 10 ⁷ Cells and 2X 10 ⁷ The cells have no tendency to proliferate. Over 24 hours, 2.5X10 ⁶ And 5X 10 ⁶ Of cellsThe cell attachment rate was higher than that of the other three groups, and the cell attachment rate (%) = (attached cells)/(initial seeding density) ×100 (fig. 3E).

At day 7, only 2.5X10 ⁶ Cells and 5X 10 ⁶ The cells were in an upward trend, indicating 2.5X10 ⁶ Cells and 5X 10 ⁶ Cells are the optimal concentration for cell proliferation.

Example 3 in vitro 3D dynamic culture by rotating culture

300. Mu.L of EpSCs cell concentrate was concentrated at 2.5X10 in a non-TC 6 well plate ⁶ cells/slice and 5×10 ⁶ concentration of cells/plate was inoculated in 3DMicrocarriers (W01-200, beijing Hua niche Biotechnology Co., ltd., 20 mg/piece), PBS or deionized water was added to the space of the well plate, and after incubation in an incubator for 2 hours, complete medium was added to 5 mL/well, and after 24 hours, the mixture was gently transferred to a 10mL RCCS culture vessel (HARVs, synthesis, U.S. A, FIG. 4A) and 10mL complete medium (KGM-gold medium) was added for cultivation.

The culture was incubated for 4 days at 5 rpm for the first three days, followed by changing the rotation rate to 10 rpm, and then increasing the rotation rate to 15 rpm until the incubation was completed on day 14, with medium being changed every 2-3 days.

After 14 days of culture, the extracellular matrix self-assembled to form clear three-dimensional cell aggregates was observed visually (fig. 4B). Further comparing 2.5X10 under RCCS dynamic culture with Static Culture (SC) cells as control ⁶ Cells and 5X 10 ⁶ Proliferation of cells. Inoculation of 2.5X10 ⁶ Cells showed higher PD (Population doubling, population doubling level) and faster PDT (population doubling time ) at days 7, 10 and 14, whereas 5X 10 ⁶ Cells were superior to static cultures in PDT (FIGS. 4C-D). RCCS dynamic culture is far from ultra-static culture whether PD or PDT.

Where N is the cell count, N0 is the cell count at seeding, t is the incubation time (hours), and Nt is the cell count at harvesting.

Cell status and proliferation of the micro-tissues in RCCS dynamic and static cultures were examined using live/dead staining (live/dead fluorescence images). The results show that from day 7, cell aggregation in RCCS appears as 3D multicellular spheroids, i.e. micro-tissues, whereas cell aggregation in SC is less pronounced. The higher the cell concentration in RCCS culture, the more and earlier the cell aggregation, but no greater cell aggregation was formed (fig. 5A). At inoculation of 5X 10 ⁶ In the dynamic cell culture group, the OD value and the cell attachment rate of the cells reach a peak value on day 10 and drop on day 14; while at inoculation of 2.5X10 ⁶ In the dynamic cell culture group, the OD value and the cell attachment rate of the cells were increased (FIGS. 5B and C), indicating that the inoculation was 2.5X10 ⁶ Individual cells are the optimal concentration for cell proliferation. The cell attachment rate in RCCS dynamic culture was significantly higher than that in static culture at this inoculation number (FIG. 5D).

Furthermore, at 5X 10 ⁶ At cell concentrations, the cell attachment rate and OD values of RCCS cultured cells were also consistently greater than static cultures (fig. 6). After RCCS culture, the cell microstructures were placed in a planar culture plate for culture, and cells were able to migrate to the culture plate, indicating that cells had good cell migration ability (fig. 7).

In summary, when the cell inoculum size was 2.5X10 ⁶ When cells are cultured in a rotating manner, the cells can be promoted to proliferate and induce spontaneous formation of larger micro-tissues.

Example 4 characterization and characterization of the rotating cultured micro-tissue

To further characterize the apparent appearance of the cell aggregates in the rotating cultures, live/dead fluorescence tests and scanning electron microscopy were performed. The results show that the 7 th day is opened under the dual actions of the rapid proliferation period of the cells and simulated microgravityInitial cell aggregation was gradually increased, day 14, 2.5X10 ⁶ The cells showed the greatest cell aggregation (fig. 5A and 8A). SEM images visually showed the morphology of cell adhesion and three-dimensional cell aggregation (fig. 8B). Monolayer flat cells initially adhere to MC (microcarriers) surfaces and pores; on day 7, 1 microcarrier was almost fully occupied, and 2 layers of cells appeared; many multilaminate cells appeared until day 10, more evident at day 14 (fig. 8B).

The process of culturing and forming the micro-tissue is illustrated in FIG. 9A. Laser confocal three-dimensional imaging of the microtissue was performed (fig. 9B). Compared with 2D culture, RCCS-induced expression of epidermal progenitor markers K5, K14, COL17A1, ki67 was significantly increased in the micro-tissues. The expression levels of K14, COL17A1 and Ki67 in RCCS were significantly higher than those in static culture. The expression of the epidermal stem cell markers ITGA6 and ITGB1 and the differentiation markers K10, K1 and ceiling element were not significantly different in RCCS and 2D culture. However, other genes in RCCS were significantly higher than SC except for K10 and INL (fig. 9C). The results of cellular immunofluorescence showed that epidermal stem/progenitor markers p63, CK19 and K5 were continuously expressed in large amounts, while differentiation marker K10 was rarely expressed (fig. 9D).

The above results demonstrate that RCCS-induced EpSCs retain their dryness after proliferation in micro-tissues, promoting their proliferation and differentiation potential.

Example 5 application of micro-tissue in skin wound model

To study the effect of EpSC-loaded 3D micro-tissue on total wound repair in vivo, a total skin defect model was constructed using 7-8 week old BALB/c nude mice. Animal experiments were performed according to standard guidelines approved by the animal ethics committee of the general hospital of the release army.

Mice were anesthetized with inhaled Isoflurane (INH) alone and their back skin was sterilized with 75% alcohol. Then, a full lesion wound having a diameter of 1 cm was formed on the back skin of each nude mouse. The wound edges were trimmed using iris scissors and the wound covered with a 3M tenaderm transparent dressing.

24 nude mice were randomly divided into 4 groups (n=6/group): sham surgery (PBS), epSCs, MC (microcarriers) and mc+epscs (micro-tissue).

200 μl of 1×10 was subcutaneously injected in the center and edge of wounds for EpSCs group and MC+EpSCs group ⁶ A cell; for the PBS and MC groups, 200. Mu.L of PBS solution and microcarriers dispersed in PBS were injected with a 1mL syringe and a 9-gauge needle, respectively, and all injection sites were evenly distributed.

The wounds of the mice were observed and measured daily, and each group of nude mice was sacrificed by day 14. The wound healing time was recorded and analyzed using image J, and the residual wound area ratio was calculated as (An/A0) ×100% (A0 is the initial wound area on day 0, an is the wound area on day N).

Wound healing was observed at 0, 3, 7, 10, 14 days after injection, respectively (fig. 10A). The difference of the residual wound surface areas of the treatment groups has statistical significance. The residual area of the wound surface of the MC group on days 3, 7 and 10 is obviously smaller than that of the other groups, and the residual area of the wound surface on day 14 is obviously smaller than that of the sham operation group. The EpSCs group was smaller than the MC group and the sham group on day 10 after wound surface. These results indicate that the EpSCs-loaded micro-tissue group significantly accelerated wound healing, especially on days 3 and 7 (fig. 10B).

After 14 days of healing, wound fresh tissue samples were fixed in 4% pfa for 24 hours and embedded in paraffin; prepared 4 μm thick paraffin-embedded sample sections were subjected to H & E and Masson staining. Sections were dehydrated, fixed, stained and imaged using a slide scanner. The results showed that each group restored the layered epithelial structure after complete wound closure, while the mc+epscs group had thicker epithelium than the other groups (fig. 10C-11). Meanwhile, masson staining detected that collagen fibers (collagen was blue) were more in the mc+epscs group than in the other groups, and that the defect repair site collagen content was increased in the mc+epscs group (fig. 10C, fig. 12).

The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.

Claims (10)

1. A method for preparing a micro-tissue for repairing skin damage, the method comprising the steps of loading epidermal stem cells on a microcarrier and performing a rotation culture;

preferably, the rotation culture is performed at a rotation speed of 15 rpm after 1-3 days 5 rpm, 4-7 days 10 rpm, and 8 days;

preferably, the rotational culture lasts for 14 days or more in total.

2. The method of claim 1, wherein the culture system of the rotary culture is about 5-20mL;

preferably, the culture system of the rotary culture is about 10mL;

preferably, a serum-free medium is used in the culture;

preferably, a keratinocyte growth medium is used in the culture;

preferably, the medium is changed every 2-3 days.

3. The method of claim 1, wherein the microcarrier is a solid;

preferably, the microcarrier is a tablet;

preferably, the microcarrier forms a porous structure of 100-300 μm when exposed to liquid;

preferably, the microcarriers are present in a dose of 20mg per tablet;

preferably, the microcarrier is a 3D TableTrix.

4. The method of claim 1, wherein the epidermal stem cells are present in an amount of 2.5X10 ⁶ -5×10 ⁶ A dose of 20mg microcarriers is loaded on the microcarriers;

preferably, the epidermal stem cells are present in an amount of 2.5X10 ⁶ A dose of 20mg of microcarrier was loaded on the microcarrier.

5. The method of claim 1, wherein the epidermal stem cells are isolated from a skin sample discarded after circumcision, or wherein the epidermal stem cells are induced to differentiate from stem cells, or wherein the epidermal stem cells are commercial epidermal stem cell lines.

6. The method of manufacture of claim 1, wherein the skin injury comprises an abrasion, a cut, a tear, a blister, or a pressure ulcer;

more specifically, the skin injury is a skin defect.

7. A microstructure prepared by the preparation method of claim 1;

preferably, the microstructure is preserved in a liquid;

preferably, the micro-tissue is preserved in phosphate buffered saline, saline used clinically.

8. Use of a microcarrier or a micro-tissue obtained by loading epidermal stem cells on a microcarrier for repairing skin lesions and promoting wound healing;

more preferably, the micro-tissue is the micro-tissue of claim 7.

9. The use of claim 8, wherein the micro-tissue is administered to the subject by a subcutaneous administration, a transdermal administration, an intradermal administration, a subdermal administration route;

preferably, the micro-tissue is subcutaneously injected at the center or edge of the repair target.

10. The use of claim 8, wherein the skin injury comprises an abrasion, a cut, a tear, a blister, or a pressure ulcer;

more specifically, the skin injury is a skin defect.