Organ-like microsphere / hydrogel compound for repairing skin injury and preparation method of organ-like microsphere / hydrogel compound

Abstract

The invention belongs to the field of stem cell and tissue engineering, and particularly provides an organoid microsphere-hydrogel compound and a preparation method thereof. The compound is prepared from organ-like microspheres and a hydrogel material, wherein the organ-like microspheres comprise mesenchymal stem cells and fibroblasts. The basic preparation method comprises the following steps: forming dense three-dimensional organ-like microspheres consisting of mesenchymal stem cells and fibroblasts on the surface of an ultra-low adsorption medium or in a shaking culture device according to a ratio of (1-10): (1-10), centrifuging at a low speed (300-500 revolutions for 3-5 minutes), and collecting the microspheres, thereby obtaining the three-dimensional organ-like microspheres. And mixing the organoid microsphere with a hydrogel material to form the organoid microsphere-hydrogel compound. The organoid microsphere-hydrogel compound prepared by the method has the characteristics of high cell viability and good skin regeneration and repair effect, and provides a new material for regeneration and repair of large-area or difficult-to-heal skin injury.

Description

Technical Field

[0001] The invention belongs to the field of stem cells and tissue engineering, and relates to an organoid microsphere / hydrogel complex for repairing skin damage and a preparation method thereof. Background Art

[0002] The skin is the largest organ in the human body and constitutes the body's first barrier to the external environment. Skin wound healing is a common problem, but large-area injuries, burns and non-healing (diabetic skin injuries) are still a problem. Therapeutic strategies based on stem cells or skin cell types have good potential to promote tissue regeneration and promote wound healing. Transplanting mesenchymal stem cells into the site of skin damage can accelerate wound healing. Further studies have shown that the combined transplantation of mesenchymal stem cells and fibroblasts into the injured site can not only promote wound healing, but also reduce scar formation. The above results show that this strategy has good application prospects in solving skin healing problems.

[0003] Since skin wounds lack an environment that can nourish transplanted cells, how to improve the survival and function of transplanted cells remains a key issue faced in the process of stem cell and fibroblast transplantation and repair. Existing methods mainly transplant single cell suspensions or complexes with biomaterials to the site of injury, and the implanted cells quickly die due to lack of nutrients. The patent "An injectable thermosensitive composite hydrogel containing adipose mesenchymal stem cells and its preparation method and application (CN202110295199.5)" provides a method for compounding adipose mesenchymal stem cells with hydrogel materials to provide them with a better environment, but its cell components are single.

[0004] Therefore, how to obtain a new material with skin damage repairing function to solve the problem of skin wound healing has become an urgent problem to be solved. Summary of the invention

[0005] In order to solve the above problems, the purpose of the present invention is to provide an organoid microsphere / hydrogel complex for skin damage repair, which can maintain the activity of mesenchymal stem cells and fibroblasts through the three-dimensional organoid microspheres formed by mesenchymal stem cells and fibroblasts and the organoid microsphere / hydrogel complex formed by the hydrogel material, thereby improving the wound healing effect.

[0006] In order to achieve the above-mentioned object, the present invention provides an organoid microsphere-hydrogel complex for skin damage repair, characterized in that it is made of organoid microspheres and hydrogel materials, and the organoid microspheres include mesenchymal stem cells and fibroblasts.

[0007] As an improvement, the ratio of the number of mesenchymal stem cells and fibroblasts in the organoid microspheres is (1-10):(1-10), the mesenchymal stem cells are any one of human umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, endometrial mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, and bone marrow mesenchymal stem cells; the fibroblasts include fibroblasts of human skin origin.

[0008] As a further improvement, the organoid microspheres are microspheres in which the mesenchymal stem cells and the fibroblasts are uniformly mixed; or the organoid microspheres are microspheres with the mesenchymal stem cells as the inner core and the fibroblasts as the outer core; or the organoid microspheres are microspheres with the fibroblasts as the inner core and the mesenchymal stem cells as the outer core.

[0009] As a further improvement, the ratio of organoid microsphere cell number to hydrogel material volume is: 0.1-5×10 7 The hydrogel material is a natural hydrogel material or a synthetic hydrogel material. The natural hydrogel material is any one of type I collagen, chitosan or fibrin glue. The synthetic hydrogel material includes polyvinyl alcohol.

[0010] Another aspect of the present invention provides a method for preparing an organoid microsphere-hydrogel complex, comprising the following steps:

[0011] Taking the organoid microspheres and the hydrogel solution made of the hydrogel material;

[0012] The organoid microspheres are centrifuged at 300-500 r / min for 3 minutes, the supernatant is discarded, the precipitated organoid microspheres are collected, and then resuspended with 100-300 μL of culture medium, and the resuspended organoid microspheres are added to 700-900 μL of hydrogel solution. After blowing and mixing, the hydrogel solution is converted into a non-flowing gel state to obtain an organoid microsphere-hydrogel complex.

[0013] As an improvement, the invention further comprises: the hydrogel solution comprises a natural hydrogel solution and a synthetic hydrogel solution;

[0014] Preparation of natural hydrogel solution: dissolving type I collagen in 0.1% acetic acid solution to obtain a collagen solution with a mass volume concentration of 0.1-1.5 mg / ml; or dissolving chitosan in 2% acetic acid solution to obtain a chitosan solution with a mass volume concentration of 1-3 mg / ml; or dissolving fibrin glue in water to obtain a fibrin glue solution with a concentration of 30-50 mg / ml;

[0015] Preparation of synthetic hydrogel solution: polyvinyl alcohol is dissolved in water to prepare a polyvinyl alcohol aqueous solution with a mass volume concentration of 5 to 10 mg / ml.

[0016] Another aspect of the present invention provides a method for preparing organoid microspheres, comprising the following steps:

[0017] The mesenchymal stem cells are prepared into a concentration of 0.5 to 1×10 7 / ml of mesenchymal stem cell single cell suspension, the fibroblasts are prepared to a concentration of 0.5 to 1×10 7 / ml single cell suspension of fibroblasts;

[0018] The single cell suspension of mesenchymal stem cells and the single cell suspension of fibroblasts are inoculated in a culture medium containing a culture medium in proportion, and then the culture medium is placed in a 5% CO2 incubator for culturing at 37°C for 3 to 5 days or the culture medium is placed in a shaking culture device and placed in a 5% CO2 incubator for culturing at 37°C for 3 to 5 days to form organoid microspheres mixed with mesenchymal stem cells and fibroblasts;

[0019] Or the single cell suspension of fibroblasts is inoculated into a culture medium containing culture medium, and cultured in a 5% CO2 incubator at 37°C for 3-5 days to form fibroblast microspheres, and then the single cell suspension of mesenchymal stem cells is added in proportion, and the culture medium is placed in a 5% CO2 incubator for conventional culture at 37°C or the culture medium is placed in a shaking culture device for shaking culture for 2-3 days to obtain organoid microspheres with fibroblasts as the inner core and mesenchymal stem cells as the outer layer;

[0020] Alternatively, a single cell suspension of mesenchymal stem cells is inoculated into a culture medium containing culture medium, and cultured in a 5% CO2 incubator at 37°C for 3 to 5 days to form mesenchymal stem cell microspheres, and then a single cell suspension of fibroblasts is added in proportion, and the culture is placed in a 5% CO2 incubator for conventional culture at 37°C, or the culture medium is placed in a shaking culture device for shaking culture for 2 to 3 days to obtain organoid microspheres with mesenchymal stem cells as the inner core and fibroblasts as the outer layer.

[0021] As a further improvement, the culture medium is any one of DMEM culture medium + 10% serum, DMEM / F12 culture medium + 10% serum or MEM + 10% serum.

[0022] As a further improvement, the culture dish includes any one of an ultra-low adsorption culture dish, an ultra-low adsorption culture bottle, an agarose-coated culture dish, an agarose-coated culture bottle or an ordinary culture dish, and the shaking culture device is a decolorizing shaker with a shaking frequency of 0.05 to 0.5 Hz.

[0023] On the other hand, the present invention provides a use of an organoid microsphere-hydrogel complex in preparing a material for repairing skin damage.

[0024] After forming microspheres, mesenchymal stem cells and fibroblasts can better maintain cell activity and secrete more cytokines. Compared with single cell suspensions, mesenchymal stem cells and fibroblasts have better therapeutic effects in forming organoids.

[0025] The hydrogel material provides a natural-like microenvironment for the organoid microspheres, enabling them to better play a repair role in this environment where the skin is fully exposed.

[0026] The beneficial effects of the present invention are:

[0027] The present invention provides an organoid microsphere / hydrogel complex for skin damage repair and a preparation method thereof, wherein mesenchymal stem cells and fibroblasts that can promote skin regeneration are formed into organoid microspheres, and the organoid microspheres are then combined with a hydrogel material to form an organoid / hydrogel complex, thereby effectively improving the viability and regeneration function of the cells, enhancing the effect of promoting the regeneration and repair of skin damage, and being significantly superior to existing complexes that promote skin regeneration in terms of structure and function. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 After 3-5 days of culture, organoid microspheres composed of umbilical cord mesenchymal stem cells and fibroblasts are formed;

[0029] Figure 2 Experimental results of organoid microsphere-hydrogel complex repairing rat skin injuries. DETAILED DESCRIPTION

[0030] The embodiments of the present invention will be described in detail and comprehensively below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0031] Cell microspheres are three-dimensional cell culture aggregates formed under specific conditions. When forming microspheres, mesenchymal stem cells secrete more key transcription factors HIF-1 (hypoxia-inducible factor), which enhances the synthesis of pro-activating proteins and the adaptability of cells; increases the secretion of growth factors VEGF, HGF, and FGF2, thereby enhancing their potential to promote angiogenesis; upregulates the expression of TSG-6 (TNFα-induced gene / protein 6), STC-1 (staniocalcin-1), and PGE2 (prostaglandin E2), and enhances anti-inflammatory and anti-apoptotic abilities. It has been reported in the literature that compared with single cell suspensions, mesenchymal stem cells transplanted in the form of microspheres have a higher survival rate and more significant therapeutic effects. Transplanting cells in the form of microspheres is expected to enhance their activity and function.

[0032] The present invention further improves the survival of transplanted cells and promotes the effect of wound healing by creating a stem cell and fibroblast three-dimensional microsphere / hydrogel complex that is closely related to skin regeneration.

[0033] Material:

[0034] 1. Type I collagen: Homemade. After removing the skin from the rat tail, obtain the white fascia, cut it into pieces, and dissolve it in 0.1% acetic acid solution to obtain type I collagen solution.

[0035] 2. Chitosan was purchased from Sigma-Aldrich (C3646) and dissolved in 1% acetic acid solution to obtain chitosan gel.

[0036] 3. Fibrin glue was purchased from Hualan Bioengineering Co., Ltd.

[0037] 4. T75 culture flasks were purchased from Sigma-Aldrich, with the catalog number Z707503. 5. Mesenchymal stem cell serum-free culture medium (StemPro TM MSC SFM) was purchased from Thermo Fisher Scientific, product number: A1033201.

[0038] 6. High-glucose DMEM (Advanced DMEM) was purchased from Thermo Fisher Scientific, product number: 0030034DJ.

[0039] 7. Human platelet lysate was purchased from Wuhan Jiecheng Yuheng Technology Co., Ltd., with the catalog number PLTGOLD500R. 8. DMEM was purchased from HyClone, with the catalog number SH30022.02.

[0040] 9. Serum was purchased from HyClone, SH30413.02.

[0041] An organoid microsphere-hydrogel complex for skin damage repair is made of organoid microspheres and hydrogel materials. The organoid microspheres include mesenchymal stem cells and fibroblasts. The ratio of the number of mesenchymal stem cells and fibroblasts in the organoid microspheres is (1-10): (1-10). The mesenchymal stem cells are any one of human umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, endometrial mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, and bone marrow mesenchymal stem cells; the fibroblasts include fibroblasts from human skin. The organoid microspheres are microspheres formed by a uniform mixture of the mesenchymal stem cells and the fibroblasts; or the organoid microspheres are microspheres with the mesenchymal stem cells as the core and the fibroblasts as the outer layer; or the organoid microspheres are microspheres with the fibroblasts as the core and the mesenchymal stem cells as the outer layer. The ratio of the number of organoid microspheres to the volume of hydrogel material is: 0.1-5×10 7 The hydrogel material is a natural hydrogel material or a synthetic hydrogel material. The natural hydrogel material is any one of type I collagen, chitosan or fibrin glue. The synthetic hydrogel material includes polyvinyl alcohol.

[0042] Example 1 Preparation of human umbilical cord mesenchymal stem cell suspension and single cell suspension of fibroblasts

[0043] Take the umbilical cord of a healthy perinatal fetus, obtain Wharton's jelly under sterile conditions, and cut it into 1-2 mm 3 The cells were inoculated into T75 culture flasks, and serum-free medium for mesenchymal stem cells was added for culture at 37°C and 5% CO2. When the cells grew to 80% confluence, they were subcultured and expanded to obtain single-cell suspension of umbilical cord mesenchymal stem cells.

[0044] Take plastic skin fragments and cut them into 1-2mm pieces 3 The size of the cells was inoculated into a T75 culture flask, and high-glucose DMEM and human platelet lysate were added for culture at 37°C and 5% CO2. When the cells grew to 80% confluence, they were subcultured and expanded to obtain a single-cell suspension of fibroblasts.

[0045] Example 2 Preparation of human umbilical cord mesenchymal stem cells and fibroblast organoid microspheres

[0046] The umbilical cord mesenchymal stem cell suspension cultured to passage 3-5 was mixed with the single cell suspension of fibroblasts at a ratio of 1:1, inoculated on an agarose-coated culture dish, the culture medium was DMEM supplemented with 10% serum, and cultured in an incubator at 37°C and 5% CO2. After 3-5 days of culture, organoid microspheres composed of two types of cells, umbilical cord mesenchymal stem cells and fibroblasts, were formed. Figure 1 shown.

[0047] Example 3 Preparation of human umbilical cord mesenchymal stem cells and fibroblast organoid microspheres

[0048] The single cell suspension of fibroblasts cultured to the 3rd to 5th generation is inoculated on an ultra-low adsorption culture flask, the culture medium is MEM + 10% serum, and the flask is cultured in a 37°C, 5% CO2 incubator. After 3-5 days of culture, fibroblast microspheres are formed; then, the single cell suspension of mesenchymal stem cells is added at a ratio of 10:1 between the number of fibroblasts and mesenchymal stem cells, and the flask is placed in a 5% CO2 incubator and cultured at 37°C for 2-3 days to obtain organoid microspheres with fibroblasts as the inner core and mesenchymal stem cells as the outer shell.

[0049] Example 4 Preparation of human umbilical cord mesenchymal stem cells and fibroblast organoid microspheres

[0050] The umbilical cord mesenchymal stem cell suspension cultured to the 3rd to 5th generation is inoculated on an ultra-low adsorption culture dish, the culture medium is DMEM / F12 culture medium + 10% serum, and cultured in a 37°C, 5% CO2 incubator. After 3-5 days of culture, mesenchymal stem cell microspheres are formed; then, a single cell suspension of fibroblasts is added at a ratio of 1:10 between the number of fibroblasts and the number of mesenchymal stem cells, and the dish is placed in a 5% CO2 incubator and cultured at 37°C for 2-3 days to obtain organoid microspheres with mesenchymal stem cells as the inner core and fibroblasts as the outer layer.

[0051] Example 5

[0052] The difference from Example 2 is that the cells were inoculated on agarose-coated culture flasks.

[0053] Example 6

[0054] The difference from Example 2 is that the ratio of the number of fibroblasts to the number of mesenchymal stem cells is 1:5.

[0055] Example 7

[0056] The difference from Example 2 is that the ratio of the number of fibroblasts to the number of mesenchymal stem cells is 5:1.

[0057] Example 8

[0058] Different from Example 2, the cells were inoculated in a common culture dish, which was then placed in a shaking culture device, and the ultra-low adsorption medium culture dish or the shaking culture device was then placed in a 5% CO2 incubator at 37°C for 3-5 days.

[0059] The shaking culture device is a decolorizing shaker with a shaking frequency of 0.05-0.5 Hz.

[0060] It should be noted that the preparation method of the agarose-coated culture dish or agarose-coated culture bottle is: agarose and water are prepared into a 1-2% agarose aqueous solution, after high temperature and high pressure sterilization, a 1-3 mm thick agarose layer is spread on the surface of the culture dish or culture bottle, and then cooled and solidified.

[0061] Example 9

[0062] Preparation of natural hydrogel solution: dissolving type I collagen in 0.1% by mass acetic acid solution to prepare a collagen solution with a mass volume concentration of 0.1 to 1.5 mg / ml;

[0063] Example 10

[0064] Preparation of natural hydrogel solution: dissolving chitosan in acetic acid solution with a mass percentage concentration of 2% to prepare a chitosan solution with a mass volume concentration of 1-3% mg / ml;

[0065] Embodiment 11

[0066] Preparation of natural hydrogel solution: dissolving fibrin glue in water to prepare a fibrin glue aqueous solution with a concentration of 30-50 mg / ml;

[0067] Example 12

[0068] Preparation of synthetic hydrogel solution: polyvinyl alcohol is dissolved in water to prepare a polyvinyl alcohol aqueous solution with a mass volume concentration of 5-10% mg / ml.

[0069] Example 13 Preparation of human umbilical cord mesenchymal stem cells, fibroblast organoid microspheres / collagen hydrogel complex

[0070] The organoid microspheres cultured in Example 1 were centrifuged at low speed (300 rpm for 3 minutes) and the supernatant was discarded. The precipitated organoid microspheres were collected and resuspended with 100 μL of culture medium. The resuspended organoid microspheres were added to 700 μL of hydrogel solution prepared in Example 9, mixed by pipetting, and then incubated in an incubator at 37° C. and 5% CO2 for 10 minutes to turn them into a gel to obtain an organoid microsphere-hydrogel complex.

[0071] Example 14 Preparation of human umbilical cord mesenchymal stem cells, fibroblast organoid microspheres / collagen hydrogel composites

[0072] The organoid microspheres cultured in Example 1 were centrifuged at low speed (400 rpm for 3 minutes) and the supernatant was discarded. The precipitated organoid microspheres were collected and resuspended with 200 μL of culture medium. The resuspended organoid microspheres were added to 800 μL of hydrogel solution prepared in Example 10, mixed by pipetting, and then incubated in an incubator at 37° C. and 5% CO2 for 10 minutes to turn them into a gel to obtain an organoid microsphere-hydrogel complex.

[0073] Example 15 Preparation of human umbilical cord mesenchymal stem cells, fibroblast organoid microspheres / collagen hydrogel complex

[0074] The organoid microspheres cultured in Example 1 were centrifuged at low speed (500 rpm for 3 minutes) and the supernatant was discarded. The precipitated organoid microspheres were collected and resuspended with 300 μL of culture medium. The resuspended organoid microspheres were added to 900 μL of hydrogel solution prepared in Example 11, mixed by pipetting, and then incubated in an incubator at 37° C. and 5% CO2 for 10 minutes to turn them into a gel to obtain the organoid microsphere-hydrogel complex.

[0075] Example 16 Preparation of human umbilical cord mesenchymal stem cells, fibroblast organoid microspheres / collagen hydrogel composites

[0076] Different from Example 15, the resuspended organoid microspheres were added to 900 μL of the hydrogel solution prepared in Example 12.

[0077] Comparative Example 1 Application publication number is CN 113384753 A, and its name is an injectable thermosensitive composite hydrogel containing adipose mesenchymal stem cells and its preparation method and application

[0078] S1. The chitosan powder was dissolved in a composite alkaline solution of 11wt% sodium hydroxide and 4wt% urea, and propylene oxide was added. The mixture was first reacted in an ice bath with mechanical stirring for 2h, then heated to 5°C for 24h, then heated to 15°C for 6h, and finally cooled to 4°C for 2h to prepare hydroxypropyl chitosan. A 2.5wt% hydroxypropyl chitosan solution was obtained by dialysis, freeze drying and dissolution.

[0079] S2. Mixing the hydroxypropyl chitosan solution with the collagen solution and the sodium hyaluronate solution to prepare a composite solution, wherein the concentration of the collagen solution is 1wt%, the concentration of the sodium hyaluronate solution is 1wt%, and the mass ratio of hydroxypropyl chitosan, collagen and sodium hyaluronate in the composite solution is 20:1:2.

[0080] S3. Resuspend the adipose-derived mesenchymal stem cells using the composite solution prepared in S2, and obtain an injectable thermosensitive composite solution containing adipose-derived mesenchymal stem cells under stirring conditions, wherein the cell concentration of the adipose-derived mesenchymal stem cells is 1×10 6 Pieces / mL.

[0081] S4. Add genipin to the injectable thermosensitive composite solution of S3, and then place it at 37°C to obtain an injectable thermosensitive composite hydrogel containing adipose mesenchymal stem cells, wherein the concentration of genipin in the obtained composite hydrogel is 0.02wt%.

[0082] Experimental example: organoid microsphere-hydrogel complex repairs rat skin damage

[0083] After depilating the back skin of rats, circular skin lesions of the same size were made on both sides. Hydrogel, stem cell microspheres, injectable thermosensitive composite hydrogel containing adipose mesenchymal stem cells prepared in Control Example 1, and organoid microsphere-hydrogel complex prepared in Example 13 were applied to the wounds, and a transparent bandage was applied to observe the wound repair and record the repair time.

[0084] Compared with the repair time of the control example 1 and the organoid microsphere-hydrogel complex prepared in Example 13, the organoid microsphere-hydrogel complex prepared in Example 13 only takes 10-12 days to repair skin damage and leaves no scars.

[0085] It can be seen from the above examples that the organoid microsphere / hydrogel complex for skin damage repair provided by the present invention and its preparation method are composed of mesenchymal stem cells, fibroblasts and hydrogel materials that can promote skin damage regeneration, wherein the mesenchymal stem cells and fibroblasts are first formed into three-dimensional organoid microspheres with a diameter of 100-500um, and then compounded with the hydrogel material to form an organoid microsphere / hydrogel complex. Figure 2 As shown, the complex effectively improves the viability and regeneration function of cells and enhances the effect of promoting the regeneration and repair of skin damage.

[0086] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An organoid microsphere-hydrogel complex for skin damage repair, characterized in that: The organoid microspheres are made of organoid microspheres and hydrogel materials, wherein the organoid microspheres include mesenchymal stem cells and fibroblasts.

2. The organoid microsphere-hydrogel complex according to claim 1, characterized in that The ratio of the number of mesenchymal stem cells and fibroblasts in the organoid microspheres is (1-10):(1-10), the mesenchymal stem cells are any one of human umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, endometrial mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, and bone marrow mesenchymal stem cells; the fibroblasts include fibroblasts from human skin.

3. The organoid microsphere-hydrogel complex according to claim 2, characterized in that: The organoid microspheres are microspheres in which the mesenchymal stem cells and the fibroblasts are uniformly mixed; or the organoid microspheres are microspheres with the mesenchymal stem cells as the inner core and the fibroblasts as the outer core; or the organoid microspheres are microspheres with the fibroblasts as the inner core and the mesenchymal stem cells as the outer core.

4. The organoid microsphere-hydrogel complex according to claim 1, characterized in that The ratio of organoid microsphere cell number to hydrogel material volume is: 0.1~5×10 7 The hydrogel material is a natural hydrogel material or a synthetic hydrogel material. The natural hydrogel material is any one of type I collagen, chitosan or fibrin glue. The synthetic hydrogel material includes polyvinyl alcohol.

5. A method for preparing the organoid microsphere-hydrogel complex according to any one of claims 1 to 4, characterized in that: The steps include: Taking the organoid microspheres and the hydrogel solution made of the hydrogel material; The organoid microspheres are centrifuged at 300-500 r / min for 3 minutes, the supernatant is discarded, the precipitated organoid microspheres are collected, and then resuspended with 100-300 μL of culture medium, and the resuspended organoid microspheres are added to 700-900 μL of hydrogel solution. After blowing and mixing, the hydrogel solution is converted into a non-flowing gel state to obtain an organoid microsphere-hydrogel complex.

6. A method for preparing the organoid microsphere-hydrogel complex according to claim 5, characterized in that: Also includes: The hydrogel solution includes a natural hydrogel solution and a synthetic hydrogel solution; Preparation of natural hydrogel solution: dissolving type I collagen in 0.1% acetic acid solution to obtain a collagen solution with a mass volume concentration of 0.1-1.5 mg / ml; or dissolving chitosan in 2% acetic acid solution to obtain a chitosan solution with a mass volume concentration of 1-3 mg / ml; or dissolving fibrin glue in water to obtain a fibrin glue solution with a concentration of 30-50 mg / ml; Preparation of synthetic hydrogel solution: polyvinyl alcohol is dissolved in water to prepare a polyvinyl alcohol aqueous solution with a mass volume concentration of 5 to 10 mg / ml.

7. A method for preparing organoid microspheres according to any one of claims 1 to 4, characterized in that: The steps include: The mesenchymal stem cells are prepared into a concentration of 0.5 to 1×10 7 / ml of mesenchymal stem cell single cell suspension, the fibroblasts are prepared to a concentration of 0.5 to 1×10 7 / ml single cell suspension of fibroblasts; The single cell suspension of mesenchymal stem cells and the single cell suspension of fibroblasts are inoculated in a culture medium containing a culture medium in proportion, and then the culture medium is placed in a 5% CO2 incubator for culturing at 37°C for 3 to 5 days or the culture medium is placed in a shaking culture device and placed in a 5% CO2 incubator for culturing at 37°C for 3 to 5 days to form organoid microspheres mixed with mesenchymal stem cells and fibroblasts; Or the single cell suspension of fibroblasts is inoculated into a culture medium containing culture medium, and cultured in a 5% CO2 incubator at 37°C for 3-5 days to form fibroblast microspheres, and then the single cell suspension of mesenchymal stem cells is added in proportion, and the culture medium is placed in a 5% CO2 incubator for conventional culture at 37°C or the culture medium is placed in a shaking culture device for shaking culture for 2-3 days to obtain organoid microspheres with fibroblasts as the inner core and mesenchymal stem cells as the outer layer; Alternatively, a single cell suspension of mesenchymal stem cells is inoculated into a culture medium containing culture medium, and cultured in a 5% CO2 incubator at 37°C for 3 to 5 days to form mesenchymal stem cell microspheres, and then a single cell suspension of fibroblasts is added in proportion, and the culture is placed in a 5% CO2 incubator for conventional culture at 37°C, or the culture medium is placed in a shaking culture device for shaking culture for 2 to 3 days to obtain organoid microspheres with mesenchymal stem cells as the inner core and fibroblasts as the outer layer.

8. The method for preparing organoid microspheres according to any one of claims 7, characterized in that: The culture medium is any one of DMEM culture medium + 10% serum, DMEM / F12 culture medium + 10% serum or MEM + 10% serum.

9. The method for preparing organoid microspheres according to any one of claims 7, characterized in that: The culture dish comprises any one of an ultra-low adsorption culture dish, an ultra-low adsorption culture bottle, an agarose-coated culture dish, an agarose-coated culture bottle or an ordinary culture dish; the shaking culture device is a decolorizing shaker with a shaking frequency of 0.05 to 0.5 Hz.

10. Use of the organoid microsphere-hydrogel complex according to any one of claims 1 to 6 in preparing materials for repairing skin damage.

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