Microcarrier, preparation method thereof and application of microcarrier in amplification of hair papilla cells

Abstract

The invention relates to the technical field of microcarriers, in particular to a microcarrier, a preparation method of the microcarrier and application of the microcarrier in amplification of hair papilla cells. The microcarrier is composed of calcium alginate microspheres and gelatin crosslinked on the surfaces of the calcium alginate microspheres; the calcium alginate gel is prepared from the following raw materials in parts by weight: calcium alginate microspheres and gelatin, and the weight part ratio of the calcium alginate microspheres to the gelatin is 81: (2000-4000). The porous microcarrier is prepared, the surface-to-body ratio of the porous microcarrier is large, a larger growth area is provided for cells, the cells preferentially enter the microspheres to grow, and then the cells are attached to the surfaces of the microspheres, so that higher cell density is achieved. Microcarrier culture simulates an in-vivo microenvironment for DP cell growth, and DP cells with hair follicle induction characteristics are amplified on a large scale and cannot be differentiated into other cells.

Description

Technical Field

[0001] The invention relates to the technical field of microcarriers, in particular to a microcarrier and a preparation method thereof and an application thereof in amplifying hair papilla cells. Background Art

[0002] At present, the treatment of hair loss mainly adopts two methods: drug therapy and autologous hair follicle transplantation. Although drugs have certain effects, they cannot promote the generation of new hair follicles, so the indicators are not a cure. Autologous hair follicle transplantation often requires a large number of hair papilla cells (DP) with hair follicle regeneration function, but the source of autologous human scalp hair papilla for transplantation in patients with severe hair loss is limited, so it is unlikely to obtain enough DP from patients for surgical transplantation in clinical practice.

[0003] Prior art CN 213113172 U discloses a process system for preparing microcarrier particles, which can be used to prepare microcarrier particles suitable for three-dimensional cell culture on a large scale at low temperature. However, the preparation process involves mixing and stirring the oil phase and the water phase. After the reaction is completed, the oil phase is difficult to remove, the cleaning process is cumbersome, and the droplet size is uneven, so the microcarrier yield after screening is only 30%.

[0004] Prior art CN 116656591 A discloses a microcarrier comprising calcium alginate microspheres, and chitosan and gelatin cross-linked on the surface of the calcium alginate microspheres; the curvature of the microcarrier is 1 / 1000 -1 / 100μm -1 Among them, chitosan can significantly improve the adhesion of the microsphere surface to cells. However, since the invention only considers increasing the adhesion of the microcarrier surface, the prepared microspheres are solid spheres and swell before inoculating cells, so the microcarriers cannot absorb more liquid. When adsorbing cells, the cells can only adhere to the surface of the microspheres, and the cell adsorption and growth area is small.

[0005] Prior art CN 109136168 B discloses a method for obtaining artificial dermal papilla (DP)-like cells, comprising the following steps: 1) preparing a mixed solution of sodium alginate and D-mannitol, filtering and sterilizing; 2) extracting human dermal fibroblasts and culturing them in vitro in large quantities; 3) obtaining sodium alginate microspheres encapsulating human dermal fibroblasts by electrostatic spraying; 4) treating the sodium alginate microspheres to obtain APA microspheres; 5) culturing the APA microspheres in vitro to induce human dermal fibroblasts into human dermal papilla-like cells. The invention uses sodium alginate microspheres to encapsulate human dermal fibroblasts to simulate the characteristics of DP cells that aggregate and grow in the human body. Through this method, human dermal fibroblasts are transdifferentiated into human dermal papilla cells, and a large number of human dermal papilla-like cells with the ability to induce hair production are obtained. However, the growth space of cells encapsulated in microspheres is limited, and the transportation of oxygen and nutrients to cells in microspheres and the simple diffusion of metabolic waste are difficult, resulting in low oxygen and nutrient concentrations inside the spheres, which is not conducive to DP proliferation, so the proliferation efficiency is very low.

[0006] The only method for expanding DP is the traditional plate culture method, which loses the ability to induce hair growth after several generations of culture. The reason is that the environment in which cells grow is quite different from the microenvironment in the body. The plate culture method is to culture DP on a plastic matrix, while DP grows on a rich ECM in the body. The culture scale of traditional culture dishes limits the efficient expansion of cells in the short term. Summary of the invention

[0007] The purpose of the present invention is to provide a microcarrier capable of large-scale amplification of DP cells, a preparation method thereof and an application thereof in amplifying dermal papilla cells.

[0008] In order to achieve the above object, the present invention adopts the following technical solution: A microcarrier is composed of calcium alginate microspheres and gelatin cross-linked on the surface of the calcium alginate microspheres; the raw materials include calcium alginate microspheres and gelatin, and the weight ratio of the calcium alginate microspheres to the gelatin is 81:2000-4000.

[0009] If the ratio is too low, the microcarrier will be too hard, which may change the biological behavior of DP cells and cause the microcarrier pore size to be too small, reducing the area where cells can adhere. If the ratio is too high, the microcarrier will have poor mechanical strength, uneven pore size and low porosity. The microcarrier will be easily degraded during cell culture and will not be able to support the attachment and proliferation of DP cells for a long time.

[0010] Based on the same inventive concept, the present invention also claims a method for preparing the microcarrier, comprising the following steps: S1, spraying alginate into a calcium source solution by high-voltage electrostatic spraying, reacting, and obtaining calcium alginate microspheres; S2, mixing calcium alginate microspheres, gelatin solution and cross-linking agent, and freeze-drying after reaction to obtain microcarriers.

[0011] In a preferred embodiment, freeze drying is as follows: first freezing at sub-zero temperature, and then transferring to a freeze dryer for freeze drying at -40°C or below for 20-28 hours.

[0012] In a preferred embodiment, the mass ratio of alginate to calcium source is 1:10-15. If the ratio is too low, the alginate core is prone to collapse during the freeze-drying process. If the ratio is too high, it is difficult for the alginate to be completely cross-linked.

[0013] In a preferred embodiment, the concentration of the calcium source solution is 1-3 wt %, preferably 2-3 wt %.

[0014] In one preferred embodiment, the alginate includes at least one of sodium alginate and potassium alginate. Sodium alginate is preferred, which is a natural high molecular linear polysaccharide with good biocompatibility and stability. The carboxyl group of guluronic acid in sodium alginate reacts with hydroxyl groups and calcium ions to form a stable chelate. However, alginate lacks surface adhesion properties. Gelatin is also a biocompatible hydrogel that plays an important role in cell adhesion and growth. Gelatin molecules are physically cross-linked to form a hydrogel layer at a low temperature of 4°C, and then chemically cross-linked with glutaraldehyde through a Schiff base reaction, using its unique RGD sequence to increase its cell loading capacity.

[0015] In a preferred embodiment, the calcium source is any water-soluble calcium salt.

[0016] In a preferred embodiment, the calcium source includes any one or more of calcium gluconate, calcium hydrogen phosphate, calcium lactate and calcium chloride.

[0017] In one preferred embodiment, the process of spraying alginate into a calcium source solution by high-voltage electrostatic spraying includes: injecting alginate solution into a syringe, connecting the syringe needle tip to the positive electrode of a power source, placing a receiving device under the positive electrode of the power source and connecting it to a ground electrode, the electrode distance is 6-10 cm, the flow rate is 2-5 ml / h, the applied voltage is 5-9 kV, and the alginate solution is formed into a jet and added to the calcium source solution.

[0018] Voltage, electrode distance and flow rate have a significant effect on the size of the microspheres.

[0019] As the voltage increases, the size of the microspheres decreases. As the electrode distance increases, the size of the microspheres increases.

[0020] In a preferred embodiment, the particle size of the microcarrier is 100-350 μm, the pore size is 10-33 μm, the porosity is 80-95%, and the specific surface area (area / volume m 2 / m 3 ) is 17646-42858.

[0021] Microcarriers that are too small cannot provide sufficient surface area for cell adhesion and proliferation, while microcarriers that are too large have a small specific surface area.

[0022] In a preferred embodiment, the average pore size of the microcarrier is 15-22 μm; the average particle size is 220-260 μm.

[0023] In a preferred embodiment, the syringe has a 26G blunt needle.

[0024] In a preferred embodiment, the diameter of the droplets ejected from the syringe is less than 300 microns.

[0025] Droplets with diameters less than 300 microns have the advantage of being monodisperse and easy to scale up.

[0026] In a preferred embodiment, the alginate solution is sprayed into the calcium source solution by high-voltage electrostatic spraying, and the reaction is carried out for 20-40 minutes to obtain calcium alginate microspheres.

[0027] In a preferred embodiment, the concentration of the alginate solution is 0.5-1.5 wt %, preferably 0.8-1.2 wt %.

[0028] In a preferred embodiment, the alginate solution is an aqueous alginate solution.

[0029] In a preferred embodiment, the concentration of the gelatin solution is 1-8wt%, preferably 4-8wt%, more preferably 4-6wt%, and further preferably 6-8wt%.

[0030] In a preferred embodiment, the cross-linking agent is glutaraldehyde solution.

[0031] In a preferred embodiment, the mass concentration of the cross-linking agent is 1-3 wt %, preferably 2-3 wt %.

[0032] In a preferred embodiment, the reaction time of calcium alginate microspheres, gelatin solution and cross-linking agent is 8-20 min.

[0033] Based on the same inventive concept, the present invention also claims to protect the use of the microcarrier in amplifying DP cells.

[0034] Compared with tissue culture plates, the three-dimensional shape of microcarriers significantly increases the available surface area for cell adhesion and provides a higher surface-to-volume ratio. In addition, the microcarrier matrix is ​​composed of ECM biomacromolecules, which is closer to the in vivo microenvironment and is conducive to the formation of sufficient cell-ECM and cell-cell contacts, thereby maintaining the phenotype.

[0035] Based on the same inventive concept, the present invention also claims a method for expanding DP cells, which uses the microcarrier for expansion.

[0036] In a preferred embodiment, DP cells are expanded by intermittent culture.

[0037] In a preferred embodiment, the method for expanding DP cells comprises the following steps: adding microcarriers into a rotary bioreactor, inoculating DP cells, stirring intermittently within the first 20-28 hours, then replenishing culture medium, and continuing to culture at a stirring speed of 30-50 rpm / min for 3-7 days.

[0038] In a preferred embodiment, the intermittent stirring is: stirring for 5-10 minutes after no stirring for 1-3 hours, with a stirring speed of 20-30 rpm; and the alternating cycle is 20-28 hours.

[0039] Too long or too short an intermittent stirring time is not conducive to cell growth. If the intermittent stirring time is too long, the effect is close to static culture, and if the intermittent stirring time is too short, the effect is close to continuous stirring.

[0040] In a preferred embodiment, the seeding density of DP cells is 2×10 3 -8×10 3 cells / mgMC, preferably 4×10 3 -6×10 3 cell / mgMC.

[0041] The present invention provides a method for preparing microcarriers by electrostatic spraying, which produces droplets of suitable diameter under a high-voltage electric field, and has the advantages of monodispersity, high yield, and easy scale-up. The operation is simple, the raw materials are easily available, the cost is saved, the amount of organic solvent used is minimized, and there is almost no cytotoxicity. The porous microcarrier has a large surface-to-body ratio, which provides more growth area for cells. The cells preferentially enter the interior of the microspheres for growth, and then adhere to the surface of the microspheres, thereby achieving a higher cell density. In addition, the present invention provides a DP culture method combining microcarriers with a rotating culture system, which creates more uniform culture conditions compared to monolayer culture such as tissue culture dishes, and is easier to monitor and control culture parameters, thereby making it easier to mass-produce cells. Microcarrier culture simulates the in vivo microenvironment in which DP cells grow, forming sufficient and multidirectional cell-cell and cell-ECM interactions, so as to maintain the ability of DP to induce hair follicle regeneration in vitro, and to amplify DP cells with hair follicle induction properties on a large scale without differentiating into other cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a scanning electron micrograph of alginate-gelatin core-shell microcarrier; Figure 2 The bar graph is a size analysis of alginate-gelatin core-shell microcarriers; Figure 3 The bar graph is a pore size analysis bar graph of alginate-gelatin core-shell microcarriers; Figure 4 The SEM images of microcarriers with different concentrations of gelatin coating layers; Figure 5 SEM images of alginate core microcarriers with different concentrations; Figure 6 SEM images of microcarriers cross-linked with glutaraldehyde at different concentrations; Figure 7 This is a schematic diagram of the process of using the microcarrier of the present invention for DP cell expansion; Figure 8 This is the result of fluorescence staining of microcarrier / cell complex; Fig. 9 This is a bar graph showing the cell proliferation levels under two culture methods for CCK8 testing; Fig.10 This is a comparison chart of cell proliferation results of dynamic culture and static culture; Fig.11 The bar graph is a comparison of the attachment rates of cells and microcarriers under different treatments; Fig.12 This is a bar graph showing the proliferation multiples of DP cells under different gelatin concentrations; Fig.13 is the transfer growth curve of DP on microcarriers; Fig.14The bar graphs are the qPCR results of DP marker genes ALP, Versican, Sox-2, β-catenin, and α-SMA under different treatments; Fig.15 Immunofluorescence staining of DP marker genes ALP, Versican, Sox-2, β-catenin, and α-SMA under different treatments; Fig.16 This is the result of observing the hair follicle regeneration in different treatment groups under optical microscope; Fig.17 This is the result of H&E staining to observe the hair follicle regeneration in different treatment groups. DETAILED DESCRIPTION

[0043] The present invention is not limited to the following specific embodiments. A person skilled in the art can implement the present invention in various other specific embodiments according to the contents disclosed in the present invention, or any simple changes or modifications made to the design structure and ideas of the present invention fall within the protection scope of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict. Unless otherwise specified, the following percentages are all mass concentrations. Example 1

[0044] Preparation of microcarriers 1. Preparation of calcium alginate gel microspheres Sodium alginate was dissolved in sterile water to prepare a 1% sodium alginate solution. 1.5 ml of sodium alginate solution was injected into a 5 ml syringe with a 26G blunt needle. The needle tip of the syringe was connected to the positive electrode of the power supply, and a receiving device was placed directly below and connected to the ground electrode. The electrode distance was 8 cm, the flow rate was adjusted to 3 ml / h, and the applied voltage was 7 kV. The 1.5 ml sodium alginate droplets produced by jet decomposition were gelled in 10 ml of 2% calcium chloride for 30 minutes. The calcium alginate microspheres were collected by centrifugation and washed three times with sterile water.

[0045] 2. Preparation of core-shell microcarriers Dissolve gelatin in sterile water to prepare a gelatin solution with a mass concentration of 6%. Evenly mix the above-obtained 0.081g calcium alginate microspheres with 50ml gelatin solution and place at 4°C for 24 hours. After gelatin coating, collect the microspheres and wash them three times with sterile water. Soak the alginate-gelatin microspheres in 10ml 2% glutaraldehyde solution for 10 minutes for chemical cross-linking. Collect the obtained microspheres, wash them three times with sterile water, and pre-freeze them in a -20°C refrigerator. After pre-freezing, transfer them to a vacuum freeze dryer and freeze-dry them at -40°C for 24 hours to obtain alginate-gelatin core-shell microcarriers.

[0046] Scanning electron microscopy observation of alginate-gelatin core-shell microcarriers showed that: Figure 1 As shown, the microcarriers present a regular spherical shape, and a nanofiber porous structure is formed on the surface of the sphere. The diameter of the obtained alginate-gelatin core-shell microcarriers is 100-350 microns, and the porosity of the microcarriers reaches 85-95%.

[0047] The size and pore size analysis were performed on it, and the results were as follows: Figure 2 and Figure 3 As shown, the results show that the average diameter of the microcarriers is about 243.6µm, the pores are uniform, the average pore size of the micropores is about 18.2µm, and the long axis of DP cells is about 8-10µm, so they can enter the pores of the microcarriers. The porous surface of the microcarriers further increases the surface area available for cell culture. Example 2

[0048] The difference between Example 2 and Example 1 is that the solubility of the gelatin solution is 0%. Example 3

[0049] The difference between Example 3 and Example 1 is that the solubility of the gelatin solution is 1%. Example 4

[0050] The difference between Example 4 and Example 1 is that the solubility of the gelatin solution is 2%. Example 5

[0051] The difference between Example 5 and Example 1 is that the solubility of the gelatin solution is 4%. Example 6

[0052] The difference between Example 6 and Example 1 is that the solubility of the gelatin solution is 8%.

[0053] The alginate-gelatin core-shell microcarriers prepared in Examples 1-6 were compared by SEM. Figure 4 As shown. The results showed that the microcarriers were all based on 1% alginate as the core, and chemically cross-linked gelatin (1%, 2%, 4%, 6%, 8%) as the coating layer. Alginate spheres without gelatin were used as controls. It was found that when the gelatin concentration was too low, below 4%, the morphology of the microcarriers was irregular and collapsed. When the gelatin concentration increased to 4%-8%, the morphology of the microcarriers tended to be a regular sphere, rather than a collapsed irregular shape, and a nanofiber porous structure was formed on the surface of the sphere.

[0054] The CCK8 experiment was performed on alginate-gelatin core-shell microcarriers prepared with 1% alginate as the core and chemically cross-linked gelatin (4%, 6%, 8%, 10%) as the coating layer. The experimental process is as follows: Cell seeding and expansion Vaccination method: Sterilize the microcarriers with UV for more than 2 hours, take 100 mg of microcarriers and spread them on the bottom of a 150 ml rotating bioreactor. Mix the cell suspension with about 20 ml of MSCM complete medium and add 4 × 10 3 DP was inoculated at a density of 10 cells / mgMC, and stirred intermittently in the first 24 hours, alternating between no stirring for 1 hour and stirring at 25 rpm / min for 5 minutes to allow the cells to fully adhere to the microcarriers. The culture medium was supplemented to about 50 ml, and the culture was continued with stirring at 40 rpm / min.

[0055] 2. Cell Passaging After 7 days of culture, add new 100 mg microcarriers to the culture flask, change half of the medium and replenish the medium to 100 ml. Adjust the speed to the corresponding mode 24 hours before inoculation, alternately without stirring for 1 hour and stirring at 25 rpm / min for 5 minutes, and continue stirring and culturing at 40 rpm / min after the cells are fully attached.

[0056] 3. Cell Harvest Seven days after cell seeding, allow the cell-loaded microcarriers to settle and transfer to a centrifuge tube. Wash the microcarriers with PBS to remove all unattached cells. Add 5 ml of 0.25% trypsin / EDTA to the microcarriers and incubate at 37°C for 2 min. Pipette thoroughly to separate the cells from the microcarriers, add culture medium to inactivate trypsin, and collect the harvested cells by centrifugation.

[0057] The cell proliferation level was quantified by Cell Counting Kit-8 (CCK8). On days 1, 3, 5, and 7, 1 ml of culture medium was drawn from the spinner flask and added to a 96-well plate. The culture medium was prepared with MSCM to contain 10% CCK-8 solution and added to each well in the form of liquid exchange. Avoid sucking away the microcarriers during the liquid exchange. 100 µL was added to each well, and bubbles were avoided during the addition process. Then the culture plate was placed in the incubator and incubated for 2 hours. The 96-well plate was removed, and 100 µL of culture medium was drawn and added to a new 96-well plate, avoiding sucking away the microcarriers. The absorbance of each sample was measured at 450 nm using a microplate reader.

[0058] The results are as follows Fig.12 As shown in the figure, compared with the OD value on the first day, the proliferation multiples of the microcarriers coated with 4%-8% gelatin were 3.8-6 times on the seventh day, among which the microcarriers coated with 6% gelatin had the highest proliferation efficiency, which could reach 5-6 times. The proliferation multiples of the microcarriers coated with 10% gelatin were only 2.5 times on the seventh day, which was relatively low in efficiency. Example 7

[0059] The difference between Example 7 and Example 1 is that the solubility of the gelatin solution is 4%, and the concentration of the alginate is 0.5%. Example 8

[0060] The difference between Example 8 and Example 1 is that the solubility of the gelatin solution is 4%, and the concentration of the alginate is 1.5%. Example 9

[0061] The difference between Example 9 and Example 1 is that the solubility of the gelatin solution is 4%, and the concentration of the alginate is 1.5%.

[0062] The alginate-gelatin core-shell microcarriers prepared in Examples 7-9 were compared by SEM. Figure 5 As shown. The results showed that the microcarriers were coated with 4% gelatin and encapsulated with alginate (0.5%, 1%, 1.5%) as the core. The results showed that when the concentration of sodium alginate was 0.5%, the structure was relatively loose, and when the concentration of sodium alginate was 1%, a nanofiber network structure was formed on the surface of the sphere, and the shape was relatively regular. When the concentration continued to increase to 1.5%, the pores of the sphere were few and uneven. Example 10

[0063] The difference between Example 10 and Example 1 is that the concentration of glutaraldehyde is 0%. Embodiment 11

[0064] The difference between Example 11 and Example 1 is that the concentration of glutaraldehyde is 4%. Example 12

[0065] The difference between Example 12 and Example 1 is that the concentration of glutaraldehyde is 6%.

[0066] The alginate-gelatin core-shell microcarriers prepared in Examples 1 and 10-12 were compared by SEM. Figure 6 As shown. The results showed that the above four groups of microcarriers all used 1% alginate as the core and 4% gelatin as the coating layer. The microspheres without crosslinking agents had poor mechanical strength and were easy to collapse. Under the action of glutaraldehyde (2%, 4%, 6%), spheres with uniform micron-scale pore structures were formed. Since glutaraldehyde has certain cytotoxicity, a concentration of 2% was used in subsequent experiments.

[0067] Embodiment 13 according to Figure 7 The process schematic diagram shown in the figure shows that the microcarrier prepared in the example is used for DP cell expansion, and the process is as follows: Cell seeding and expansion Vaccination method 1: Sterilize the microcarriers with UV for more than 2 hours, take 100 mg of microcarriers and spread them on the bottom of a 150 ml rotating bioreactor. Mix the cell suspension with about 20 ml of MSCM complete medium and add 4 × 10 3Inoculate DP at a density of 10 cells / mgMC, stir intermittently in the first 24 hours, alternately without stirring for 1 hour and stirring at 25 rpm / min for 5 minutes, so that the cells can fully attach to the microcarriers. Supplement the culture medium to about 50 ml, and continue to stir at 40 rpm / min.

[0068] Vaccination method 2: Take 10 mg of microcarriers and spread them on the bottom of a 24-well plate. Irradiate with ultraviolet light for 2 h to achieve sterilization. Collect the microcarriers and 1 mL of cell suspension (4 × 10 4 The cells were mixed with 100 cells / mL and placed in an ultra-low adhesion EP tube for pre-culture for 2 h. Then, the cells were transferred to a 24-well plate without dynamic culture.

[0069] Vaccination method 3: Sterilize the microcarriers with UV for more than 2 hours, take 100 mg of microcarriers and spread them on the bottom of a 150 ml rotating bioreactor. Mix the cell suspension with about 50 ml of MSCM complete medium at 4 × 10 3 DP was inoculated at a density of 10 cells / mgMC and cultured at 40 rpm / min during the first 24 h and subsequent culture.

[0070] Vaccination method 4: Sterilize the microcarriers with UV for more than 2 hours, take 100 mg of microcarriers and spread them on the bottom of a 150 ml rotating bioreactor. Mix the cell suspension with about 20 ml of MSCM complete medium and add 4 × 10 3 Inoculate DP at a density of 10 cells / mgMC, do not stir for the first 24 hours to allow the cells to fully attach to the microcarriers, add culture medium to about 50 ml, and continue to stir at 40 rpm / min.

[0071] 2. Cell Passaging After 7 days of culture, add new 100 mg microcarriers to the culture flask, replace half of the medium and replenish the medium to 100 ml. Adjust the rotation speed to the mode corresponding to 24 hours before inoculation, and continue stirring and culturing at 40 rpm / min after the cells are fully attached.

[0072] 3. Cell Harvest Seven days after cell seeding, allow the cell-loaded microcarriers to settle and transfer to a centrifuge tube. Wash the microcarriers with PBS to remove all unattached cells. Add 5 ml of 0.25% trypsin / EDTA to the microcarriers and incubate at 37°C for 2 min. Pipette thoroughly to separate the cells from the microcarriers, add culture medium to inactivate trypsin, and collect the harvested cells by centrifugation.

[0073] After 1, 3, 5, and 7 days of DP culture, the microcarrier / cell complex was stained with a live / dead staining kit and observed under a fluorescence microscope. Figure 8 The results showed that 2 days after DP was inoculated on the microcarriers, sporadic green fluorescence appeared on the surface of the microcarriers, indicating that the microcarriers can provide attachment sites for cells. After 4 days, the green fluorescence increased. On the 6th day, most of the microcarriers were covered with strong green fluorescence, without red fluorescence, and no dead cells were found, indicating that the microcarriers were non-cytotoxic and DP could survive and proliferate normally on the microcarriers.

[0074] DP of the same generation cultured in a culture dish were inoculated on 3D TableTrix (Huakan Biotechnology Co., Ltd., China) and the microcarrier of the present invention, respectively, and cultured under the same culture conditions as inoculation method 1 until the 6th day, and the cells were digested and counted. The results are as follows: Fig. 9 As shown, 3D TableTrix culture can expand cells by about 5 times, and the microcarrier culture of the present invention significantly improves the yield of DP cells, expanding DP by about 9.7 times.

[0075] The cell proliferation level under the conditions of inoculation method 1 and inoculation method 2 at the same temperature and time was quantified by CellCounting Kit-8 (CCK8). At different time points, 10 mg of microcarriers were aspirated from the roller bottle and 24-well plate, added to the 96-well plate, and the culture medium containing 10% CCK-8 solution was prepared with MSCM and added to each well in the form of liquid replacement. Avoid sucking away the microcarriers during the liquid replacement process. Add 100 µL to each well, and avoid generating bubbles during the addition process. Then place the culture plate in the incubator and incubate for 42 hours. Take out the 96-well plate, aspirate 100 µL of culture medium and add it to the new 96-well plate, avoiding sucking out the microcarriers. Use an enzyme reader to measure the absorbance of each sample at 450 nm. The comparison results of the two are shown as follows. Fig.10 As shown, the proliferation efficiency of cells cultured in inoculation method 1 was significantly higher than that of cells cultured in inoculation method 2.

[0076] The attachment rate of cells on the microcarriers was tested 24 hours after inoculation. The method was as follows: 1 mL of sample was taken from the side arm with a pipette and the cells were counted. To ensure uniform sampling, the microcarriers were kept suspended when sampling. The attachment efficiency was evaluated by measuring the number of cells in the supernatant of the sample 24 hours after inoculation. Attachment efficiency = (total number of inoculated cells - number of cells in the supernatant) / total number of inoculated cells. The results are shown in Fig.11As shown in the figure, the adhesion rate of microcarriers is significantly different when DP is not stirred in a rotating bottle (inoculation method 4), continuously stirred for 40 revolutions (inoculation method 3), and alternately not stirred for 1h and 25 revolutions for 5 minutes (inoculation method 1). The adhesion force is significantly improved under the process of alternating not stirring for 1h and 25 revolutions for 5 minutes (intermittent) compared with inoculation method 3. Cells can only proliferate when attached to the surface of a solid matrix, so the attachment of cells to the surface of microcarriers is the key to further spreading and growth. Adhesion mainly relies on electrostatic attraction and van der Waals force. Whether cells can adhere to the surface of microcarriers depends mainly on the contact probability and compatibility between cells and microcarriers. Since animal cells have no cell walls and are sensitive to shear forces, it is impossible to increase the contact probability by increasing the stirring speed.

[0077] To investigate the transfer ability of DP on microcarriers, new microcarriers were added on day 7, and the proliferation level was measured by CCK8 and a growth curve was plotted. Fig.13 As shown, DP cells can achieve "sphere-to-sphere" behavior on microcarriers without the need for trypsin digestion, which simplifies cell passaging operations and avoids trypsin damage to cells.

[0078] After the culture was completed, the harvested DP was characterized. To study its hair follicle regeneration potential, the cells on the microcarriers were digested and qRT-PCR was performed. [1] and immunofluorescence staining [2] , detect the expression of ALP, Versican, Sox-2, β-catenin, and α-SMA. [2] The same experiment was performed on cells grown on the same generation as the control group.

[0079] qPCR results ( Fig.14 ) showed that the expression of stemness genes ALP, Versican, Sox-2, and β-catenin in DP cultured on microcarriers was significantly upregulated, while the expression of differentiation gene α-SMA was significantly downregulated. Immunofluorescence staining results ( Fig.15 ) showed that after microcarrier culture, the fluorescence signals of ALP, Versican, Sox-2, and β-catenin of DP were enhanced, and the fluorescence signal of α-SMA was weakened, which was consistent with the qPCR results. The above results indicate that microcarrier culture can restore the hair follicle induction properties of DP.

[0080] Hair follicle regeneration experimental design: 1) Positive group: 1*10 cells from each of dermis and epidermis 6 The suspensions of different amounts were mixed, centrifuged to obtain cell pellets, resuspended with 30 μL Metrigel + 70 μL PBS, transferred to 1.5 mL EP tubes, and placed on ice for later use.

[0081] 2) Control group: the number is 2*10 6 Mouse epidermal cell suspension was centrifuged to obtain the precipitate, which was resuspended in 30 μL Metrigel + 70 μL PBS, and then transferred to a 1.5 mL EP tube and placed on ice for later use.

[0082] 3) Traditional plate culture group: P3 DP cells cultured in a culture dish were digested and counted, and 1*10 6 The cell suspension of the same number was mixed with the epidermal cells of the same number and centrifuged. The precipitate was resuspended in 100 μL of 30 μL Metrigel + 70 μL PBS and placed on ice for later use.

[0083] 4) Microcarrier culture group: P3 DP cells cultured on microcarriers were digested and counted, and 1*10 6 The cell suspension of the same number was mixed with the epidermal cells of the same number and centrifuged, and the pellet was resuspended in 30 μL Metrigel + 70 μL PBS.

[0084] Three weeks after the cell mixture was injected subcutaneously into nude mice, the skin tissue was cut off for observation, the back skin was fixed with 4% paraformaldehyde for 24 hours, then embedded in paraffin blocks, and 10µm thick sections were prepared according to the longitudinal section of the hair follicles. Then, the sections were stained with HE, rehydrated, stained, dehydrated, transparent, and sealed, and observed under a microscope.

[0085] The results of the observation of hair regeneration in different groups of the hair follicle regeneration experiment under an optical microscope showed that only melanin deposition was observed in the traditional plate culture group, and no hair follicle structure was formed. In the microcarrier culture group, more hair shafts were formed, accompanied by a large amount of fat. Compared with the positive group, the hair shafts were thicker ( Fig.16 ). The excised skin tissue was sliced ​​and stained with H&E after paraffin sectioning. An elliptical area was formed at the subcutaneous injection site in the microcarrier culture group, and a large number of hair follicles were formed. Since the newly formed hair cannot penetrate the skin, the growth direction of the newly formed hair is irregular. A clearer cross-section of the hair follicle can be seen in the figure. The traditional flat plate culture group did not generate obvious hair shafts, and only a small number of hair follicle-like structures were formed ( Fig.17 ).

[0086] Comparative Example 1 Reference to prior art CN 213113172 U Oil ratio 1.1 Inspection device; 1.2 Take 80L of chloroform, 20L of petroleum ether, and 10L of Tween 20 reagent and add them to the mixing tank in sequence using a peristaltic pump. Turn on the mixing tank and stir at 60rpm for 1 hour; 1.3 Seal the mixing tank and leave only the compressed air port open until the internal pressure of the tank reaches 0.1MP, then pump the oil phase into the pre-cooling tank. Use the continuous cooling effect of the chiller to reduce the oil phase temperature to -30℃ for standby use. During this period, the stirring of the pre-cooling tank can be maintained at 40rpm.

[0087] Water ratio 2.1 Inspection device; 2.2 Weigh 50g of gelatin and 10g of sodium alginate and put them into 1.5L of deionized water. Use a stirrer set at 60rpm to stir the gelatin solution until it is fully dissolved. Use a peristaltic pump to add it to the water phase tank, then add 100g of sodium chloride, heat to 60℃, set the speed to 150rpm and stir for 120 minutes for later use; mix 3.1 Inspection device; 3.2 The reaction tank is continuously acted by a chiller to reduce the tank temperature to below -30°C. The lower the temperature, the smaller the pore size of the prepared carrier material. The pore size of the microcarrier prepared at -30°C is about 5μm-20μm. After the temperature is reduced to the preset temperature, 5ml of 75% formaldehyde solution is added to the water phase and the speed is set to 80rpm for stirring for 5 minutes. Then the oil phase and the water phase are mixed at a certain flow rate through a microsphere forming machine and pumped into the reaction tank. The oil phase is driven by a gear pump (pump speed is 1000ml / min). The lower the pump speed, the larger the particle size of the prepared granular material, and vice versa. The average particle size of the prepared material under this condition is about 400μm. The water phase pressure is set to 10KPa using nitrogen boost. The larger the pressure, the larger the microsphere particle size, and vice versa. The average particle size under this condition is 400μm. The micropore diameter of the porous plate selected by the microsphere forming machine is 50μm.

[0088] 3.3 After the material enters the reaction tank, turn on the stirring immediately and set the speed to 30rpm for stirring reaction for 48 hours. During this period, regularly observe and record the reaction status and various parameters of the tank material.

[0089] Cleaning 4.1 Inspection device; 4.2 The materials after the reaction are pumped into the oil filter tank through 0.15MP nitrogen, and the excess liquid in the materials is removed by continuous action of the vacuum pump. Then, cleaning agents are added to the materials, which are 50L acetone, 20kg anhydrous magnesium sulfate, 100L medical alcohol, and 100L anhydrous ethanol. The stirring speed is 300rpm, and each time for 20min. After each cleaning, the excess liquid is removed by vacuum filtration. Use 0.10MP compressed air to pump the effective materials into the water washing tank; 4.3 Add 20L of deionized water to the water washing tank, stir at 100rpm for 50 minutes, and remove the injection water by vacuum pump. Repeat the washing 3-5 times.

[0090] Freeze-drying 5.1 Inspection device; 5.2 Add the cleaned material into the freeze-drying box according to the ratio of 50 g wet weight to 300 mL injection water, and mix them. Freeze at -40℃ for 96 hours.

[0091] 5.3 Put the frozen material into the freeze dryer and perform freeze drying according to the freeze dryer operating procedures for more than 72 hours. Weigh the total weight of the freeze-dried material and transfer it to the material temporary storage box for the next process.

[0092] Screening 6.1 First check the cleanliness of the screen and the screening machine and the clearance records. They can only be used if they are clean and within the validity period; 6.2 The freeze-dried material is sieved in the range of 50-500 microns. The sieved material is collected to obtain porous microcarriers with a certain particle size range.

[0093] Results: The average particle size of the obtained microcarriers was 400 μm, the average pore size was 15 μm, the porosity was 90%, the yield was about 20 g, and the yield was about 30%.

[0094] The prepared microcarriers were used for DP cell expansion, and the process was as follows: Vaccination method: Sterilize the microcarriers with UV for more than 2 hours, take 100 mg of microcarriers and spread them on the bottom of a 150 ml rotating bioreactor. Mix the cell suspension with about 20 ml of MSCM complete medium and add 4 × 10 3 Inoculate DP at a density of 10 cells / mgMC, stir intermittently in the first 24 hours, alternately without stirring for 1 hour and stirring at 25 rpm / min for 5 minutes, so that the cells can fully attach to the microcarriers. Supplement the culture medium to about 50 ml, and continue to stir at 40 rpm / min.

[0095] As a result, the number of DP cells on day 6 increased by about 5 times compared with that on day 1, which was much lower than that of the microcarriers of the present invention.

[0096] Comparative Example 2: Reference to prior art CN 116656591 A A microcarrier comprises calcium alginate microspheres, and chitosan and gelatin cross-linked on the surface of the calcium alginate microspheres.

[0097] The curvature of the microcarrier is 1 / 600 -1 / 200μm -1 , corresponding to a particle size of 200-600μm.

[0098] The method for preparing the above microcarrier comprises the following steps: S1 Use an infusion pump to push out a 30 mg / mL sodium alginate solution, adjust the voltage to the point where the electric field force can break through the surface tension of the sodium alginate solution, and spray it as droplets onto the surface of a 10 mg / mL calcium chloride solution. The volume ratio of the sodium alginate solution to the calcium chloride solution is 1:1. The reaction ends after 0.5 h and is washed with deionized water to obtain calcium alginate microspheres with a particle size of 200-600 μm and a corresponding curvature of 1 / 600. -1 / 200μm -1 ; S2 Mix chitosan with a mass concentration of 5 mg / mL and gelatin with a mass concentration of 5 mg / mL to obtain a coating solution, mix calcium alginate microspheres, coating solution and citral with a mass concentration of 2 mg / mL, react for 1 hour, wash with deionized water, add glycine with a mass concentration of 1 mg / mL for neutralization, wash the microcarriers with deionized water again after 1 hour to obtain microcarriers, place the microcarriers in a high pressure sterilizer at 121°C for 20 minutes, and obtain sterilized microcarriers by high pressure sterilization. Among them, the parameters of the high-voltage electrostatic spray method are: the output voltage of the high-voltage generator is 0-10kv, a 200μm needle is used, and the propulsion speed of the infusion pump is 5mL / min.

[0099] The volume ratio of calcium alginate microspheres to coating solution is 1:10.

[0100] Results: The light yellow transparent adhesive was observed on the surface of the microcarriers under a microscope, and cells could be attached and grown on the microspheres, indicating that chitosan and gelatin were cross-linked on the surface of calcium alginate microspheres.

[0101] The prepared microcarriers were used for DP cell expansion, and the process was as follows: Vaccination method: Sterilize the microcarriers with UV for more than 2 hours, take 100 mg of microcarriers and spread them on the bottom of a 150 ml rotating bioreactor. Mix the cell suspension with about 20 ml of MSCM complete medium and add 4 × 10 3 Inoculate DP at a density of 10 cells / mgMC, stir intermittently in the first 24 hours, alternately without stirring for 1 hour and stirring at 25 rpm / min for 5 minutes, so that the cells can fully attach to the microcarriers. Supplement the culture medium to about 50 ml, and continue to stir at 40 rpm / min.

[0102] As a result, the number of DP cells on day 6 increased by about 3.7 times compared with that on day 1, which was much lower than that of the microcarriers of the present invention.

[0103] Comparative Example 3: A mixed solution of 1% sodium alginate and 10% D-mannitol is prepared according to a mass volume ratio, and the mixture is fully mixed and dissolved overnight at room temperature. After the mixture is fully dissolved, the hydrogel is filtered and sterilized using a 0.22 μm pore size sterilizing filter and a sterile syringe in a biological safety cabinet to obtain a sterile sodium alginate hydrogel; DP cells are cultured in large quantities in vitro; The DP cells were suspended in the sodium alginate solution prepared in step 2) to form a suspension containing 6×10 6 DP cells; The hydrogel suspension mixed with DP cells at an appropriate concentration was transferred into a 5 ml sterile syringe, and the syringe pillow was replaced with a 23G point needle; The cell suspension in 4) was formed into a microdroplet state with a diameter of about 350-550 μm by using a high-voltage electric field microcapsule generator, and then dropped into a 2% CaCl2 solution to make 4×10 3 The density of cell / mgMC is DP cells in relatively stable hydrogel microspheres, and solid microsphere precipitates are obtained; the electrostatic spray parameters of the microspheres are: the flow rate of the electrostatic propulsion pump is 3ml / h, the voltage of the electrostatic generating device is 5-6kV, and the electrostatic spray time shall not be longer than 20min.

[0104] The sodium alginate microspheres obtained above were washed twice with sterile saline to remove a large amount of CaCl2 in the receiving device during the spraying process, and then DMEM culture medium containing 0.5 mg / ml PLL was added at 37°C for 10 minutes to allow PLL to form a thin film on the surface of the microspheres, which helped to fix the morphology of the microspheres; Remove the PLL solution and add 0.1% sodium alginate-1% mannitol solution for 4 minutes to remove the excess PLL around the microspheres and form a loose sodium alginate protective layer on the outside of the microspheres. After the reaction, add 5.5mM sodium citrate solution for 3 minutes to remove the residual Ca 2+ And slightly dissolve sodium alginate and Ca 2+ The formed film makes the membrane structure around the microsphere looser. At this time, due to the presence of the PLL protective film, the microsphere can be supported to maintain its spherical shape. After proper dissolution, the spatial structure inside the sodium alginate microsphere becomes looser, which can reduce the resistance of cells moving in the microsphere, which is conducive to the discharge of cell metabolic waste and the entry of external nutrients. After the reaction is completed, the microspheres are gently washed with a small amount of DMEM.

[0105] The prepared microcarriers were used for DP cell expansion, and the process was as follows: Take 100 mg of microcarriers + DP cells and spread them on the bottom of a 150 ml rotary bioreactor. Mix with about 20 ml of MSCM complete medium and stir intermittently for the first 24 hours, alternately without stirring for 1 hour and stirring at 25 rpm / min for 5 minutes. Then continue to stir and cultivate at 40 rpm / min.

[0106] As a result, the number of DP cells on the 6th day was basically not increased compared with that on the 1st day, and the cell viability was poor, which was far lower than that of the microcarriers of the present invention.

[0107] 【1】Wu Y, Zheng Y, Jin Z, et al. Controllable manipulation ofalginate-gelatin core-shell microcarriers for HUMSCs expansion. Int J BiolMacromol. 2022 Sep 1;216:1-13. 【2】Zhang Y, Yin P, Huang J, et al. Scalable and high-throughputproduction of an injectable platelet-rich plasma (PRP) / cell-ladenmicrocarrier / hydrogel composite system for hair follicle tissue engineering. J Nanobiotechnology. 2022 Nov 3;20(1):465. It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A microcarrier, characterized in that The invention is composed of calcium alginate microspheres and gelatin cross-linked on the surface of the calcium alginate microspheres; the raw materials include calcium alginate microspheres and gelatin in a weight ratio of 81:2000-4000.

2. The method for preparing a microcarrier according to claim 1, characterized in that: The following steps are involved: S1, spraying alginate into a calcium source solution by high-voltage electrostatic spraying, reacting, and obtaining calcium alginate microspheres; S2, mixing calcium alginate microspheres, gelatin solution and cross-linking agent, transferring to a freezing device for freeze drying after reaction, and obtaining microcarriers.

3. The preparation method according to claim 2, characterized in that: The mass ratio of the alginate to the calcium source is 1:10-15.

4. The preparation method according to claim 2, characterized in that: The concentration of the alginate solution is 0.5-1.5wt%; the concentration of the gelatin solution is 4-8wt%.

5. The preparation method according to claim 2, characterized in that: The process of spraying alginate into a calcium source solution by high-voltage electrostatic spraying includes: injecting alginate solution into a syringe, connecting the syringe needle tip to the positive electrode of a power source, placing a receiving device under the positive electrode of the power source and connecting it to a ground electrode, the electrode distance is 6-10 cm, the flow rate is 2-5 ml / h, the applied voltage is 5-9 kV, and the alginate solution is formed into a jet and added to the calcium source solution.

6. The preparation method according to any one of claims 2 to 5, characterized in that: The particle size of the microcarrier is 100-350 μm, the pore size is 10-33 μm, the porosity is 80-95%, and the specific surface area is 17646-42858.

7. The preparation method according to claim 6, characterized in that: The average pore size of the microcarriers is 15-22µm; the average particle size is 220-260µm.

8. A method for expanding DP cells, characterized in that: The microcarrier according to claim 1 is used for amplification.

9. The method according to claim 8, characterized in that The method for expanding DP cells comprises the following steps: adding microcarriers into a rotary bioreactor, inoculating DP cells, stirring intermittently within the first 20-28 hours, then replenishing culture medium, and continuing to culture at a stirring speed of 30-50 rpm / min for 3-7 days.

10. The method according to claim 9, characterized in that Intermittent stirring is: 1-3 hours without stirring, then stirring for 5-10 minutes, with a stirring speed of 20-30 rpm; alternating cycle for 20-28 hours.

Images