A serum-free cell cryoprotective solution for epidermal stem cells, and a preparation method and application thereof

Abstract

The application provides a serum-free cell cryopreservation solution for epidermal stem cells and a preparation method and application thereof, and specifically belongs to the technical field of stem cell cryopreservation.The serum-free cell cryopreservation solution for epidermal stem cells comprises the following components in the following amounts: 0 g / L to 50 g / L raffinose, 3 g / L to 30 g / L dextran, 0% to 30% (volume percent) glycerol, 0% to 10% (volume percent) DMSO, 0% to 30% (volume percent) platelet lysate, and the balance of Ham's F-12; wherein the values of DMSO and glycerol are not both 0%. The serum-free cell cryopreservation solution can significantly improve the proliferation activity and colony formation ability of epidermal stem cells after resuscitation.

Description

Technical Field

[0001] This invention belongs to the field of stem cell cryopreservation technology, specifically relating to a serum-free cell cryopreservation solution for epidermal stem cells, its preparation method, and its application. Background Technology

[0002] Epidermal stem cells (EPSCs) are a type of stem cell located in the basal layer of the skin's epidermis. They possess high repair and regeneration capabilities and can differentiate into keratinocytes. Unlike traditional cell line cryopreservation techniques, cryopreservation methods for EPSCs require protecting the stem cells' self-replication ability and undifferentiated state. This allows for rapid proliferation after thawing, enabling them to repair target tissues or be further induced into keratinocytes. Maintaining the proliferation capacity and function of these cells during cryopreservation is a key research challenge.

[0003] Traditional cell cryopreservation solutions not only introduce exogenous animal serum (FBS), but their cryopreservation methods often only focus on reducing cell death during cryopreservation, neglecting the functional state of cells after survival. For example, patent CN111838139A also protects a serum-free cell cryopreservation solution, but its beneficial effects are limited to maintaining the cell recovery rate after cryopreservation and reducing cell death within 24 hours of thawing. It cannot be used in stem cell cryopreservation applications that require the protection of self-replication and clonogenic capabilities, and it cannot achieve efficient preservation of epidermal stem cells, ensuring their expansion capacity and function after preservation. Summary of the Invention

[0004] The purpose of this invention is to provide a serum-free cryopreservation solution for epidermal stem cells, its preparation method, and its application. The serum-free cryopreservation solution of this invention enables efficient preservation of epidermal stem cells, ensuring a high cell recovery rate shortly after cryopreservation, and protecting cell viability and proliferative capacity after thawing.

[0005] This invention provides a serum-free cryopreservation solution for epidermal stem cells, comprising the following components in the following amounts: 0 g / L to 50 g / L raffinose, 3 g / L to 30 g / L dextran, 0% to 30% glycerol (volume percentage), 0% to 10% DMSO (volume percentage), 0% to 30% platelet lysate (volume percentage), and the balance being Ham's F-12; wherein the values ​​of DMSO and glycerol are not both 0%.

[0006] Preferably, the component comprises the following components in the following amounts: 0 g / L to 50 g / L raffinose, 3 g / L to 22 g / L dextran, 0% to 7.5% glycerol by volume, 5% to 10% DMSO by volume, 0% to 30% platelet lysate by volume, and the balance being Ham's F-12.

[0007] Preferably, the component comprises the following amounts: 20 g / L raffinose, 22 g / L dextran, 7.5% glycerol by volume, 5% DMSO by volume, 15% platelet lysate by volume, and the balance being Ham's F-12.

[0008] This invention also provides a method for preparing the serum-free cell cryopreservation solution described in the above technical solution, comprising the following steps: The serum-free cell cryopreservation solution is prepared by mixing 0 g / L to 50 g / L of raffinose, 3 g / L to 30 g / L of dextran, 0% to 30% (v / v) of glycerol, 0% to 10% (v / v) of DMSO, 0% to 30% (v / v) of platelet lysate, and the remainder of Ham's F-12.

[0009] The present invention also provides the application of the serum-free cell cryopreservation solution described above in the cryopreservation of epidermal stem cells.

[0010] The present invention also provides the application of the serum-free cell cryopreservation solution described above in improving the cell viability of cryopreserved epidermal stem cells after thawing.

[0011] The present invention also provides the application of the serum-free cell cryopreservation solution described above in increasing the number of viable cells after cryopreserved epidermal stem cells are thawed.

[0012] The present invention also provides the application of the serum-free cell cryopreservation solution described above in protecting the cell expansion capacity of cryopreserved epidermal stem cells after thawing.

[0013] The present invention also provides the application of the serum-free cell cryopreservation solution described above in protecting the clonogenic ability of cryopreserved epidermal stem cells after thawing.

[0014] This invention also provides a method for cryopreserving epidermal stem cells using the serum-free cell cryopreservation solution described above, comprising the following steps: The epidermal stem cells were mixed with the serum-free cell cryopreservation solution described in the above technical solution, and then frozen and stored.

[0015] This invention provides a serum-free cryopreservation solution for epidermal stem cells. By carefully selecting the composition and dosage of each component (crystalt), this invention increases the success rate of deep cryopreservation and thawing of epidermal stem cells. Specifically, it uses natural biocompatible materials as cryoprotectants, avoiding the introduction of exogenous animal serum (FBS) in traditional cryopreservation solutions. This serum-free cryopreservation solution not only effectively protects cell survival during freezing and thawing but also offers advantages such as low toxicity and being serum-free. Experimental results show that this serum-free cryopreservation solution achieves efficient preservation of epidermal stem cells, ensuring a high cell recovery rate shortly after cryopreservation and protecting cell viability and proliferation capacity after thawing. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Figure 1 shows the cell recovery rate and number of viable cells of epidermal stem cells after 24 hours of cryopreservation using different cryoprotectants provided by the present invention. Figure 2 The number of epidermal stem cells increased after thawing for 2 days using different cryopreservatives provided in this invention. Figure 3 The results of cell recovery rate and number of viable cells after 24 hours of cryopreservation of epidermal stem cells in different cryopreservation solutions provided by the present invention are shown in the figure. Figure 4 The image shows the colony morphology of epidermal stem cells cultured for 2 days, as provided by this invention; where A is experimental group 16; B is experimental group 12; C is experimental group 4; D is experimental group 6; E is experimental group 17; and F is experimental group 13. Figure 5 The image shows the colony morphology of epidermal stem cells cultured for 2 days, stained with P63 DAB. A represents experimental group 16; B represents experimental group 12; C represents experimental group 4; D represents experimental group 6; E represents experimental group 17; F represents experimental group 13; and G represents the blank control. Figure 6 The image shows the colony morphology of epidermal stem cells cultured for 2 days, stained with P63 hematoxylin and eosin. A represents experimental group 16; B represents experimental group 12; C represents experimental group 4; D represents experimental group 6; E represents experimental group 17; F represents experimental group 13; and G represents the blank control. Figure 7Figure 1 shows the cell recovery rate and number of viable cells of epidermal stem cells after 24 hours of cryopreservation using different cryoprotectants provided by the present invention. Figure 8 Image showing the cell expansion results of epidermal stem cells thawed 2 days after being cryopreserved using different cryoprotectants provided by this invention. Figure 9 The cryopreservation curve results provided by this invention; Figure 10 Figure 1 shows the cell recovery rate and viable cell count of 3T3 cells after 24 hours of cryopreservation in different cryopreservation solutions provided by the present invention. Figure 11 The graph shows the cell expansion results of 3T3 cells after three days of thawing following cryopreservation in different cryopreservation solutions provided by this invention. Figure 12 Figure 1 shows the cell recovery rate and number of viable cells after 24 hours of cryopreservation of hair follicle stem cells in different cryopreservation solutions provided by the present invention. Figure 13 The colony morphology diagram of hair follicle stem cells after 7 days of culture provided by the present invention; wherein, A, experimental group 16; B, experimental group 12; C, experimental group 4; D, experimental group 6; E, experimental group 17; F, experimental group 13; Figure 14 The graph shows the cell expansion results after seven days of thawing of hair follicle stem cells cryopreserved in different cryopreservation solutions provided by this invention. Detailed Implementation

[0018] This invention provides a serum-free cryopreservation solution for epidermal stem cells, comprising the following components in the following amounts: 0 g / L to 50 g / L raffinose, 3 g / L to 30 g / L dextran, 0% to 30% glycerol (v / v), 0% to 10% DMSO (v / v), 0% to 30% platelet lysate (v / v), and the balance being Ham's F-12; wherein the values ​​of DMSO and glycerol are not both 0%. In a specific embodiment, the solution comprises the following components in the following amounts: 0 g / L to 50 g / L raffinose, 3 g / L to 22 g / L dextran, 0% to 7.5% glycerol (v / v), 5% to 10% DMSO (v / v), 0% to 30% platelet lysate (v / v), and the balance being Ham's F-12. In a specific embodiment, the component may include the following components in the following amounts: 15 g / L to 30 g / L raffinose, 20 g / L to 22 g / L dextran, 5% to 7.5% glycerol (volume percentage), 5% to 8% DMSO (volume percentage), 12% to 20% platelet lysate (volume percentage), and the balance Ham's F-12. In another specific embodiment, the component may include the following components in the following amounts: 20 g / L raffinose, 22 g / L dextran, 7.5% glycerol (volume percentage), 5% DMSO (volume percentage), 15% platelet lysate (volume percentage), and the balance Ham's F-12.

[0019] In this invention, raffinose and dextran are non-permeable cryoprotectants; a variety of permeable lyophilizing cryoprotectants, characterized by glycerol and DMSO, leverage their small molecular weight to penetrate into cells before complete freezing, thereby reducing the electrolyte concentration difference between the intracellular and extracellular solutions, preventing intracellular water from escaping, and avoiding ice crystal formation that could damage cells; Ham's F-12 is selected as the basal culture medium to regulate cell osmotic pressure; platelet lysate can replace serum components such as fetal bovine serum, offering the advantage of being free of foreign proteins; reducing the amount of DMSO and increasing glycerol as a mixed permeable cryoprotectant provides cells with a less toxic and milder cryopreservation environment, which is beneficial for maintaining the original state and function of cells. This invention also provides a method for preparing the serum-free cell cryopreservation solution described above, including the following steps: The serum-free cell cryopreservation solution is prepared by mixing 0 g / L to 50 g / L of raffinose, 3 g / L to 30 g / L of dextran, 0% to 30% (v / v) of glycerol, 0% to 10% (v / v) of DMSO, 0% to 30% (v / v) of platelet lysate, and the remainder of Ham's F-12.

[0020] In a specific embodiment, the present invention first mixes raffinose, dextran, and Ham's F-12 to obtain a first mixture; after obtaining the first mixture, the present invention mixes the first mixture with glycerol and DMSO to obtain a second mixture; after obtaining the second mixture, the present invention mixes platelet lysate with the second mixture to obtain a serum-free cell cryopreservation solution. In a specific embodiment, the mixing is carried out by thorough stirring to ensure that all components are completely dissolved and homogeneous. When the values ​​of raffinose, glycerol, DMSO, or platelet lysate are 0, the addition of these components is omitted accordingly. After obtaining the serum-free cell cryopreservation solution, the present invention performs sterilization by 0.22 μm filtration and tests whether its various indicators meet the expected requirements, such as endotoxin content ≤0.5 EU / ml and absence of fungi, bacteria, mycoplasma, and other microorganisms. After passing the test, the serum-free cryopreservation solution is aliquoted, sealed, and stored at -20℃. Before subsequent use, it should be placed at 4℃ to allow it to thaw completely before use.

[0021] The present invention also provides the application of the serum-free cell cryopreservation solution described above in the cryopreservation of epidermal stem cells.

[0022] The present invention also provides the application of the serum-free cell cryopreservation solution described above in improving the cell viability of cryopreserved epidermal stem cells after thawing.

[0023] The present invention also provides the application of the serum-free cell cryopreservation solution described above in increasing the number of viable cells after cryopreserved epidermal stem cells are thawed.

[0024] The present invention also provides the application of the serum-free cell cryopreservation solution described above in protecting the cell expansion capacity of cryopreserved epidermal stem cells after thawing.

[0025] The present invention also provides the application of the serum-free cell cryopreservation solution described above in protecting the clonogenic ability of cryopreserved epidermal stem cells after thawing.

[0026] This invention also provides a method for cryopreserving epidermal stem cells using the serum-free cell cryopreservation solution described above, comprising the following steps: Epidermal stem cells are mixed with the serum-free cell cryopreservation solution described in the above technical solution, frozen, and then stored. In a specific embodiment, the epidermal stem cells of the present invention can be harvested from trophoblast cells. The present invention uses a serum-free cell cryopreservation solution to suspend epidermal stem cells, obtaining a single-cell suspension for cell counting. In a specific embodiment, the present invention can adjust the cell density of epidermal stem cells to 1×10⁻⁶. 6 / ml and aliquoted. After aliquoting, the cells can be stored in cryovials. In a specific embodiment, the cryovials can be stored in a programmed cooling box. In a specific embodiment, the programmed cooling box can be used for cryopreservation at -80°C for 24 hours, after which the cells can be transferred to a liquid nitrogen tank or stored at -80°C for long-term storage.

[0027] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a serum-free cell cryopreservation solution for epidermal stem cells, its preparation method, and its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 The effects of different cryopreservatives on the cryopreservation of epidermal stem cells.

[0029] 1. Prepare cell cryopreservation solution.

[0030] Experimental group 1: 0% DMSO + 7.5% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 2: 5% DMSO + 7.5% glycerol + 0% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 3: 0% DMSO + 15% glycerol + 0% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 4: 10% DMSO + 7.5% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 5: 0% DMSO + 0% glycerol + 0% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 6: 5% DMSO + 7.5% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 7: 5% DMSO + 15% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 8: 5% DMSO + 7.5% glycerol + 30% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 9: 10% DMSO + 15% glycerol + 0% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 10: 0% DMSO + 15% glycerol + 30% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 11: 10% DMSO + 0% glycerol + 30% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 12: 5% DMSO + 0% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 13: 10% DMSO + 0% glycerol + 0% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 14: 0% DMSO + 0% glycerol + 30% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 15: 10% DMSO + 15% glycerol + 30% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 16: Commercial cryopreservation solution (CryoStor® CS10, STEMCELL, catalog number: 07930).

[0031] The percentage (%) is the volume percentage. Each component in the experimental group was accurately measured and prepared into a 10 ml system in a 15 ml centrifuge tube. The system was then filtered through a 0.22 μm filter to remove bacteria and stored in a 15 ml sterile centrifuge tube for later use.

[0032] II. Experimental Methods (1) Epidermal stem cells are trophoblast (3T3 cells) dependent adherent cells. Epidermal stem cells with good growth status on trophoblast cells as a substrate were selected, washed once with PBS buffer, and 0.25% trypsin was added to digest the cells in a 37°C incubator. After the cells were completely digested, twice the volume of epidermal stem cell culture medium was added to stop the digestion. The cell suspension was transferred into a 50 ml sterile centrifuge tube, centrifuged at 450 g for 5 min to remove the supernatant, and trophoblast cells were removed using magnetic beads (Feeder Removal MicroBeads Mouse, Miltenyi) according to the instructions. The cell pellet was resuspended in 2-3 ml of epidermal stem cell culture medium and the cells were counted. (2) Supplement the epidermal stem cell culture medium according to the cell number, and replenish the cell suspension to 1×10⁻⁶. 6 At a cell density of / ml, 1ml of the well-mixed cell suspension was pipetted into 15ml sterile centrifuge tubes, centrifuged at 350g for 5 min to remove the supernatant, and 600 μl of cryopreservation solution for each experimental group was added and transferred to cryopreservation tubes that were labeled accordingly. The cryopreservation tubes were then transferred to a programmed cooling box and stored at -80℃. (3) After the cells are cryopreserved for 24 hours, the cell cryopreservation tubes are taken out of the programmed cooling box and quickly transferred to a 37°C water bath. The tubes are gently shaken continuously until the cell suspension melts. The cells are then transferred to a 15 ml centrifuge tube, an appropriate amount of epidermal stem cell culture medium is added, and the cells are slowly aspirated and mixed by pipetting. (4) Centrifuge at 350 g for 5 min, remove the supernatant, add 1 ml of epidermal stem cell culture medium, gently pipette to resuspend the cell pellet, mix well, and then take 20 μl of cell suspension for trypan blue staining. Add the remaining cell suspension to a six-well plate with feeder cells laid one day in advance and place it in a 37℃ incubator for further culture.

[0033] (5) After culturing for 2 days, wash once with PBS buffer, add 0.25% trypsin and digest the cells in a 37°C incubator. After the cells are completely digested, add twice the volume of epidermal stem cell culture medium to stop the digestion. Transfer the cell suspension into a 50 ml sterile centrifuge tube, centrifuge at 450 g for 5 min to remove the supernatant, and use magnetic beads (Feeder Removal MicroBeadsMouse, Miten) to remove the trophoblast cells according to the instructions to eliminate the influence on the epidermal stem cell count. Add 1 ml of epidermal stem cell culture medium to resuspend the cell pellet and count the cells.

[0034] III. Experimental Results The cell viability and number of viable cells of epidermal stem cells after 24 hours of cryopreservation are shown in the figure. Figure 1 .

[0035] The number of viable cells in epidermal stem cells two days after resuscitation is shown in [the figure]. Figure 2 .

[0036] The data above show that, using a cryopreservation buffer system based on Ham's F-12 medium, and adding 22 g / L dextran to prevent cell adhesion and 20 g / L raffinose as a non-permeable cryopreservative, the experimental groups can achieve good short-term and long-term cell counts after resuscitation without the introduction of serum components. Experimental group 6 showed the best results, achieving the optimal short-term and long-term cell counts after resuscitation using a formulation of 5% DMSO + 7.5% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + the remainder Ham's F-12 medium. In other words, the medium of this invention not only protects cells during cryopreservation and achieves a high cell recovery rate in the short term after cryopreservation, but also protects cell viability and proliferation capacity after resuscitation. The formulation of experimental group 6 showed the best results.

[0037] Example 2 The effects of different cryopreservation solutions on the cryopreservation of epidermal stem cells.

[0038] 1. Prepare cell cryopreservation solution.

[0039] Experimental group 16: Commercial cryopreservation solution (CryoStor® CS10, STEMCELL, catalog number: 07930). Experimental group 12: 5% DMSO + 0% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 4: 10% DMSO + 7.5% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 6: 5% DMSO + 7.5% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 17: 5% DMSO + 15% glycerol + 0% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 13: 10% DMSO + 0% glycerol + 0% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium.

[0040] The percentage (%) is the volume percentage. Each component in the experimental group was accurately measured and prepared into a 10 ml system in a 15 ml centrifuge tube. The system was then filtered through a 0.22 μm filter to remove bacteria and stored in a 15 ml sterile centrifuge tube for later use.

[0041] II. Experimental Methods (1) Epidermal stem cells are trophoblast (3T3 cells) dependent adherent cells. Epidermal stem cells with good growth status on trophoblast cells as a substrate were selected, washed once with PBS buffer, and 0.25% trypsin was added to digest the cells in a 37°C incubator. After the cells were completely digested, twice the volume of epidermal stem cell culture medium was added to stop the digestion. The cell suspension was transferred into a 50 ml sterile centrifuge tube, centrifuged at 450 g for 5 min to remove the supernatant, and trophoblast cells were removed using magnetic beads (Feeder Removal MicroBeads Mouse, Miltenyi) according to the instructions. The cell pellet was resuspended in 2-3 ml of epidermal stem cell culture medium and the cells were counted. (2) Supplement the epidermal stem cell culture medium according to the cell number, and replenish the cell suspension to 1×10⁻⁶. 6 At a cell density of / ml, 1ml of the well-mixed cell suspension was pipetted into 15ml sterile centrifuge tubes, centrifuged at 350g for 5 min to remove the supernatant, and 600 μl of cryopreservation solution for each experimental group was added and transferred to cryopreservation tubes that were labeled accordingly. The cryopreservation tubes were then transferred to a programmed cooling box and stored at -80℃. (3) After the cells are cryopreserved for 24 hours, the cell cryopreservation tubes are taken out of the programmed cooling box and quickly transferred to a 37°C water bath. The tubes are gently shaken continuously until the cell suspension melts. The cells are then transferred to a 15 ml centrifuge tube, an appropriate amount of epidermal stem cell culture medium is added, and the cells are slowly aspirated and mixed by pipetting. (4) Centrifuge at 350 g for 5 min, remove the supernatant, add 1 ml of epidermal stem cell culture medium, gently pipette to resuspend the cell pellet, mix well, and then take 20 μl of cell suspension for trypan blue staining. Add the remaining cell suspension to a six-well plate with feeder cells laid one day in advance and place it in a 37℃ incubator for further culture.

[0042] (5) After culturing for 2 days, wash once with PBS buffer, observe the colony formation of epidermal stem cells, and take photos. Figure 4 .

[0043] (6) In addition, cell culture plates containing epidermal stem cells 2 days after resuscitation were cultured in the same manner. Following the reagent instructions, immunostaining was performed using anti-p63 mouse monoclonal antibody (Beyotime brand) and DAB chromogenic solution (Biosharp brand), as shown below. Figure 5 The cell nuclei were counterstained with hematoxylin (Biosharp) to turn them blue-purple, and p63-positive stem cell clones were observed. Figure 6 .

[0044] III. Experimental Results The cell viability and number of viable cells of epidermal stem cells after 24 hours of cryopreservation are shown in the figure. Figure 3 .

[0045] The colony formation status of epidermal stem cells 2 days after resuscitation is shown in the figure. Figure 4 , Figure 5 and Figure 6 .

[0046] The results showed that, compared with the four superior cryopreservation solutions, the newly added experimental group 17, and commercial cryopreservation solutions, experimental group 6 achieved a higher cell recovery rate and cell viability after cryopreservation and thawing. Meanwhile, experimental group 6 ( Figure 6 In group D), there was obvious colony formation with deep p63 staining, and it was significantly larger than that in experimental groups 16, 12, 4, 17, and 13. The blank control group consisting only of trophoblast cells (…) Figure 5 G and Figure 6 G in the study group did not show stem cell clonal formation. It is evident that the cryopreservation solution formulation experimental group 6 ( Figure 6 D) in the formula not only protects the cell's proliferative capacity, but also facilitates the re-formation of clonal proliferation of epidermal stem cells on the trophoblast.

[0047] Example 3 The effects of different cryopreservatives on the cryopreservation of epidermal stem cells.

[0048] 1. Prepare cell cryopreservation solution.

[0049] Experimental group 1: 0% DMSO + 0% glycerol + 3 g / L dextran + 0 g / L raffinose + balance Ham's F-12 medium; Experimental group 2: 10% DMSO + 30% glycerol + 3 g / L dextran + 0 g / L raffinose + balance Ham's F-12 medium; Experimental group 3: 10% DMSO + 15% glycerol + 30 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 4: 5% DMSO + 15% glycerol + 30 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 5: 0% DMSO + 0% glycerol + 30 g / L dextran + 0 g / L raffinose + balance Ham's F-12 medium; Experimental group 6: 10% DMSO + 0% glycerol + 22 g / L dextran + 0 g / L raffinose + balance Ham's F-12 medium; Experimental group 7: 5% DMSO + 0% glycerol + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 8: 0% DMSO + 0% glycerol + 22 g / L dextran + 50 g / L raffinose + balance Ham's F-12 medium; Experimental group 9: 5% DMSO + 30% glycerol + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 10: 10% DMSO + 0% glycerol + 3 g / L dextran + 50 g / L raffinose + balance Ham's F-12 medium; Experimental group 11: 0% DMSO + 15% glycerol + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 12: 5% DMSO + 15% glycerol + 3 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 13: 10% DMSO + 0% glycerol + 22 g / L dextran + 50 g / L raffinose + balance Ham's F-12 medium; Experimental group 14: 10% DMSO + 30% glycerol + 30 g / L dextran + 50 g / L raffinose + balance Ham's F-12 medium; Experimental group 15: 5% DMSO + 15% glycerol + 22 g / L dextran + 0 g / L raffinose + balance Ham's F-12 medium; Experimental group 16: 0% DMSO + 30% glycerol + 22 g / L dextran + 0 g / L raffinose + balance Ham's F-12 medium; Experimental group 17: 10% DMSO + 30% glycerol + 30 g / L dextran + 0 g / L raffinose + balance Ham's F-12 medium; Experimental group 18: 0% DMSO + 30% glycerol + 30 g / L dextran + 50 g / L raffinose + balance Ham's F-12 medium; Experimental group 19: 5% DMSO + 15% glycerol + 22 g / L dextran + 50 g / L raffinose + balance Ham's F-12 medium; Experimental group 20: 0% DMSO + 30% glycerol + 3g / L dextran + 50g / L raffinose + balance Ham's F-12 medium; Experimental group 21: 10% DMSO + 15% glycerol + 1.28% dextran + 20 g / L raffinose + balance Ham's F-12 medium.

[0050] The percentage (%) is the volume percentage. Each component in the experimental group was accurately measured and prepared into 10 ml systems in 15 ml centrifuge tubes. The systems were then filtered through a 0.22 μm filter to remove bacteria and stored in clean 15 ml sterile centrifuge tubes for later use.

[0051] II. Experimental Methods (1) Epidermal stem cells are trophoblast (3T3 cells) dependent adherent cells. Epidermal stem cells with good growth status on trophoblast cells as a substrate were selected, washed once with PBS buffer, and 0.25% trypsin was added to digest the cells in a 37°C incubator. After the cells were completely digested, twice the volume of epidermal stem cell culture medium was added to stop the digestion. The cell suspension was transferred into a 50 ml sterile centrifuge tube, centrifuged at 450 g for 5 min to remove the supernatant, and trophoblast cells were removed using magnetic beads (Feeder Removal MicroBeads Mouse, Miltenyi) according to the instructions. The cell pellet was resuspended in 2-3 ml of epidermal stem cell culture medium and the cells were counted. (2) Supplement the epidermal stem cell culture medium according to the cell number, and replenish the cell suspension to 1×10⁻⁶. 6 At a cell density of / ml, 1ml of the well-mixed cell suspension was pipetted into 15ml sterile centrifuge tubes, centrifuged at 350g for 5 min to remove the supernatant, and 600 μl of cryopreservation solution for each experimental group was added and transferred to cryopreservation tubes that were labeled accordingly. The cryopreservation tubes were then transferred to a programmed cooling box and stored at -80℃. (3) After the cells are cryopreserved for 24 hours, the cell cryopreservation tubes are taken out of the programmed cooling box and quickly transferred to a 37°C water bath. The tubes are gently shaken continuously until the cell suspension melts. The cells are then transferred to a 15 ml centrifuge tube, an appropriate amount of epidermal stem cell culture medium is added, and the cells are slowly aspirated and mixed by pipetting. (4) Centrifuge at 350 g for 5 min, remove the supernatant, add 1 ml of epidermal stem cell culture medium, gently pipette to resuspend the cell pellet, mix well, and then take 20 μl of cell suspension for trypan blue staining. Add the remaining cell suspension to a six-well plate with feeder cells laid one day in advance and place it in a 37℃ incubator for further culture.

[0052] (5) After culturing for 2 days, wash once with PBS buffer, add 0.25% trypsin and digest the cells in a 37°C incubator. After the cells are completely digested, add twice the volume of epidermal stem cell culture medium to stop the digestion. Transfer the cell suspension into a 50 ml sterile centrifuge tube, centrifuge at 450 g for 5 min to remove the supernatant, and use magnetic beads (Feeder Removal MicroBeadsMouse, Miten) to remove the trophoblast cells according to the instructions to eliminate the influence on the epidermal stem cell count. Add 1 ml of epidermal stem cell culture medium to resuspend the cell pellet and count the cells.

[0053] III. Experimental Results The cell viability and number of viable cells of epidermal stem cells after 24 hours of cryopreservation are shown in the figure. Figure 7 .

[0054] The number of viable cells in epidermal stem cells two days after resuscitation is shown in [the figure]. Figure 8 .

[0055] The results showed that cryopreservation solutions with no or low raffinose and dextran content had poor cryopreservation effects (Experimental Groups 1 and 2). However, cryopreservation solutions with the highest raffinose and dextran content (Experimental Groups 14 and 18) also failed to achieve ideal results. Furthermore, there were complex relationships between the components of each cryopreservation solution. For example, increasing the glycerol content, even with appropriate raffinose and dextran concentrations, did not yield ideal experimental results (as demonstrated in Experimental Group 9). Therefore, statistical calculations suggest that optimizing other components based on 22 g / L dextran and 20 g / L raffinose is a more robust approach.

[0056] Example 4 Determination of cryopreservation curves in programmed cooling boxes Cell cryovials prepared with a formulation of 5% DMSO + 7.5% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + the remainder Ham's F-12 medium were placed in a programmed cooling box. The temperature profiles of the cell cryovials were measured using a digital temperature recorder. Simultaneously, the cryovial profiles of water were measured under the same conditions for comparison. Figure 9 ).

[0057] Figure 9 The cryopreservation profile of the cell cryoprotectant is shown (with pure water as a reference). It can be seen that cell cryovials prepared with the formula 5% DMSO + 7.5% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + the balance Ham's F-12 medium exhibit a more gradual cooling process in a programmed cooling chamber than with pure water, reaching a eutectic point at around -20°C. This cryopreservation profile is well-suited for existing commercially available programmed cooling equipment, requiring no special devices.

[0058] Comparative Example 1 The effects of different cryopreservation solutions on the cryopreservation of 3T3 cells.

[0059] 1. Prepare cell cryopreservation solution.

[0060] Experimental group 16: Commercial cryopreservation solution (CryoStor® CS10, STEMCELL, catalog number: 07930). Experimental group 12: 5% DMSO + 0% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 4: 10% DMSO + 7.5% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 6: 5% DMSO + 7.5% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 17: 5% DMSO + 15% glycerol + 0% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 13: 10% DMSO + 0% glycerol + 0% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; The percentages are volume percentages. Each component in the experimental group was accurately measured and prepared into 10 ml systems in 15 ml centrifuge tubes. The systems were then filtered through a 0.22 μm filter to remove bacteria and stored in 15 ml sterile centrifuge tubes for later use.

[0061] II. Experimental Methods (1) Select 3T3 cells in good growth condition (purchased from the Cell Bank of Chinese Academy of Sciences, catalog number SCSP-5012), wash once with an appropriate amount of PBS buffer, add 0.25% trypsin and digest the cells in a 37℃ incubator. After the cells are completely digested, add twice the volume of 3T3 cell culture medium to stop the digestion. Transfer the cell suspension into a 50 ml sterile centrifuge tube, centrifuge at 450 g for 5 min to remove the supernatant, add 2~3 ml of 3T3 cell culture medium to resuspend the cell pellet and count the cells. (2) Supplement 3T3 cell culture medium according to the cell number, and bring the cell suspension to 1×10⁻⁶. 6 At a cell density of / ml, 1ml of the well-mixed cell suspension was pipetted into 15ml sterile centrifuge tubes, centrifuged at 350g for 5 min to remove the supernatant, and 600 μl of cryopreservation solution for each experimental group was added and transferred to cryopreservation tubes that were labeled accordingly. The cryopreservation tubes were then transferred to a programmed cooling box and stored at -80℃. (3) After the cells are cryopreserved for 24 hours, the cell cryopreservation tubes are taken out of the programmed cooling box and quickly transferred to a 37°C water bath. The tubes are gently shaken continuously until the cell suspension melts. The cells are then transferred to a 15 ml centrifuge tube, an appropriate amount of 3T3 cell culture medium is added, and the cells are slowly aspirated and mixed by pipetting. (4) Centrifuge at 350 g for 5 min, remove the supernatant, add 1 ml of 3T3 cell culture medium, gently pipette to resuspend the cell pellet, mix well, and then take 20 μl of cell suspension for trypan blue staining. Add the remaining cell suspension to a six-well plate and place it in a 37℃ incubator for further culture.

[0062] (5) After culturing for 3 days, wash once with an appropriate amount of PBS buffer and observe the proliferation of 3T3 cells. After observation, add 0.25% trypsin to digest the cells in a 37℃ incubator. After the cells are completely digested, add twice the volume of 3T3 cell culture medium to stop the digestion. Transfer the cell suspension to a 50 ml sterile centrifuge tube, centrifuge at 450 g for 5 min to remove the supernatant, add 1 ml of 3T3 cell culture medium to resuspend the cell pellet and count the cells.

[0063] III. Experimental Results The cell viability and number of viable cells of 3T3 cells after 24 hours of cryopreservation are shown in the figure. Figure 10 .

[0064] The number of 3T3 cells expanded 3 days after resuscitation is shown in the figure. Figure 11 .

[0065] The results showed that the protective effect of experimental group 6 on the cryopreservation of 3T3 cells was not as significant as that of commercial cryopreservation solution, indicating that the patented formula has a poor protective effect on the cryopreservation of 3T3 cells.

[0066] Comparative Example 2 The effects of different cryopreservation solutions on the cryopreservation of hair follicle stem cells.

[0067] 1. Prepare cell cryopreservation solution.

[0068] Experimental group 16: Commercial cryopreservation solution (CryoStor® CS10, STEMCELL, catalog number: 07930). Experimental group 12: 5% DMSO + 0% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 4: 10% DMSO + 7.5% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 6: 5% DMSO + 7.5% glycerol + 15% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 17: 5% DMSO + 15% glycerol + 0% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium; Experimental group 13: 10% DMSO + 0% glycerol + 0% platelet lysate + 22 g / L dextran + 20 g / L raffinose + balance Ham's F-12 medium.

[0069] The percentages are volume percentages. Each component in the experimental group was accurately measured and prepared into 10 ml systems in 15 ml centrifuge tubes. The systems were then filtered through a 0.22 μm filter to remove bacteria and stored in 15 ml sterile centrifuge tubes for later use.

[0070] II. Experimental Methods (1) Hair follicle stem cells are trophoblast (3T3 cells) dependent adherent cells. Select hair follicle stem cells that are growing well on the trophoblast cells as a substrate, wash once with PBS buffer, add 0.25% trypsin and digest the cells in a 37℃ incubator. After the cells are completely digested, add twice the volume of hair follicle stem cell culture medium to stop the digestion. Transfer the cell suspension into a 50 ml sterile centrifuge tube, centrifuge at 450 g for 5 min to remove the supernatant, use magnetic beads (Feeder Removal MicroBeads Mouse, Miltenyi) to remove the trophoblast cells according to the instructions, add 2~3 ml of hair follicle stem cell culture medium to resuspend the cell pellet and count the cells. (2) Replenish the hair follicle stem cell culture medium according to the number of cells, and replenish the cell suspension to 1×10⁻⁶. 6 At a cell density of / ml, 1ml of the well-mixed cell suspension was pipetted into 15ml sterile centrifuge tubes, centrifuged at 350g for 5 min to remove the supernatant, and 600 μl of cryopreservation solution for each experimental group was added and transferred to cryopreservation tubes that were labeled accordingly. The cryopreservation tubes were then transferred to a programmed cooling box and stored at -80℃. (3) After the cells are cryopreserved for 24 hours, the cell cryopreservation tubes are taken out of the programmed cooling box and quickly transferred to a 37°C water bath. The tubes are gently shaken continuously until the cell suspension melts. The cells are then transferred to a 15 ml centrifuge tube, an appropriate amount of hair follicle stem cell culture medium is added, and the cells are slowly aspirated and mixed by pipetting. (4) Centrifuge at 350 g for 5 min, remove the supernatant, add 1 ml of hair follicle stem cell culture medium, gently pipette to resuspend the cell pellet, mix well, and then take 20 μl of cell suspension for trypan blue staining. Add the remaining cell suspension to a six-well plate with a feeder layer prepared one day in advance and place it in a 37℃ incubator for further culture.

[0071] (5) After culturing for 2 days, wash once with PBS buffer, add 0.25% trypsin and digest the cells in a 37°C incubator. After the cells are completely digested, add twice the volume of hair follicle stem cell culture medium to stop the digestion. Transfer the cell suspension into a 50 ml sterile centrifuge tube, centrifuge at 450 g for 5 min to remove the supernatant, and use magnetic beads (Feeder Removal MicroBeadsMouse, Miten) to remove the trophoblast cells according to the instructions to eliminate the influence on the hair follicle stem cell count. Add 1 ml of hair follicle stem cell culture medium to resuspend the cell pellet and count the cells.

[0072] III. Experimental Results The viability and number of live cells of hair follicle stem cells after 24 hours of cryopreservation are shown in the figure. Figure 12 .

[0073] The colony morphology and number of hair follicle stem cells after 7 days of resuscitation are shown in the figure. Figure 13 and Figure 14 .

[0074] The results showed that although the resuscitation viability and cell number of hair follicle stem cells after cryopreservation and thawing in experimental group 6 were better than those in commercial cryopreservation solutions, the cell proliferation capacity during culture was far less significant than that of hair follicle stem cells cryopreserved and thawing in commercial cryopreservation solutions. This indicates that the patented formula (experimental group 6) is not suitable for the cryopreservation of hair follicle stem cells.

[0075] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A serum-free cryopreservation solution for epidermal stem cells, characterized in that, The components include the following: raffinose at 0 g / L to 50 g / L, dextran at 3 g / L to 30 g / L, glycerol at 0% to 30% by volume, DMSO at 0% to 10% by volume, platelet lysate at 0% to 30% by volume, and the balance being Ham's F-12; wherein the values ​​of DMSO and glycerol are not both 0%.

2. The serum-free cell cryopreservation solution according to claim 1, characterized in that, It includes the following components in the following amounts: raffinose 0 g / L to 50 g / L, dextran 3 g / L to 22 g / L, glycerol 0% to 7.5% by volume, DMSO 5% to 10% by volume, platelet lysate 0% to 30% by volume, and the balance Ham's F-12.

3. The serum-free cell cryopreservation solution according to claim 1, characterized in that, It includes the following components by volume percentage: 20 g / L raffinose, 22 g / L dextran, 7.5% glycerol by volume percentage, 5% DMSO by volume percentage, 15% platelet lysate by volume percentage, and the balance Ham's F-12.

4. A method for preparing the serum-free cell cryopreservation solution according to any one of claims 1 to 3, comprising the following steps: The serum-free cell cryopreservation solution is prepared by mixing 0 g / L to 50 g / L of raffinose, 3 g / L to 30 g / L of dextran, 0% to 30% (v / v) of glycerol, 0% to 10% (v / v) of DMSO, 0% to 30% (v / v) of platelet lysate, and the remainder of Ham's F-12.

5. The use of the serum-free cell cryopreservation solution according to any one of claims 1 to 3 in the cryopreservation of epidermal stem cells.

6. The use of the serum-free cell cryopreservation solution according to any one of claims 1 to 3 in improving the cell viability of cryopreserved epidermal stem cells after thawing.

7. The use of the serum-free cell cryopreservation solution according to any one of claims 1 to 3 in increasing the number of viable cells after thawing cryopreserved epidermal stem cells.

8. The use of the serum-free cell cryopreservation solution according to any one of claims 1 to 3 in protecting the cell expansion capacity of cryopreserved epidermal stem cells after thawing.

9. The use of the serum-free cell cryopreservation solution according to any one of claims 1 to 3 in protecting the clonogenic ability of cryopreserved epidermal stem cells after thawing.

10. A method for cryopreserving epidermal stem cells using the serum-free cell cryopreservation solution according to any one of claims 1 to 3, comprising the following steps: Epidermal stem cells are mixed with the serum-free cell cryopreservation solution described in any one of claims 1 to 3, and then frozen and stored.

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