CN107114357A - The frozen stock solution and its cryopreservation methods of a kind of periodontal ligament stem cell - Google Patents

Abstract

The invention belongs to the frozen stock solution and its cryopreservation methods in cell cryopreservation field, more particularly to a kind of periodontal ligament stem cell.The frozen stock solution of the present invention includes glycerine, mesenchymal stem cell serum-free nutrient solution and human serum albumin.It is poor that the invention can effectively solve existing frozen stock solution using effect, the low technological deficiency of periodontal ligament stem cell motility rate after recovery.The periodontal ligament stem cell frozen stock solution of the present invention can not only keep freeze-stored cell activity, and periodontal ligament stem cell motility rate is significantly improved after recovery, not influence expression and the differentiation potential of stem cell of Stem cell surface marker thing also.

Description

A kind of cryopreservation solution and cryopreservation method of periodontal ligament stem cells

technical field

The invention belongs to the field of cell cryopreservation, and in particular relates to a cryopreservation solution and a cryopreservation method for periodontal ligament stem cells.

Background technique

Periodontal disease is not only the main cause of tooth loss in adults, but also a major oral disease that endangers the health of human oral cavity and even the whole body. Periodontal disease is an infectious disease stimulated by chronic inflammation of periodontal tissue. It is an inflammation caused by microorganisms in dental plaque in a corresponding microenvironment. It damages periodontal tissue, especially the destruction of alveolar bone and periodontal tissue. Loss of tissue, eventually leading to loosening of teeth and resorption of alveolar bone. The goal of periodontal treatment is to treat periodontal inflammation, improve the microenvironment to regenerate new connective tissue attachment and support tissue, and complete the construction of alveolar bone, cementum and periodontal ligament. Commonly used periodontal regeneration methods include guided tissue regeneration, root planing, etc. These methods are simple and easy to implement, but they cannot fully achieve functional and structural periodontal tissue regeneration, and there are corresponding limitations. The emergence and development of periodontal tissue engineering technology provides a new idea and method for periodontal regeneration.

Periodontal ligament is a layer of connective tissue between the tooth root and alveolar bone, and is an important periodontal support tissue, mainly composed of fibroblasts, cementoblasts and some undifferentiated mesenchymal cells. Under normal circumstances, periodontal tissue has its own ability to renew and repair, and its repair activity is realized through the self-renewal of periodontal ligament cells. During disease or when receiving external stimuli, the tissue is regenerated through the continuous proliferation and differentiation of cells in the periodontal ligament. In 2004, Seo et al isolated periodontal ligament stem cells (Periodontal Ligament Sem Cells, PDLSCs) from the extracted third molar periodontal ligament tissue. In addition, PDLSCs also existed on the inner surface of the alveolar socket after tooth extraction. Recently, highly purified PDLSCs clones were obtained from adult periodontal tissues. It is worth noting that more studies have found that PDLSCs can also be isolated from inflamed periodontal ligament tissue, and proved that they still maintain the potential of regenerating cementum and periodontal ligament tissue, which also makes up for the lack of its source to a certain extent. disadvantages. Studies have shown that PDLSCs express the markers STRO-1 and CD146/MUC18 of mesenchymal stem cells, which means that PDLSCs are likely to be derived from the perivascular cell population. And this is very similar to Bone Marrow Mesenchymal Stem Cells (BMMSCs) that also express STRO-1 and CD146/MUC18. PDLSCs also have a multi-directional differentiation ability similar to BMSCs, and can differentiate into a variety of cells in vitro, such as adipocytes, chondrocytes, nerve cells, etc., and play a role in maintaining the stability of periodontal tissue and maintaining the metabolism of alveolar bone. important role.

Although some studies have used adult stem cells from a variety of tissue sources, including fat and bone marrow, for periodontal regeneration, the invasiveness and ethical issues of the materials still restrict its development. Therefore, PDLSCs are expected to be the preferred seed cells for periodontal tissue regeneration.

In recent years, the need to establish a stem cell bank and the clinical demand for stem cells have increased, and research on stem cell cryopreservation and recovery has attracted more and more attention. Like other sources of MSCs, PDLSCs are used as seed cells for tissue engineering research and potential clinical applications. Over-passaging will show obvious senescence or apoptosis, and long-term in vitro culture is prone to spontaneous differentiation, loss of multi-differentiation potential, and decreased proliferation and adhesion capabilities. , the apoptosis rate increases, so cryopreservation of PDLSCs is also one of the important links in its research and application.

Cell cryopreservation solution is a solution used for cell cryopreservation. It can supply the nutrients necessary for cell life and metabolism, and at the same time prevent or reduce the damage of frozen ice crystals to cells. The currently commonly used cell cryopreservation solution or commercially available cell cryopreservation solution is usually composed of a mixture of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS). DMSO is a chemical substance with small molecular weight and strong solubility and penetration ability. In many studies, DMSO is the most commonly used cell cryoprotectant, and the cells preserved with it have similar phenotypes, cell surface markers and growth rates after thawing compared with fresh cells. The mechanism of action of DMSO is to penetrate the cell membrane into the cell during the cooling process, reduce the electrolyte concentration in the unfrozen solution inside and outside the cell, thereby protecting the cell from high-concentration electrolyte damage, and at the same time, the water in the cell will not leak excessively, avoiding Excessive dehydration and shrinkage of cells. However, DMSO has a certain toxic effect on cells, and too high concentration will cause high osmotic pressure, which is not conducive to cell recovery. Therefore, the current routine use concentration of DMSO is 5%-15%. FBS is a heterogeneous substance with complex components and the risk of introducing contamination and allergens, so it is not suitable for clinical application. Especially in cell therapy, the presence of heterologous proteins may cause unnecessary or even unknown adverse reactions, seriously affecting the treatment results.

In summary, the traditional cell cryopreservation solution will produce toxic and immunogenic reactions to periodontal ligament stem cells. Therefore, the development of a solution that does not produce cytotoxicity and immunogenicity to periodontal ligament stem cells can also significantly improve cell cryopreservation. The cryopreservation solution of post-resuscitation viability is a technical problem to be solved urgently by those skilled in the art.

Contents of the invention

In view of this, the present invention provides a cryopreservation solution of periodontal ligament stem cells and a cryopreservation method thereof, which can effectively solve the poor cryopreservation effect caused by the toxicity and immunogenic reaction of traditional cryopreservation solutions to periodontal ligament stem cells, Technical flaw of low cell viability after thawing.

The cryopreservation solution of periodontal ligament stem cells provided by the invention comprises glycerin, serum-free culture solution of mesenchymal stem cells and human serum albumin.

Preferably, the cryopreservation solution includes: 10% by volume of glycerol, 30-60% by volume of mesenchymal stem cell serum-free culture medium, and 30% by volume of human serum albumin. -60%.

Preferably, the cryopreservation solution includes: 10% by volume of glycerol, 40% by volume of mesenchymal stem cell serum-free culture medium, and 50% by volume of human albumin.

Preferably, the mass concentration of human serum albumin is 20%.

Further, the present invention also discloses a method for cryopreserving periodontal ligament stem cells, which includes the following steps: adding the cryopreservation solution to the periodontal ligament stem cells, mixing them and then freezing them.

Preferably, the cryopreservation solution is added in an amount until the density of periodontal ligament stem cells is 1.0×10 ⁶ cells/mL-5×10 ⁶ cells/mL.

As a preference, the cryopreservation specifically includes mixing the cryopreservation solution with the periodontal ligament stem cells and then distributing them into cryopreservation tubes, putting them into a programmed cooling box that restores room temperature in advance, and placing them in a -80°C ultra-low temperature refrigerator for 2 - After 3 days, transfer to liquid nitrogen for storage.

The invention discloses a periodontal ligament stem cell cryopreservation solution, which comprises glycerin, mesenchymal stem cell serum-free culture solution and human serum albumin. Through the synergistic effect of glycerol, mesenchymal stem cell serum-free culture medium and human serum albumin. Among them, glycerin can be used as an osmotic cryoprotectant. Before the frozen cell suspension is completely solidified, it penetrates into the cell, generates a certain molar concentration inside and outside the cell, and reduces the concentration of electrolyte in the unfrozen solution inside and outside the cell, thereby protecting the cell from freezing. High-concentration electrolyte damage, while the intracellular water will not leak excessively, avoiding excessive dehydration and shrinkage of cells; serum-free culture medium and human serum albumin of mesenchymal stem cells can maintain normal osmotic pressure of cells and maintain the integrity of cell membranes. Maintain normal PH value and protect the normal function of cell membrane surface protein. The invention can greatly improve the permeability of the cell membrane to water, make the water in the cell seep out of the cell, reduce the formation of ice crystals in the cells, and thereby reduce the cell damage caused by the formation of ice crystals. This cryopreservation solution does not contain DMSO, which avoids its toxic effect on cells, and does not contain animal-derived serum, which avoids the risk of introducing contamination and allergens. Compared with conventional cell cryopreservation solutions, it has higher clinical safety. At the same time, the periodontal ligament stem cell cryopreservation solution and the cryopreservation method of the present invention can maintain the activity of cryopreserved periodontal ligament stem cells. Affects its differentiation potential, especially into adipocytes and osteoblasts.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art.

Figure 1 shows a graph of the expression rate of periodontal ligament stem cell surface markers;

Fig. 2 shows the cell resuscitation result and cell viability statistics of the resuscitated cells in the embodiment;

Fig. 3 shows the cell growth curve chart adopting comparative example culture;

Fig. 4 shows the cell growth curve that adopts embodiment 3 to cultivate;

Fig. 5 shows the image of the cell growth form adopting comparative example and embodiment 3 culture (the left column is the comparative example, the right column is the embodiment 3, the first, second, and third horizontal lines are the cell growth diagrams of the first, second, and third days);

Fig. 6 shows the image of the cell growth morphology that adopts comparative example and embodiment 3 to cultivate (the left column is comparative example, the right column is embodiment 3, and the 1st, 2nd, 3rd rows are the cell growth diagrams of the fourth, fifth, and sixth days);

Fig. 7 shows the image of the cell growth morphology cultivated by the comparative example and embodiment 3 (the left column is the comparative example, the right column is the embodiment 3, and the first row is the cell growth diagram on the seventh day);

Fig. 8 shows the PDLSCs surface marker expression rate figure of embodiment 3;

Fig. 9 shows comparative example and embodiment 3 osteogenic differentiation effect figure (200×);

Fig. 10 shows the effects of adipogenic differentiation in Comparative Example and Example 3 (200×).

Among them, CD146-A, CD105-A, CD73-A, HLA-DR-A, CD34-A, and CD45-A in Figure 1 and Figure 8 are surface antigens CD146, CD105, CD73, HLA-DR, and CD34 , CD45, and the vertical axis is the antigen expression rate.

detailed description

The invention provides a periodontal ligament stem cell cryopreservation solution and a cryopreservation method, which can effectively solve the problem of poor cryopreservation effect caused by the toxicity and immunogenic reaction of traditional cryopreservation solutions to periodontal ligament stem cells, and the low cell viability after resuscitation technical flaws.

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Wherein, the serum-free culture medium of mesenchymal stem cells used in the present invention is a commercially available product: LONZA12-725F, UltraCULTURE Serum-free Medium (SFM).

Example 1

Primary isolation, culture, identification and passage of PDLSCs were carried out.

Primary isolation and culture of PDLSCs includes the following steps:

1. Material collection: Take the third molars of 12-18-year-old healthy donors that need to be extracted for orthodontics, and quickly immerse them in PBS that has been pre-cooled at 4°C and contains 3 times the volume of double antibodies.

2. Rinsing: Rinse the tooth body with PBS containing 3 times the double antibody from the root to the crown, thoroughly remove blood stains, repeat 3 times.

3. Scraping of periodontal tissue: Scrape the periodontal ligament tissue of the middle and lower 1/3 section of the root with a scalpel blade in one direction, transfer it into a centrifuge tube, and cut the tissue block into a size of about 1 mm ³ with ophthalmic scissors.

4. Digestion: add an appropriate amount of collagenase-dispase enzyme 1:1 mixed digestion solution (collagenase 3g/L, dispase enzyme 4g/L), and place in a constant temperature shaker at 37°C for digestion for 30-35min. After digestion, centrifuge at 1000r/min for 5min and discard the supernatant. Add an appropriate amount of PBS to resuspend, centrifuge at 1000r/min for 5min, and discard the supernatant.

5. Inoculation: Add culture medium (DMEM culture medium containing 10% FBS by volume) to resuspend the cells, inoculate in a six-well plate, and culture in a 5% _CO2 incubator at 37°C.

6. Purification: After the cells cover 80%-90% of the cell plate area, collect the cells, and purify the cells by magnetic bead sorting. After incubating the collected cells with STRO-1 monoclonal antibody at 4°C for 1 hour, and The beads were mixed and incubated at 4°C for 30 min, and STRO-1 and PDLSCs were screened out under the action of magnetic equipment, and PDLSCs were seeded in a new six-well plate and cultured at 37°C in a 5% CO ₂ incubator.

PDLSCs were identified, including the following steps: after the cells covered 80-90% of the plate area, the cells (about 1×10 ⁶ ) were collected, soaked in 3g/L Triton for 20min, washed 3 times with PBS, 10% goat The serum was blocked at room temperature for 2 hours, and 50 μL of CD146, CD105, CD73, HLA-DR, CD34, and CD45 antibodies diluted at 1:200 were added dropwise, while the control group (negative group without antibody incubation) was added dropwise with 50 μL of pure PBS, and treated at 4°C for 16 hours The next day, place at room temperature for 30 minutes, rinse with PBS three times, each group was added dropwise 1:500 diluted FITC-labeled secondary antibody IgG, incubate at room temperature in the dark for 1 hour, rinse with PBS twice, fix the cells in 10% formalin, and flow The positive rate of antigen was determined by cytometer.

The results showed that compared with the control group, the surface antigens of the isolated PDLSCs were positive for CD146, CD105, and CD73, while HLA-DR, CD34, and CD45 were negative, indicating that PDLSCs were successfully isolated from teeth.

Table 1 Expression rate of cell surface markers in PDLSCs

cell phenotype CD146 CD105 CD73 HLA-DR CD34 CD45 Positive expression rate (%) 99.80 99.80 99.80 0.20 0.20 0.30

Passage of PDLSCs includes the following steps: PDLSCs to be identified as positive are plated, and after covering 80-90% of the plate area, use a straw to discard the old culture medium, add 0.25% trypsin, digest for 1-3 minutes, and observe under the microscope When the cells shrink and become round, immediately add DMEM culture solution containing 10% by volume FBS to stop the digestion, collect the cells, centrifuge at 1000r/min for 5min, and discard the supernatant. Add DMEM culture solution containing 10% volume percentage FBS, count the cells, inoculate in a culture dish at a density of ⁵ ×104/mL for subculture, and culture at 37°C in a 5% CO ₂ incubator, every 2-3 Change the solution every day.

comparative example

Preparation of the cryopreservation solution of the comparative example: DMSO with a volume percentage content of 10% of the cryopreservation solution and FBS with a volume percentage content of 90% of the cryopreservation solution were mixed to prepare the cryopreservation solution of the comparative example, and refrigerated at 4°C spare.

Example 2

The preparation of the cryopreservation liquid of embodiment 2: the glycerol with the volume percentage content of the cryopreservation liquid of embodiment 2 being 10%, the mesenchymal stem cell serum-free culture medium with the volume percentage content of the freezing liquid of embodiment 2 of 30% and the volume percentage of the frozen storage solution of Example 2 is 60% human albumin (human serum albumin mass concentration is 20%), after mixing, the frozen storage solution of Example 2 is prepared, and it is refrigerated at 4°C for subsequent use .

Example 3

Preparation of the cryopreservation solution in Example 3: Serum-free culture of the mesenchymal stem cells in which the volume percentage of the cryopreservation solution in Example 3 is 10% glycerol and 40% by volume solution and the volume percentage of the frozen storage solution of Example 3 is 50% human albumin (the mass concentration of human serum albumin is 20%), and after mixing, the frozen storage solution of Example 3 is prepared, and the frozen storage solution of Example 3 is prepared at 4°C in a refrigerator Refrigerate and set aside.

Example 4

Example 4 Preparation of freezing solution: 10% by volume of glycerol, 50% by volume of mesenchymal stem cell serum-free culture medium and 40% by volume of human serum albumin ( The mass concentration of human serum albumin is 20%), after mixing, the cryopreservation solution of Example 4 is prepared, and it is refrigerated in a refrigerator at 4°C for subsequent use.

Example 5

Preparation of the frozen storage solution in Example 5: Glycerin with a volume percentage of 10% of the frozen storage solution in Example 3, and serum-free mesenchymal stem cells with a volume percentage of 60% in the frozen storage solution in Example 3 The culture fluid and the volume percentage of the frozen storage solution of Example 3 are 30% human albumin (human serum albumin mass concentration is 20%), mixed to prepare the frozen storage solution of Example 5, at 4°C Refrigerate for later use.

Example 6

The steps for cell freezing are as follows:

1. Cell collection: Select the third-generation PDLSCs, wait until the cells cover 80-90% of the plate, use a pipette to discard the old culture medium, add an appropriate amount of 0.25% trypsin, digest for 1-3 minutes, and observe the shrinkage and roundness of the cells under the microscope Immediately add an appropriate amount of serum-free medium for mesenchymal stem cells to terminate the digestion, collect the cells, centrifuge at 1000r/min for 5min, and discard the supernatant.

2. Cryopreservation: cryopreserve the cells with the prepared cryopreservation solution of each example and comparative example, the amount of the cryopreservation solution is such that the density of periodontal ligament stem cells is 1.0×10 ⁶ cells/mL, and the setting is repeated 3 times. Dispense into 2mL cryopreservation tubes, 1.5mL/tube, put them into a programmed cooling box that restores room temperature in advance, and place in a -80°C ultra-low temperature refrigerator. The programmed cooling box can ensure that the temperature drops at a rate of 1°C/min; transfer to liquid after 2 days nitrogen preservation. Example 7

Resuscitating the cells of each embodiment and comparative example includes the following steps: after the frozen cells of the comparative examples and each embodiment are frozen in liquid nitrogen for one month, three groups are taken out to repeat the cell recovery, and the cryopreservation tubes are taken out and put away immediately. Dissolve in a 37°C water bath, and shake the cryotube constantly during the dissolution process. 1-2min after the liquid melted, dilute the cell suspension with 10mL of mesenchymal stem cell serum-free medium (wash the cryotube once with the medium), mix well and take 0.5mL of the cell suspension for cell counting and activity detection. Experimental results show that the cryopreservation effect of the PDLSCs cryopreservation solution of the present invention is significantly better than that of conventional cell cryopreservation solutions (Table 2, Figure 2).

Table 2 adopts the cell resuscitation result and cell viability statistics of embodiment resuscitated cells

Example 8

Comparing the cell growth curves of Example 3 and Comparative Example includes the following steps: after the frozen cells of Comparative Example and Example 3 are frozen in liquid nitrogen for one month, the cryopreservation tube is taken out and immediately put into a 37°C water bath to dissolve, Shake the cryovial constantly during the thawing process. After thawing the liquid for 1-2 minutes, dilute the cell suspension with 10 mL of serum-free medium for mesenchymal stem cells (wash the cryopreservation tube once with the medium), centrifuge at 1000 r/min for 5 minutes, and discard the supernatant. After being resuspended in serum-free medium for mesenchymal stem cells containing 10 ng/ml EGF, the cells were seeded in a 24-well plate at a density of 1×10 ⁴ cells/mL, and cultured in a 5% CO ₂ incubator at 37°C. Cells were collected every day for cell counting, and three replicates (well 1, well 2, and well 3) were randomly collected for each random collection, and the cell growth curve was drawn for 7 consecutive days. The results show that using the human PDLSCs cryopreservation solution of the present invention to freeze PDLSCs, the cultured cells grow normally after resuscitation, and their proliferation activity is higher than that of conventional cell cryopreservation solutions for freezing PDLSCs, as shown in Table 3 and Table 4, Figure 3, and Figure 3. 4. The growth morphology of the two groups of cells was continuously observed and images were collected under an inverted microscope every day before cell collection. The results are shown in Figures 5-7. The results in Fig. 3-Fig. 7 all show that the cryopreservation solution of Example 3 of the present invention can promote the proliferation of cells after resuscitation.

Table 3 The daily counting results of the frozen MDSCs recovered and cultured in the comparison

Table 4 Example 3 freeze-preserved MDSCs recovery culture counting results every day

Example 9

The determination of cell surface markers in Example 3 includes the following steps: after freezing the frozen cells in Example 3 for one month in liquid nitrogen, take out the frozen storage tube and immediately put it in a 37°C water bath for dissolution. Shake the cryovial. After thawing the liquid for 1-2 minutes, dilute the cell suspension with 10 mL of serum-free medium for mesenchymal stem cells (wash the cryopreservation tube once with the medium), centrifuge at 1000 r/min for 5 minutes, and discard the supernatant. After resuspending in 10ml of mesenchymal stem cell serum-free medium containing 10ng/ml EGF, the cells were seeded in a 10cm culture dish and cultured in a 5% CO ₂ incubator at 37°C. Cells were collected after 48 hours, and the expression of surface markers CD146, CD105, CD73, HLA-DR, CD34 and CD45 were detected by flow cytometry. The results are shown in Table 5 and Figure 8.

The detection results show that, using the periodontal ligament stem cell cryopreservation medium of the present invention to freeze the cells, the recovered PDLSCs expresses typical surface markers of stem cells, which is not significantly different from that of the PDLSCs before freezing. It shows that the PDLSCs cryopreservation solution of the present invention will not affect the expression of cell surface markers of PDLSCs.

Table 5 Example 3PDLSCs cell surface marker expression rate

Example 10

Detection of multi-directional differentiation potential, including the following steps: the cells frozen in Comparative Example and Example 3 were frozen in liquid nitrogen for one month, and the frozen tube was taken out and immediately put into a 37°C water bath to dissolve. During the dissolution process, the frozen cells should be shaken continuously Depository. After thawing the liquid for 1-2 minutes, dilute the cell suspension with 10 mL of serum-free medium for mesenchymal stem cells (wash the cryopreservation tube once with the medium), centrifuge at 1000 r/min for 5 minutes, and discard the supernatant. After being resuspended with mesenchymal stem cell serum-free medium containing 10 ng/mL EGF, the cells were seeded in a 6-well plate at a density of 1×10 ⁵ cells/mL, and cultured in a 5% CO ₂ incubator at 37°C. When the degree of cell confluence reaches over 80%, osteogenic differentiation and adipogenic differentiation are induced on the normal periodontal ligament stem cells that have been cryopreserved and the periodontal ligament stem cells after cryopreservation and recovery in Example 3. After 14 days, the cells of the two adipogenic differentiation groups were stained with Oil Red O, and after 21 days, the cells of the two groups of osteogenic differentiation groups were stained with Alizarin Red. As shown in Figure 9, the negative control of the comparative example and the adipogenic induction group are similar to the staining results of the negative control of the embodiment 3 and the adipogenic induction group, and Figure 10 shows that the negative control of the comparative example and the osteogenic induction group are similar to those of the embodiment 3. The staining results of the negative control group and the osteogenic induction group were similar, and the experimental results showed that the periodontal ligament stem cell cryopreservation solution of the present invention would not affect the adipogenic and osteogenic differentiation potentials of PDLSCs ( FIG. 9 , FIG. 10 ).

To sum up, the invention can effectively solve the technical defects of the existing cryopreservation solution, which has poor cryopreservation effect on periodontal ligament stem cells and low cell viability after resuscitation. The cryopreservation solution for periodontal ligament stem cells of the present invention can well maintain the activity of cryopreserved cells, and the viability of periodontal ligament stem cells is significantly improved after recovery, and does not affect the expression of cell surface markers and their adipogenic and osteogenic differentiation potentials .

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (7)

1. a kind of frozen stock solution of periodontal ligament stem cell, it is characterised in that including glycerine, mesenchymal stem cell serum-free nutrient solution and Human serum albumin.

2. the frozen stock solution of periodontal ligament stem cell according to claim 1, it is characterised in that the frozen stock solution includes：Glycerine Volumn concentration be that the 10%, volumn concentration of mesenchymal stem cell serum-free nutrient solution is 30-60%, the white egg of human blood White volumn concentration is 30-60%.

3. the frozen stock solution of periodontal ligament stem cell according to claim 2, it is characterised in that the frozen stock solution includes：Glycerine Volumn concentration be that the 10%, volumn concentration of mesenchymal stem cell serum-free nutrient solution is 40%, human serum albumin Volumn concentration be 50%.

4. the frozen stock solution of periodontal ligament stem cell according to claim 3, it is characterised in that the human serum albumin quality is dense Spend for 20%.

5. a kind of periodontal ligament stem cell cryopreservation methods, add described in Claims 1-4 any one into periodontal ligament stem cell and freeze Frozen after liquid storage, mixing.

6. cryopreservation methods according to claim 5, it is characterised in that the addition of frozen stock solution is to periodontal ligament stem cell density For 1.0 × 10⁶Individual/mL-5 × 10⁶Individual/mL.

7. cryopreservation methods according to claim 6, it is characterised in that described to freeze specially described frozen stock solution and the periodontal Film stem cell is dispensed into cryopreservation tube after mixing, and is put into after the program temperature reduction box for recovering room temperature in advance, is placed -80 DEG C of ultralow temperature ices Case, is transferred to Liquid nitrogen storage after 2-3 days.