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CN111363717B - Preparation method and application of decidua sub-totipotent stem cells - Google Patents

Preparation method and application of decidua sub-totipotent stem cells Download PDF

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CN111363717B
CN111363717B CN202010166332.2A CN202010166332A CN111363717B CN 111363717 B CN111363717 B CN 111363717B CN 202010166332 A CN202010166332 A CN 202010166332A CN 111363717 B CN111363717 B CN 111363717B
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sub
decidua
totipotent stem
digestion
stem cells
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CN111363717A (en
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刘小翠
李静静
赵蓝
孙灿兴
江嘉豪
褚一凡
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Guangdong Vitalife Biotechnology Co ltd
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Abstract

The invention provides a preparation method and application of a decidua sub-totipotent stem cell, and relates to the technical field of cell engineering. The preparation method comprises the following steps: pretreatment: cleaning a periostracum Cicadae membrane group, centrifuging, and removing a supernatant; digestion: shearing decidua tissue, adding a tissue digestive juice for digestion, separating supernatant and precipitate after digestion is finished, and re-suspending the precipitate to obtain a suspension; the tissue digestive juice comprises Tryple enzyme, normal saline, I-type collagenase and II-type collagenase, wherein the volume concentration of the Tryple enzyme is 40-60%, the mass concentration of the I-type collagenase is 0.8-1.2 mg/mL, and the mass concentration of the II-type collagenase is 0.8-1.2 mg/mL; separation: and adding the lymphocyte separation liquid into the suspension, centrifuging, separating mononuclear cells after layering, centrifuging, retaining precipitates, adding a culture solution, and uniformly mixing to obtain the decidua-parietalis sub-totipotent stem cell separation liquid. The method can successfully extract the sub-totipotent stem cells from the decidua tissue of the wall, and has good cell activity and strong differentiation capability.

Description

Preparation method and application of decidua sub-totipotent stem cells
Technical Field
The invention relates to the technical field of cell engineering, in particular to a preparation method and application of a decidua sub-totipotent stem cell.
Background
Stem cells are cells with the potential of self-replication, renewal and multidirectional differentiation, can be differentiated into various tissues and organs, and have become a research hotspot in the field of life science. Scientists have found that a group of primitive stem cells exists in various adult tissues, and the primitive stem cells are different from multipotent stem cells such as hematopoietic stem cells, neural stem cells and the like, the multipotent stem cells can only differentiate into cells of a specific germ layer, and the primitive stem cells can differentiate into histiocytes of different germ layers. The primary stem cells are also different from embryonic stem cells, gradually lose partial differentiation potential in the growth and development process of individuals, and can present some special phenotypes or molecular markers, which are called as adult sub-totipotent stem cells. The sub-totipotent stem cells have the following characteristics: the stem cells can be differentiated into cells with different germ layers, the sub-totipotent stem cells in the embryonic stage can be differentiated into pluripotent stem cells with different types, and the balance is maintained in growth and metabolism; the tissue-specific cells can be differentiated into the tissue-specific cells of the organ, and can be used for organ remodeling, and the damaged tissue can be remotely transferred and repaired under the chemotactic action of inflammatory factors or growth factors. Therefore, the adult sub-totipotent stem cells play an important role in participating in tissue damage repair and making up for the shortage of the number of stem cells, and the adult sub-totipotent stem cells which can be separated, purified, cultured and effectively utilized have important significance.
At present, extraction of the sub-totipotent stem cells has some obstacles, on one hand, most of the existing sub-totipotent stem cells are extracted from human placenta, and have certain ethical problems, and on the other hand, the sub-totipotent stem cells obtained by the existing extraction method have low activity and limited differentiation capacity, so that the application of the sub-totipotent stem cells in clinical treatment is limited.
Disclosure of Invention
Therefore, it is necessary to provide a method for preparing decidua pluripotency stem cells, which has the advantages of high yield, good activity and strong differentiation capacity, and provides a basis for clinical treatment.
A preparation method of decidua sub-totipotent stem cells comprises the following steps:
pretreatment: cleaning decidua tissue of the wall, centrifuging and removing supernatant;
digestion: shearing decidua tissue, adding a tissue digestive juice for digestion, separating supernatant and precipitate after digestion is finished, and re-suspending the precipitate to obtain a suspension; the tissue digestive juice comprises Tryple enzyme, normal saline, I-type collagenase and II-type collagenase, wherein the volume concentration of the Tryple enzyme is 40-60%, the mass concentration of the I-type collagenase is 0.8-1.2 mg/mL, and the mass concentration of the II-type collagenase is 0.8-1.2 mg/mL;
separation: and adding the lymphocyte separation liquid into the suspension, centrifuging, separating mononuclear cells after layering, centrifuging, retaining precipitates, adding a culture solution, and uniformly mixing to obtain the decidua-parietalis sub-totipotent stem cell separation liquid.
According to the preparation method, the tissue digestive juice is used for successfully extracting the sub-totipotent stem cells from the decidua muralis tissue, and the extracted cells are large in number, high in purity, good in activity and strong in differentiation capacity, and can be applied to clinic; the method provides a new extraction source of sub-totipotent stem cells, namely the molting tissue, and the molting tissue has rich sources, does not have ethical disputes and has wide application prospect.
In one embodiment, in the pretreatment step, the centrifugal speed is 1000-1500 r/min, and the centrifugal time is 4-6 min; the cleaning solution comprises: physiological saline, erythrocyte lysate with volume fraction of 45-55%, gentamicin sulfate with volume fraction of 8-12 mu g/ml, and amphotericin B with volume fraction of 8-12 mu g/ml. The cell lysate can be used for lysing erythrocytes, removing blood and reducing pollution.
In one embodiment, the digestion step is specifically: cutting the decidua tissue into 0.1-1cm3Adding tissue digestive juice with the volume of 0.8-1.5 times of the fragments of the cells, uniformly mixing, performing shake digestion in a constant-temperature shaking instrument for 1-4 hours, controlling the temperature to be 37 +/-0.5 ℃, controlling the shaking rotating speed to be 180-220 r/min, separating supernate and sediment after digestion is completed, and adding physiological saline into the sediment for re-suspension.
In one embodiment, the separating step specifically comprises: and adding 1.2-1.8 times of volume of lymphocyte separation liquid into the suspension, centrifuging for 8-12 min at the centrifugation rotating speed of 1000-2000r/min, separating out mononuclear cells at the third layer after layering, continuing centrifuging for 8-12 min at the centrifugation rotating speed of 1000-2000r/min, retaining the precipitate, adding 0.3-0.8 times of volume of DMEM/F12 culture solution, and uniformly mixing to obtain the decidua-muralis sub-totipotent stem cell separation liquid.
In one embodiment, the separation step further comprises a culturing step: removing wallThe membrane sub-totipotent stem cell separation solution is 2-4 multiplied by 105Inoculation was carried out at a density of one/mL, at a temperature of 37. + -. 0.5 ℃ and CO2And (3) carrying out static culture in an incubator with the concentration of 5 +/-0.5%, marking as P0 generation, and replacing 40-60% of complete culture solution containing the double antibody every 3-4 days.
In one embodiment, the culturing step further comprises a storage step: and (3) when the fusion degree of the sub-totipotent stem cells of the P3 generation reaches 80-90%, completely removing the culture solution, cleaning, adding digestive juice for digestion, centrifuging, adding a sub-totipotent stem cell freezing solution, and placing in liquid nitrogen for freezing.
In one embodiment, the storing step specifically comprises: when the fusion degree of the P3 generation sub-totipotent stem cells reaches 80-90%, completely removing the culture solution, washing with PBS (phosphate buffer solution) for 1-3 times, adding a digestion solution for digestion for 1-10min, adding a complete culture solution to stop digestion, centrifuging at the rotating speed of 500 + 1000r/min for 5-7 min, removing the supernatant, adding a sub-totipotent stem cell freezing solution into the precipitate, wherein the cell concentration is 1-6 × 106And (4) placing the cells in liquid nitrogen for freezing.
In one embodiment, the digestive juice comprises 0.2-0.3% by mass of trypsin and 0.003-0.005% by mass of EDTA.
In one embodiment, the pluripotent stem cell cryopreservation liquid comprises 28-32% by mass of glycerol, 13-17% by mass of vitamin C and 15-25% by mass of SR serum-free culture medium. The pluripotent stem cell cryopreservation liquid can enable pluripotent stem cells to still keep good activity and differentiation capacity in a long-time preservation process, and the cell survival rate is high.
The invention also provides the decidua-murphyta sub-totipotent stem cell obtained by the preparation method. The decidua-wall sub-totipotent stem cell has high purity, large quantity, good activity and strong differentiation capability, and can be applied to clinical treatment.
The invention also provides application of the decidua sub-totipotent stem cell obtained by the preparation method in preparing osteoblasts and endometrial epithelial cells.
The inventionIn one aspect, a method for differentiating the decidua sub-totipotent stem cell into the osteoblast is further provided, which comprises the following steps: performing 1-3 multiplied by 10 on the decidua sub-totipotent stem cells4Inoculating in a concentration of/mL, and replacing an osteoblast differentiation induction culture medium every 3-4 days, wherein the osteoblast differentiation induction culture medium comprises the following components: DMEM is used as a base solution, 8-12 vt% of serum substitute, 8-12 nM/mL of dexamethasone, 8-12M/mL of beta-glycerophosphate, 0.5-1.5 wt% of antibiotic, 20-30 ng/mL of cytokine IL-beta, 100-110 mM/mL of ascorbic acid and 8-12 mu g/mL of isoflavone. The osteoblast differentiation induction culture medium can effectively induce the decidua-membrane sub-totipotent stem cells to differentiate into osteoblasts.
The invention also provides a method for differentiating the decidua sub-totipotent stem cells into endometrial epithelial cells, which comprises the following steps: performing 1-2 × 10 division of the decidua sub-totipotent stem cells5Inoculating in a concentration of/mL, and replacing the endometrial epithelial cell differentiation induction culture medium every 3-4 days, wherein the endometrial epithelial cell differentiation induction culture medium comprises the following components: the DMEM culture medium is a substrate solution, 3-7 vt% of serum substitute, 0.5-1.5 wt% of vitamin C, 10-20 nM/mL of dexamethasone, 0.5-1.5 wt% of non-essential amino acid, 115-25 ng/mL of TGF-beta, 15-25 ng/mL of EGF, 15-25 ng/mL of PDGF-BB and 25-35 ng/mL of estradiol. The endometrial epithelial cell differentiation induction culture medium can effectively induce the decidua sub-totipotent stem cells to differentiate into the endometrial epithelial cells.
Compared with the prior art, the invention has the following beneficial effects:
according to the preparation method, the tissue digestive juice is used for successfully extracting the sub-totipotent stem cells from the decidua muralis tissue, and the extracted cells are large in number, high in purity, good in activity and strong in differentiation capacity, and can be applied to clinic; the method provides a new extraction source of sub-totipotent stem cells, namely the molting tissue, and the molting tissue has rich sources, does not have ethical disputes and has wide application prospect.
Drawings
FIG. 1 is a morphogram of the P3 generation pluripotent stem cell in the example;
FIG. 2 is a graph showing the results of flow measurement of pluripotent stem cells of the P3 generation in example;
FIG. 3 is a graph showing the results of the directional differentiation of pluripotent stem cells of the P3 generation into endometrial epithelial cells in the example;
FIG. 4 is an immunofluorescence assay of the pluripotent stem cells of the P3 generation oriented to differentiate into endometrial epithelial cells in the example;
FIG. 5 is a graph showing the proliferation potency of P3 generation pluripotent stem cells in the example.
Detailed Description
To facilitate an understanding of the invention, a more complete description of the invention will be given below in terms of preferred embodiments. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Example 1
A preparation method of decidua sub-totipotent stem cells comprises the following steps:
(1) pretreatment: taking the decidua tissue out of the collection bag, cleaning with a tissue cleaning solution until no blood residue is left, then putting the cleaned placenta tissue into a 50mL sterile centrifuge tube, centrifuging at 1200rpm for 5min, and removing the supernatant; wherein, the washing liquid includes: physiological saline, erythrocyte lysate with volume fraction of 50%, gentamicin sulfate with volume fraction of 10 mu g/ml, and amphotericin B with volume fraction of 10 mu g/ml.
(2) Digestion: shearing cleaned decidua tissue into 0.1-1cm with aseptic direct scissors3Adding tissue digestive juice preheated to 37 ℃ in the same volume into the fragments, uniformly mixing, performing shake digestion in a constant-temperature shaking instrument at 37 ℃ for 2 hours at the rotating speed of 200r/min, quickly and instantly centrifuging after digestion, collecting supernatant, and adding physiological saline into the precipitate for resuspensionThen quickly and instantaneously centrifuging again, collecting supernatant, and reserving sediment; wherein, the formula of the tissue digestive juice is as follows: 50% of Tryple enzyme, 15mL of physiological saline containing double antibody, 10mg of collagenase type I and 10mg of collagenase type II.
(3) Separation: combining the collected supernatants, filtering by using a 100-micron screen, centrifuging for 10min at the rotating speed of 1500rpm, combining precipitates, adding 1.5 times of volume of physiological saline into the precipitates for resuspending to obtain cell suspension, slowly adding 1.5 times of volume of lymphocyte separation fluid, centrifuging for 10min at the rotating speed of 1500rpm, taking out mononuclear cells at the third layer after layering, centrifuging for 10min at the rotating speed of 1500rpm, retaining the precipitates, adding 0.5 times of volume of DMEM/F12 culture fluid of the cell suspension, and uniformly mixing to obtain the sub-totipotent stem cell separation fluid.
(4) Culturing: taking micro (about 1-20 μ L) sub totipotent stem cells, counting cells according to 3 × 105The inoculation was carried out at a density of one/mL, two T75 bottles were inoculated, 10mL each, at 37 ℃ with 5% CO2The culture box is subjected to static culture, marked as P0 generation, a complete culture solution containing double antibodies is replaced every 3-4 days for half a time, and cells which are not attached to the wall are discarded.
(5) Subculturing and detecting: culturing for 14 days, wherein the cell fusion degree reaches about 80-90%, completely removing the culture solution, washing twice by using PBS, adding 2-10mL of digestive juice for digestion, wherein the digestive juice contains 0.25 wt% of trypsin and 0.004 wt% of EDTA, adding the complete culture solution after digestion for 1-10min to terminate digestion, subpackaging the mixture into two T75 culture bottles, carrying out passage once every 3 days when the cell fusion degree reaches 70-80%, and transferring to P3 generation. The cell morphology at the P3 generation is shown in FIG. 1, and the cell morphology is normal. The P3 generation sub totipotent stem cell surface marker was detected by flow, and the results are shown in FIG. 2.
(6) And (3) storage: when the fusion degree of the P3 generation sub-totipotent stem cells reaches 80-90%, completely removing the culture solution, washing the cells twice by PBS, adding 2-10mL of digestive juice for digestion, wherein the digestive juice contains 0.25 wt% of trypsin and 0.004 wt% of EDTA, adding the complete culture solution to stop digestion after digestion for 1-10min, slightly blowing the bottom of the culture bottle by a pipette to completely drop the cells, collecting the cells in a sterile centrifuge tube, and transferring at 700rpmCentrifuging for 6min at a high speed, removing supernatant, adding a sub-totipotent stem cell freezing medium into the sediment, wherein the sub-totipotent stem cell freezing medium contains 30% by mass of glycerol, 15% by mass of vitamin C and 20% by mass of SR serum-free medium, gently mixing, adding into a freezing tube, and storing at a concentration of 1-6 × 106one/mL. Placing the freezing tube into a programmed cooling instrument, placing into a low-temperature refrigerator at minus 80 ℃ for 24h, taking out, and placing into liquid nitrogen at minus 196 ℃ for freezing.
Comparative example 1
A method for preparing decidua sub-totipotent stem cells, which is different from the method in example 1 in that: the formula of the tissue digestive juice in the step (2) is as follows: tryple enzyme 50%, 15mL physiological saline containing double antibody, 20mg collagenase type II.
Comparative example 2
A method for preparing decidua sub-totipotent stem cells, which is different from the method in example 1 in that: the formula of the tissue digestive juice in the step (2) is as follows: tryple enzyme 50%, 15mL physiological saline containing double antibody, 20mg collagenase type I.
Comparative example 3
A method for preparing decidua sub-totipotent stem cells, which is different from the method in example 1 in that: the formula of the tissue digestive juice in the step (2) is as follows: tryple enzyme 50% and 15mL of physiological saline containing double antibody.
Experimental example 1
The cells of P0 generation and P3 generation obtained in the examples and comparative examples were counted, and the results are shown in Table 1:
TABLE 1 decidua sub-totipotent stem cell counter
Figure BDA0002407588870000051
Experimental example 2
Osteoblast differentiation capacity identification:
the P3 generation sub totipotent stem cells in example 1 were processed in 2X 104The culture medium was inoculated in a 24-well plate at a concentration of 1mL per well, and the osteoblast differentiation induction medium was replaced every 3 days, and had the following composition: DMEM as substrate10 vt% of liquid and serum substitute, 10nM/mL of dexamethasone, 10M/mL of beta-glycerophosphate, 1 wt% of antibiotic, 25ng/mL of cytokine IL-beta, 105mM/mL of ascorbic acid and 10 mu g/mL of isoflavone. Culturing to day 14, discarding the culture medium, washing with PBS for 2 times, fixing with 4% paraformaldehyde for 20min, staining with alizarin red, washing with normal saline twice after staining for 20min, and observing that calcium nodules are obviously formed, which indicates that the sub-totipotent stem cells of example 1 can be effectively differentiated into osteoblasts.
Experimental example 3
And (3) identifying the differentiation capability of the endometrial epithelial cells:
the P3 generation sub totipotent stem cells in example 1 were processed in the order of 1X 105The concentration of the culture medium is 2mL per well, and the endometrial epithelial cell differentiation induction culture medium is replaced every 3 days by inoculating the culture medium into a 12-well plate, and the components of the induction culture medium are as follows: DMEM medium is basal liquid, 5 vt% of serum substitute, vitamin C1 wt%, dexamethasone 15nM/mL, non-essential amino acid 1 wt%, TGF-beta 120ng/mL, EGF 20ng/mL, PDGF-BB 20ng/mL, estradiol 30 ng/mL.
The induction of endometrial epithelial cells was observed by day 14 of culture before treatment, as shown in figure 3. The medium was discarded, washed 2 times with PBS, fixed with 4% paraformaldehyde at room temperature for 20min, and washed 3 times with PBS, 5min each. Adding 500 μ L of 2% Triton lysate into the washed cells, lysing for 20min, washing with PBS 3 times, 5min each time, incubating at 4 deg.C for 12h, washing, adding fluorescent secondary antibody corresponding to the primary antibody, incubating at room temperature in dark for 2h, washing with PBS 3 times, 3min each time. After washing, the nuclei were counterstained in the dark, at room temperature for 5-10min, washed 3 times with PBS, 5min each time. After cleaning, residual PBS mounting pieces are blotted by absorbent paper for microscopic examination, the microscopic examination result is shown in figure 4 (in figure 4, the background is black, the green is grey, the red is white, and the blue is black), the green fluorescence is marked as protein CK18, the red fluorescence is marked as protein CK7, and the result shows that the pluripotent stem cells and the pluripotent stem cells can be successfully differentiated into active endometrial epithelial cells.
Experimental example 4
Identification of proliferation capacity of sub-totipotent stem cells
The sub-totipotent stem cells of P3 generation in example 1 were collected at 2X 10 per well4Cell number sub-totipotent stem cells were seeded in 96-well plates in 3 groups of 5 duplicate wells per group. Each well was supplemented with DMEM medium to 100. mu.L/well, and a blank control well to which a single cell culture solution was added was set. Placing in 5% CO2And culturing for 24 hours in a constant temperature incubator at 37 ℃ until the wall is completely attached. After every 24h only 10. mu.L of CCK-8 reagent was added to each well used the day and incubated at 37 ℃ for 2.5 h. And (3) placing the incubated culture plate in a microplate reader, measuring the absorbance OD450 at the wavelength of 450nm, and recording the result. The reaction was repeated for 7 days, and a curve was drawn in accordance with the OD value of absorbance.
And then taking the frozen and stored 60d P3 generation sub totipotent stem cells, recovering, carrying out cell proliferation according to the method, and drawing an absorbance curve.
The results of proliferation capacities of the sub-totipotent stem cells after non-cryopreservation and cryopreservation recovery are shown in fig. 5, and it can be seen that the number of the cells after cryopreservation recovery is basically the same as that of the cells not cryopreserved on the seventh day, which indicates that the storage method of the present invention can effectively maintain the proliferation capacity or activity of the cells.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (4)

1. A preparation method of decidua sub-totipotent stem cells is characterized by comprising the following steps:
pretreatment: cleaning decidua tissue of a wall, centrifuging at a centrifugal rotation speed of 1000-1500 r/min for 4-6 min, and removing supernatant; the cleaning liquid used in cleaning comprises: normal saline, erythrocyte lysate with volume fraction of 45-55%, 8-12 mug/mL gentamicin sulfate and 8-12 mug/mL amphotericin B;
digestion: cutting the decidua tissue into 0.1-1cm3Adding tissue digestive juice with the volume of 0.8-1.5 times of the fragments of the cells, uniformly mixing, performing shake digestion in a constant-temperature shaking instrument for 1-4 hours, controlling the temperature to be 37 +/-0.5 ℃, controlling the shake rotation speed to be 180-220 r/min, performing rapid instantaneous centrifugation after digestion, collecting supernatant and sediment, adding normal saline into the sediment for heavy suspension, performing rapid instantaneous centrifugation again, collecting the supernatant, and keeping the sediment; the formula of the tissue digestive juice is as follows: the volume concentration of the Tryple enzyme is 50 percent, 15mL of physiological saline containing double antibodies, 10mg of collagenase type I and 10mg of collagenase type II;
separation: combining the collected supernatants, filtering by using a 100-micron screen, centrifuging for 10min at the rotating speed of 1500rpm, combining precipitates, adding 1.5-fold volume of physiological saline into the precipitates for resuspending to obtain cell suspension, adding 1.2-1.8-fold volume of lymphocyte separation liquid into the cell suspension, centrifuging for 8-12 min at the rotating speed of 1000-2000r/min, separating out mononuclear cells positioned at the third layer after layering, centrifuging for 8-12 min at the rotating speed of 1000-2000r/min, retaining the precipitates, adding 0.3-0.8-fold volume of DMEM/F12 culture solution, and uniformly mixing to obtain the sub-totipotent parietal decidua stem cell separation liquid;
culturing: separating the decidua-membrane sub-totipotent stem cells into 2-4 multiplied by 105Inoculation was carried out at a density of one/mL, at a temperature of 37. + -. 0.5 ℃ and CO2Carrying out static culture in an incubator with the concentration of 5 +/-0.5%, marking as P0 generation, and replacing 40-60% of complete culture solution containing double antibodies every 3-4 days;
and (3) storage: when the fusion degree of the sub-totipotent stem cells of the generation P3 reaches 80-90%, completely removing the culture solution, cleaning, adding digestive juice for digestion, centrifuging, adding a sub-totipotent stem cell freezing solution, and placing in liquid nitrogen for freezing;
wherein, the decidua sub-totipotent stem cell is used for preparing osteoblasts and endometrial epithelial cells.
2. The method according to claim 1, wherein the storing step comprises: when the fusion degree of the P3 generation sub-totipotent stem cells reaches 80-90%, completely removing the culture solution, washing with PBS (phosphate buffer solution) for 1-3 times, adding a digestion solution for digestion for 1-10min, adding a complete culture solution to stop digestion, centrifuging at the rotating speed of 500 + 1000r/min for 5-7 min, removing the supernatant, adding a sub-totipotent stem cell freezing solution into the precipitate, wherein the cell concentration is 1-6 × 106And (4) placing the cells in liquid nitrogen for freezing.
3. The method according to claim 1 or 2, wherein the digestive juice comprises 0.2 to 0.3 mass% of trypsin and 0.003 to 0.005 mass% of EDTA.
4. The preparation method according to claim 1 or 2, wherein the pluripotent stem cell cryopreservation solution comprises 28-32% by mass of glycerol, 13-17% by mass of vitamin C and 15-25% by mass of SR serum-free medium.
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