CN115968863A - Menstrual blood preservative fluid and cryopreservation method of menstrual blood stem cells - Google Patents

Abstract

A menstrual blood preservative fluid and a method for cryopreserving menstrual blood stem cells, comprising the following steps: the preserving fluid comprises normal saline, glucose, sodium citrate and penicillin. The method for cryopreserving the blood stem cells adopts trehalose, trans-4-hydroxy-L-proline, glycerol, tetrahydro-methylpyrimidine carboxylic acid and DMEM/F12 as cryopreservation agents. The cryopreservation agent does not contain serum protein and dimethyl sulfoxide, so that not only is virus infection possibly caused by the serum protein avoided, but also the cryopreserved stem cells have high recovery survival rate and large multidirectional differentiation potential.

Description

Menstrual blood preservative fluid and cryopreservation method of menstrual blood stem cells

Technical Field

The invention belongs to the field of cell therapy, and particularly provides menstrual blood preservative fluid and a cryopreservation method of menstrual blood stem cells.

Background

Mesenchymal Stem Cells (MSCs) are important members of the stem cell family, are derived from early-developing mesoderm, and belong to pluripotent stem cells. In 1976, freidenstein discovers mesenchymal stem cells in bone marrow for the first time, and then discovers mesenchymal stem cells in various tissues such as umbilical cord blood, placenta and the like, researches show that the mesenchymal stem cells have multidirectional differentiation potential and can be differentiated into various tissue cells such as fat, bone, muscle, nerve, liver, cardiac muscle and the like under specific induction conditions in vivo or in vitro.

The mesenchymal stem cells are obtained from the spinal cord tissues in an invasive process, which causes certain pain to donors, and the obtained stem cells have limited quantity and long doubling time. The placenta tissue or umbilical cord blood stem cells can be collected only at birth and are low in source, and the clinical application of the mesenchymal stem cells is seriously influenced by the limiting factors. In comparison, the uterine blood stem cells have many advantages, the materials are very convenient to obtain, professional people are not needed, and noninvasive collection can be realized by using sanitary cotton and a menstrual cup; can be obtained periodically, once a month, and reported on time; the multiplication capacity is very strong, and the amplification can be doubled within 24 hours. These advantages make the uterine stem cells considered to be the adult stem cells with the most advantages and potential for large-scale clinical application.

Currently, several studies have shown that uterine stem cells can differentiate into tissue cells such as cardiac myocytes, lung epithelial cells, neuronal cells, myocytes, endothelial cells, islet cells, stem cells, etc. in vitro. After the uterine blood stem cells are transplanted to a stroke mouse model, the uterine blood stem cells are found to be capable of obviously reducing brain cell death. In addition, the uterine stem cells show therapeutic potential in diseases such as myocardial infarction, diabetes, liver cirrhosis and the like.

However, cryopreservation of uterine stem cells also presents a number of problems, such as: the injection of hematopoietic stem cells containing DMSO causes abdominal pain and cramps, and the common cryopreservation solution contains animal serum which may carry prion and the like, which may cause the risk of virus transmission. Therefore, the invention develops a frozen stock solution for replacing DMSO and animal serum.

Disclosure of Invention

In order to solve the problems, the invention provides a menstrual blood preservation solution and a cryopreservation method of menstrual blood stem cells, wherein when the menstrual blood of a woman is collected, the vulva of the woman is cleaned and disinfected, and the menstrual blood is collected by using an autoclaved menstrual cup and preserved in the preservation solution, so that the pollution probability is reduced from the collection source, and the cell activity in the menstrual blood is kept; the mesenchymal stem cell seed cells obtained by separating the collected menstrual blood have good cell activity after cryopreservation and recovery, the proliferation activity of the cells after primary culture is high, the survival rate of the cells after subculture is high, and the pollution rate is low.

In order to overcome the defects in the prior art, the invention provides a solution of a menstrual blood preservative fluid and a menstrual blood stem cell cryopreservation method, which comprises the following specific steps:

on one hand, the invention provides a menstrual blood preservative fluid which comprises 0.9% of normal saline, 55.5mmol/L-110mmol/L glucose, 10-12mmol/L sodium citrate, 100U/mL penicillin and 30 mu mol/L-50 mu mol/L luteolin.

On the other hand, the stem cell freezing medium comprises the following components: trehalose and trans-4-hydroxy-L-proline, glycerol, tetrahydro-methyl-pyrimidinecarboxylic acid, DMEM/F12.

Wherein said trehalose is present in said cell cryopreservation solution in an amount of 2% to 9% by volume of said DMEM/F12; the content of the glycerol in the cell freezing solution is 5-30% of the volume of the DMEM/F12; the trans-4-hydroxy-L-proline is present in the cell culture broth in an amount of 1-5% by volume of the DMEM/F12; the content of the tetrahydro-methyl pyrimidine carboxylic acid in the cell freezing medium is 10-15% of the volume of the DMEM/F12.

In another aspect, a cryopreservation method of menstrual blood-derived mesenchymal stem cells comprises the following steps: s1, preparing menstrual blood preservative fluid; s2, collecting menstrual blood; s3, extracting mesenchymal stem cells from menstrual blood; and S4, freezing the mesenchymal stem cells.

Wherein the step S1 is as follows: the base solution of the preservation solution is 0.9% physiological saline, then glucose, sodium citrate, penicillin and luteolin are added into the base solution, the final glucose concentration is 55.5mmol/L-110mmol/L, the final sodium citrate concentration is 10-12mmol/L, the final penicillin concentration is 100U/mL, and the final luteolin concentration is 30 mu mol/L-50 mu mol/L.

Wherein the step S2 is as follows: collecting menstrual blood from the second day to the third day of the menstrual period of the female by using a menstrual cup; pouring the collected menstrual blood into a sterile container filled with preservation solution, and fully and uniformly mixing the menstrual blood and the preservation solution.

The step S3 is as follows: transferring the menstrual blood obtained in the step S2 into a centrifuge tube in an operation table, centrifuging to remove supernatant, adding the residual liquid into physiological saline with the same volume as 0.9% for dilution, adding the diluted solution into the centrifuge tube filled with lymphocyte separation liquid, and centrifuging to finally obtain a red blood cell layer; and adding the red blood cell layer into a centrifugal tube, adding physiological saline for dilution, centrifuging, and removing supernatant to finally obtain menses mesenchymal cells.

The step S4 is as follows: and (4) putting the cells obtained in the step (S3) into the freezing storage solution, putting the freezing storage solution into a freezing storage box, pre-freezing the freezing storage box in a refrigerator at the temperature of-80 ℃ for 12-36h, and then storing the freezing storage box in liquid nitrogen.

Compared with the prior art, the invention has the following advantages and benefits:

(1) The common frozen stock solution containing DMSO and animal serum has the following defects: the hematopoietic stem cells injected with DMSO may cause adverse reactions such as abdominal pain and cramps; animal serum contains viruses (prions, etc.), which may cause the risk of transmitting viruses; the components, batches, etc. of serum can have different effects on cells. Therefore, the invention abandons DMSO and animal serum and adopts trehalose and trans-4-hydroxy-L-proline, glycerol, tetrahydro-methyl pyrimidine carboxylic acid and DMEM/F12 as freezing agents.

(2) Trehalose is added into the frozen stock solution to stabilize cell membranes, glycerol can protect the inside and the outside of cells from being damaged by ice crystal formation through osmosis, and the tetrahydro-methyl pyrimidine carboxylic acid can be used as an osmotic agent to protect the cells from being damaged by high salt.

(3) According to the invention, the cryopreservation from menstrual blood to menstrual blood-derived mesenchymal cells is performed in equipment or facilities with deep sterilization, so that the activity of the cells is good after the cryopreservation recovery of the seed cells, and the survival rate of the cells is high and the pollution is small after subculture.

(4) A large number of experimental researches prove that trehalose, trans-4-hydroxy-L-proline and tetrahydro-methylpyrimidine carboxylic acid have a certain coordination effect, can inhibit the expression of apoptosis-promoting proteins and can increase the cell activity.

Drawings

FIG. 1 shows the appearance of cells obtained after cryopreservation and recovery of sample 1 in example 1.

FIG. 2 is the expression level of the pro-apoptotic protein Bax in each sample in example 3.

FIG. 3 is a graph showing the expression level of p53 in each sample in example 3.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1 preparation of frozen samples

Preparation of S1 menstrual blood preservative fluid

The base solution of the preservation solution is 0.9% physiological saline, and then glucose, sodium citrate, penicillin and luteolin are added into the base solution, wherein the final glucose concentration is 55.5mmol/L, the sodium citrate concentration is 10mmol/L, the penicillin concentration is 100U/mL, and the luteolin concentration is 50 mu mol/L.

S2 Collection of menstrual blood

Collecting menstrual blood from the second day to the third day of the menstrual period of the female by using a menstrual cup; pouring the collected menstrual blood into a sterile container filled with preservation solution, and fully and uniformly mixing the menstrual blood and the preservation solution.

S3 extraction of mesenchymal stem cells from menstrual blood

The menstrual blood obtained in step S2 was transferred to a centrifuge tube in a console, centrifuged at 2000rpm/min for 10min, the supernatant was removed, an equal volume of 0.9% saline was added, and centrifuged again at 1500rpm/min for 15min. Removing supernatant, adding the residual liquid into physiological saline with the same volume of 0.9% for dilution, adding the diluted liquid into a centrifugal tube filled with lymphocyte separation liquid, wherein the volume ratio of the diluted liquid to the lymphocyte separation liquid is 1: centrifuging at 1,2100rpm/min for 20min to obtain red blood cell layer; adding the erythrocyte layer into a centrifuge tube, adding 0.9% physiological saline with the same volume for dilution, centrifuging at 2000rpm/min for 15min, discarding supernatant, sampling and counting to finally obtain menses mesenchymal cells.

Freezing of S4 mesenchymal stem cells

Every 1 × 10 in step S3 ⁷ Adding 1ml of the cryopreservation solution into each cell, placing into a cryopreservation box, pre-freezing the cryopreservation box in a refrigerator at-80 ℃ for 15h, and storing in liquid nitrogen.

The samples were prepared by the above method, the following samples were from the same menstrual blood source, and the rest steps were the same, except for the preparation of the sample frozen stock solution.

Sample 1: the frozen stock solution used for sample 1 comprises the following components: DMEM/F12 is a mixture of DMEM and F12 in a volume ratio of 3. The content of the trehalose in the cell freezing medium is 5% of the volume of DMEM/F12, and the content of the glycerol in the cell freezing medium is 10% of the volume of DMEM/F12; the content of trans-4-hydroxy-L-proline in the cell freezing medium is 1% of the volume of DMEM/F12; the content of the tetrahydro-methyl-pyrimidinecarboxylic acid in the cell culture broth was 10% by volume of DMEM/F12.

Sample 2: the frozen stock solution used for sample 2 comprises the following components: DMEM/F12 is a mixture of DMEM and F12 in a volume ratio of 3. The content of the trehalose in the cell freezing medium is 5% of the volume of DMEM/F12, and the content of the glycerol in the cell freezing medium is 12% of the volume of DMEM/F12; the content of trans-4-hydroxy-L-proline in the cell freezing medium is 3% of the volume of DMEM/F12; the content of the tetrahydro-methyl-pyrimidinecarboxylic acid in the cell culture broth was 12% by volume of DMEM/F12.

Sample 3: the frozen stock solution used in sample 3 comprises the following components: DMEM/F12 is a mixture of DMEM and F12 in a volume ratio of 3. The trehalose content in the cell freezing medium is 7% of DMEM/F12 volume, and the glycerol content in the cell freezing medium is 25% of DMEM/F12 volume; the content of the trans-4-hydroxy-L-proline in the cell freezing medium is 3% of the volume of DMEM/F12; the content of the tetrahydro-methylpyrimidine carboxylic acid in the cell freezing medium was 15% by volume of DMEM/F12.

Sample 4: the frozen stock solution used in sample 4 comprises the following components: DMEM/F12 is a mixture of DMEM and F12 in a volume ratio of 3. The content of the trehalose in the cell freezing solution is 5% of the volume of DMEM/F12, the content of the glycerol in the cell freezing solution is 10% of the volume of DMEM/F12, and the content of the tetrahydro-methyl-pyrimidinecarboxylic acid in the cell freezing solution is 10% of the volume of DMEM/F12.

Sample 5: the frozen stock solution adopted by sample 5 comprises the following components: DMEM/F12 is a mixture of DMEM and F12 in a volume ratio of 3. The glycerol content in the cell culture medium was 10% by volume of DMEM/F12, the trans-4-hydroxy-L-proline content in the cell culture medium was 1% by volume of DMEM/F12, and the tetrahydromethylpyrimidine carboxylic acid content in the cell culture medium was 10% by volume of DMEM/F12.

Sample 6 the frozen stock solution used in sample 6 comprises the following components: DMEM/F12 is a mixture of DMEM and F12 in a volume ratio of 3. The content of the trehalose in the cell freezing medium is 5% of the volume of the DMEM/F12, the content of the trans-4-hydroxy-L-proline in the cell freezing medium is 1% of the volume of the DMEM/F12, and the content of the tetrahydro-methylpyrimidine carboxylic acid in the cell freezing medium is 10% of the volume of the DMEM/F12.

Sample 7: the cryopreservation solution adopted in sample 7 comprises the following components: DMEM/F12 is a mixture of DMEM and F12 in a volume ratio of 3. The content of the trehalose in the cell freezing medium is 5% of the volume of DMEM/F12, and the content of the glycerol in the cell freezing medium is 10% of the volume of DMEM/F12; the content of trans-4-hydroxy-L-proline in the cell culture broth was 1% by volume of DMEM/F12.

Sample 8: the cryopreservation solution adopted by the sample 8 comprises the following components: 10% DMSO, 20% human serum albumin, 70% mesenchymal stem cell serum-free medium.

Sample 9: the frozen stock solution used for sample 9 had the following composition: the volume percentage concentration of DMSO is 15%, the mass volume concentration of hydroxyethyl starch is 40%, and the balance is physiological saline.

FIG. 1 is a diagram showing the appearance of cells of sample 1 after thawing and thawing. As can be seen from FIG. 1, the stem cells are uniform in size and are mostly spindle-shaped.

Example 2 cell viability assay

The samples prepared in example 1 were frozen for 4 weeks and then subjected to recovery culture, i.e., cells were thawed in a 37 ℃ water bath, transferred into a centrifuge tube containing 0.9% physiological saline, centrifuged at 1500rpm for 5min, the supernatant was discarded, 0.9% physiological saline was added, centrifuged and washed twice, resuspended in a culture medium, sampled and counted, and the survival rate was measured. The results are shown in Table 1.

TABLE 1 cell viability results

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As can be seen from the results in table 1, the cell survival rates of samples 1, 2 and 3 after thawing recovery were higher than 95%, whereas the cell survival rates of samples 8 and 9 prepared from the prior art frozen stocks were 90%. This indicates that the frozen stock solution prepared by the present invention has a high cell survival rate. The reason for the high cell survival rate of the cryopreservation agent prepared by the invention is probably that the trehalose and the trans-4-hydroxy-L-proline can change the lipid composition on the cell membrane to achieve the function of stabilizing the cell membrane, thereby ensuring the cell survival rate.

Example 3 apoptosis Gene protein expression assay

After thawing and thawing the samples prepared in example 1, total RNA was isolated from the samples using RNA isolation kit (Roche, USA), and Bax and p53 primers were designed using national primer BLAST online software. The gene level was determined by PCR. All PCR experiments were tested using an iCycler RT-PCR detection system.

The Bax and p53 expression levels of each sample are shown in fig. 2 and fig. 3.

Bax protein is distributed in cytoplasm in the form of inactive monomer, is activated after receiving an apoptosis signal, generates molecular conformation change, shifts and inserts into outer membrane of mitochondria to form Bax macropore, and destroys integrity of mitochondria so as to cause apoptosis. And p53 protein can promote the expression of a pro-apoptotic protein gene. As can be seen from FIG. 3, the expression level of the pro-apoptotic protein Bax in the cells in sample 1 is much lower than that in the samples of the other groups, and the expression level of the pro-apoptotic protein p53 is much lower than that in the samples of the other groups. This indicates that the substance in sample 1 is capable of inhibiting the expression of the pro-apoptotic protein Bax and the pro-apoptotic protein p 53. Thereby increasing the viability of the cells, which is consistent with the higher cell viability of sample 1 in example 2 compared to the other groups.

Example 4 cell flow assay

Thawing and recovering primary mesenchymal stem cells of blood source by taking samples, washing for 2 times by PBS, digesting by 0.25 percent trypsin to prepare single cell suspension, and adjusting the cell density to be 5 multiplied by 10 ⁵ And/ml, and the cells are respectively incubated for 20min in a CD73-APC flow antibody and a CD90-PE flow antibody. After the incubation was completed, the cells were washed 2 times with PBS, and unbound antibodies were washed off, and the cell samples were loaded onto a machine and detected using a flow cytometer. The results are shown in Table 2.

TABLE 2 flow assay results

The recognized surface markers of the stem cells are CD73, CD90 and the like, the expression rates of the CD73 and CD90 are higher than 95%, and the quality of the separated stem cells is proved to reach the standard. By performing the above flow detection of the markers on the stem cells, the expression rate of CD73 and CD90 in sample 1 is significantly higher than that of the markers in sample 8 and sample 9, which indicates that the cells separated in sample 1 have higher purity and contain more stem cells.

Claims (9)

1. A menstrual blood preserving fluid is characterized by comprising the following components: normal saline, glucose, sodium citrate, penicillin, and luteolin.

2. The menstrual blood preserving fluid according to claim 1, comprising the following components: 0.9% physiological saline, 55.5-110 mmol/L glucose, 10-12mmol/L sodium citrate, 100U/mL penicillin, 30-50 μmol/L luteolin.

3. The stem cell cryopreservation solution is characterized by comprising the following components: trehalose and trans-4-hydroxy-L-proline, glycerol, tetrahydro-methyl-pyrimidinecarboxylic acid, DMEM/F12.

4. The cell cryopreservation solution of claim 3 wherein the trehalose is present in the cell cryopreservation solution in an amount of from 2% to 9% by volume of the DMEM/F12; the glycerol content in the cell frozen stock solution is 5-30% of the volume of the DMEM/F12; the trans-4-hydroxy-L-proline is present in the cell culture broth in an amount of 1-5% by volume of the DMEM/F12; the content of the tetrahydro-methyl pyrimidine carboxylic acid in the cell freezing solution is 10-15% of the volume of the DMEM/F12.

5. A method for cryopreserving menstrual blood stem cells, which is characterized by comprising the following steps: s1, preparing menstrual blood preservative fluid; s2, collecting menstrual blood; s3, extracting mesenchymal stem cells from menstrual blood; and S4, freezing the mesenchymal stem cells.

6. The method for cryopreserving menstrual blood stem cells according to claim 5, wherein the step S1: the base solution of the preservation solution is 0.9% physiological saline, then glucose, sodium citrate, penicillin and luteolin are added into the base solution, the final glucose concentration is 55.5mmol/L-110mmol/L, the final sodium citrate concentration is 10-12mmol/L, the final penicillin concentration is 100U/mL, and the final luteolin concentration is 30 mu mol/L-50 mu mol/L.

7. The method for cryopreserving menstrual blood stem cells according to claim 5, wherein the step S2: collecting menstrual blood from the second day to the third day of the menstrual period of the female by using a menstrual cup; pouring the collected menstrual blood into a sterile container filled with a preservation solution, and fully and uniformly mixing the menstrual blood and the preservation solution.

8. The method for cryopreserving menstrual blood stem cells according to claim 5, wherein the step S3: transferring the menstrual blood obtained in the step S2 into a centrifuge tube in an operation table, centrifuging to remove supernatant, adding the residual liquid into physiological saline with the same volume as 0.9% for dilution, adding the diluted solution into the centrifuge tube filled with lymphocyte separation liquid, and centrifuging to finally obtain a red blood cell layer; and adding the erythrocyte layer into a centrifugal tube, adding physiological saline for dilution, centrifuging, removing supernatant, and seeding to obtain menses mesenchymal cells.

9. The method for cryopreserving menstrual blood stem cells according to claim 5, wherein the step S4: and (4) putting the cells obtained in the step (S3) into the freezing storage solution, putting the freezing storage solution into a freezing storage box, pre-freezing the freezing storage box in a refrigerator at the temperature of-80 ℃ for 12-36h, and then storing the freezing storage box in liquid nitrogen.

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