CN110622956A - Umbilical cord mesenchymal stem cell preservation solution - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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Abstract
The invention discloses a preservation solution for umbilical cord mesenchymal stem cells, which comprises the following components in concentration: 5-10% (V/V) of low-sugar DMEM medium, 15-25 g/L of human serum albumin, 8-10 g/L of sodium chloride, 2000-molecular heparin sodium, 3000AXaIU/mL, 20-50 g/L of glucose, 100-150 mg/L of vitamin C and 10-30 uM of salidroside. The preservation solution can effectively preserve umbilical cord mesenchymal stem cells at 4 ℃ and maintain stable cell phenotype. The components of the preservation solution are safe and nontoxic to human bodies, so that the preservation solution is convenient for the subsequent use of umbilical cord mesenchymal stem cells, is very suitable for the preservation and transportation of umbilical cord mesenchymal stem cells, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of stem cells, and particularly relates to umbilical cord mesenchymal stem cell preservation solution.
Background
Mesenchymal Stem cells (MSC, Mesenchymal Stem cells) are derived from the mesoderm and ectoderm in the early development stage, and are increasingly receiving attention due to their characteristics of multipotentiality, hematopoietic support, promotion of Stem Cell implantation, immune regulation, self-replication and the like. Mesenchymal stem cells are initially found in bone marrow and subsequently found in many tissues during human development and development, such as bone marrow, fat, synovium, bone, muscle, lung, liver, pancreas, etc., as well as amniotic fluid, umbilical cord blood, placenta, etc. As a stem cell widely applied, the human umbilical cord mesenchymal stem cell has the advantages of convenient material acquisition, easy separation, low immunogenicity, multidirectional differentiation potential and the like, and can be applied to clinic.
At present, the human umbilical cord mesenchymal stem cells have some problems in scientific research, industry and clinical application, such as inconvenient transportation, temporary storage and the like. The mesenchymal stem cells are rapidly killed and deteriorated at normal temperature after being separated from the culture condition, are generally frozen in a liquid nitrogen tank for long-term storage in a non-culture state, and have very high cost for small-scale long-distance transportation, so that the large-scale production and long-distance supply of the mesenchymal stem cells in clinical application are greatly limited. In addition, in clinical application, the human umbilical cord mesenchymal stem cell preparation can be injected into a human body after quality tests such as sterility, activity and the like are completed, and the tests take a long time to obtain a reliable result. However, the conventional low-temperature cryopreservation has great damage to cells and can not be directly injected into a human body after being thawed, so that the proper preservation solution is the basis of clinical application during the quality inspection of the stem cell preparation.
Disclosure of Invention
Based on the above, the invention aims to overcome the defects of the prior art and provide the umbilical cord mesenchymal stem cell preservation solution, which can effectively preserve umbilical cord mesenchymal stem cells at 4 ℃, maintain high cell survival rate and is very suitable for preservation and transportation of umbilical cord mesenchymal stem cells.
In order to achieve the purpose, the invention adopts the technical scheme that: an umbilical cord mesenchymal stem cell preservation solution comprises the following components in concentration: 5-10% (V/V) of low-sugar DMEM medium, 15-25 g/L of human serum albumin, 8-10 g/L of sodium chloride, 2000-molecular heparin sodium, 3000AXaIU/mL, 20-50 g/L of glucose, 100-150 mg/L of vitamin C and 10-30 uM of salidroside.
Preferably, the concentration of the salidroside is 15-25 uM.
Preferably, the concentration of salidroside is 20 uM.
Preferably, the glucose concentration of the low-sugar DMEM medium is 1000 mg/L.
Human serum albumin: the non-permeable cryoprotectant is mainly used for regulating intracellular water dynamic balance and maintaining osmotic pressure of cell solution.
Sodium chloride: effectively adjust the osmotic pressure of the solution and keep the relative balance of the osmotic pressure of the liquid, thereby preventing the cells from being influenced by the osmotic pressure.
Low molecular weight heparin sodium: the anticoagulant has anticoagulant and antithrombotic effects and can prevent agglutination reaction after intravenous injection of cells.
Glucose: carbon source and energy source substances necessary for cell growth play an important role in the cell metabolism process, and a proper amount of glucose is added in the transient preservation process to increase the cell activity; meanwhile, the sodium alginate is also used as an impermeable cryoprotectant, can enter cells through active transportation, and finally keeps the inside and outside of the cells isotonic.
Vitamin C: helps cells maintain activities of various peroxidases, prevents damage of oxygen free radicals to cell membranes and organelles, and maintains cell activity under the condition of long-term preservation.
Salidroside: the components in the rhodiola rosea extract of a perennial herb plant have various pharmacological actions such as fatigue resistance, aging resistance, immunoregulation, free radical elimination and the like.
Through a large amount of researches and analyses, the inventor of the application finds that the preservation solution contains a proper amount of salidroside, so that the cell survival rate can be effectively improved, a better preservation effect can be obtained when the concentration of the salidroside in the preservation solution is 15-25 uM, and the effect is best when the concentration of the salidroside in the preservation solution is 20 uM.
The invention also provides application of the umbilical cord mesenchymal stem cell preservation solution in preservation of umbilical cord mesenchymal stem cells.
The invention also provides a preservation method of umbilical cord mesenchymal stem cells, which comprises the following steps: will prepareCounting the umbilical cord mesenchymal stem cells, adding the umbilical cord mesenchymal stem cell preservation solution into the umbilical cord mesenchymal stem cells, and adjusting the concentration of the umbilical cord mesenchymal stem cells to be 0.5 multiplied by 106～1×106/mL。
Preferably, the umbilical cord mesenchymal stem cell concentration is 0.8 × 106and/mL. When the concentration of the umbilical cord mesenchymal stem cells is 0.8 multiplied by 106Better preservation effect was obtained at/mL.
Preferably, the umbilical cord mesenchymal stem cell preservation solution is pre-cooled to 4 ℃ and then added into the umbilical cord mesenchymal stem cells.
Compared with the prior art, the invention has the beneficial effects that: (1) the preservation solution can effectively preserve umbilical cord mesenchymal stem cells at 4 ℃, so that the survival rate of the umbilical cord mesenchymal stem cells after being preserved for 5 days is up to more than 85%, and a stable cell phenotype is maintained. The components of the preservation solution are safe and non-toxic to a human body, so that the subsequent use of the umbilical cord mesenchymal stem cells is facilitated, the preservation solution is very suitable for the preservation and transportation of the umbilical cord mesenchymal stem cells, and the application prospect is wide; (2) through a large amount of researches and analyses, the inventor of the application finds that the low-temperature preservation solution contains a proper amount of salidroside, the survival rate of cells can be effectively improved, when the concentration of the salidroside in the preservation solution is 15-23 uM, a better preservation effect can be obtained, and particularly, the effect is best when the concentration of the salidroside in the preservation solution is 20 uM.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. 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.
Example 1
In an embodiment of the umbilical cord mesenchymal stem cell preservation solution of the present invention, the umbilical cord mesenchymal stem cell preservation solution comprises the following components in concentration: low-sugar DMEM medium 5% (V/V), human serum albumin 18g/L, sodium chloride 10g/L, low molecular weight heparin sodium 2500AXaIU/mL, glucose 40g/L, vitamin C100mg/L and salidroside 10 uM.
Example 2
In an embodiment of the umbilical cord mesenchymal stem cell preservation solution of the present invention, the umbilical cord mesenchymal stem cell preservation solution comprises the following components in concentration: 8 percent (V/V) of low-sugar DMEM medium, 15g/L of human serum albumin, 9g/L of sodium chloride, 3000AXaIU/mL of low-molecular heparin sodium, 25g/L of glucose, 120mg/L of vitamin C and 28uM of salidroside.
Example 3
In an embodiment of the umbilical cord mesenchymal stem cell preservation solution of the present invention, the umbilical cord mesenchymal stem cell preservation solution comprises the following components in concentration: 9 percent (V/V) of low-sugar DMEM medium, 20g/L of human serum albumin, 8g/L of sodium chloride, 2800AXaIU/mL of low-molecular heparin sodium, 50g/L of glucose, 150/L of vitamin C150mg and 30uM of salidroside.
Example 4
In an embodiment of the umbilical cord mesenchymal stem cell preservation solution of the present invention, the umbilical cord mesenchymal stem cell preservation solution comprises the following components in concentration: 10 percent (V/V) of low-sugar DMEM medium, 22g/L of human serum albumin, 9g/L of sodium chloride, 2300AXaIU/mL of low-molecular heparin sodium, 20g/L of glucose, 130mg/L of vitamin C and 18uM of salidroside.
Example 5
In an embodiment of the umbilical cord mesenchymal stem cell preservation solution of the present invention, the umbilical cord mesenchymal stem cell preservation solution comprises the following components in concentration: 7 percent (V/V) of low-sugar DMEM medium, 25g/L of human serum albumin, 9.5g/L of sodium chloride, 2000AXaIU/mL of low-molecular heparin sodium, 35g/L of glucose, 125mg/L of vitamin C and 20uM of salidroside.
Example 6
In an embodiment of the umbilical cord mesenchymal stem cell preservation solution of the present invention, the umbilical cord mesenchymal stem cell preservation solution comprises the following components in concentration: low-sugar DMEM medium 5.5% (V/V), human serum albumin 18g/L, sodium chloride 10g/L, low molecular weight heparin sodium 2200AXaIU/mL, glucose 30g/L, vitamin C140mg/L and salidroside 23 uM.
Example 7
In this example, the preservation effect of the umbilical cord mesenchymal stem cell preservation solution described in examples 1 to 6 on umbilical cord mesenchymal stem cells was studied.
1. Preparation of umbilical cord mesenchymal Stem cells
(1) Cleaning neonatal umbilical cord with physiological saline, cutting into small segments, peeling off artery and vein, separating to obtain Wharton's jelly, cutting into pieces, adding type II collagenase with concentration of 1mg/mL, digesting at 37 deg.C for 6h to obtain single cell suspension, adding PBS to stop digestion, centrifuging, and inoculating to DEME complete culture medium containing 10% fetal calf serum for culture;
(2) when the cell fusion degree cultured in the step (1) reaches 80-90%, carrying out subculture on the cells: removing the culture supernatant, adding 0.25% pancreatin, digesting for 2-3 min at 37 ℃, adding PBS to stop digestion after the cells become round and fall off, centrifuging, removing the supernatant, and inoculating the cells into a DEME complete culture medium containing 10% fetal calf serum for culture;
(3) and (3) taking cells which are passaged to 3-5 generations, culturing the cells by using a serum-free DEME culture medium, and collecting and counting the cells for later use when the cell fusion degree reaches 80-90%.
2. Adding the umbilical cord mesenchymal stem cell preservation solution of the embodiment 1-6 into the umbilical cord mesenchymal stem cells obtained in the step 1 respectively, and adjusting the concentration of the umbilical cord mesenchymal stem cells to be 0.8 multiplied by 106Obtaining umbilical cord mesenchymal stem cell preparation; and 3 parts of 2mL umbilical cord mesenchymal stem cell preparation in each group are respectively stored in a refrigerator at 4 ℃, and 1 part of the umbilical cord mesenchymal stem cell preparation is taken out after 1 day, 3 days and 5 days of storage to detect the storage effect.
3. And (3) storage effect detection: cell viability was detected by trypan blue staining, with non-stained cells being live cells and blue stained cells being dead cells. The results are shown in table 1:
TABLE 1 cell viability assay results
The results show that the umbilical cord mesenchymal stem cell preservation solution disclosed in embodiments 1-6 of the present invention can achieve a good preservation effect when preserving umbilical cord mesenchymal stem cells at 4 ℃, and the survival rate of the cells after being preserved for 3 days is still up to 90%, even 90% after being preserved for 5 days (embodiment 5).
4. And (3) safety detection: umbilical cord mesenchymal stem cell preparations which are stored for 5 days at the temperature of 4 ℃ in each group are respectively injected into SD rats through tail veins, and each group of umbilical cord mesenchymal stem cell preparations is injected into 10 SD rats. The results show that all SD rats injected with the umbilical cord mesenchymal stem cell preparation have no death or abnormal phenomenon, and the umbilical cord mesenchymal stem cell preparation prepared by the umbilical cord mesenchymal stem cell preservation solution disclosed by the embodiments 1-6 of the invention is safe and nontoxic.
Example 8
Through a large amount of researches and analyses, the inventor discovers that the survival rate of cells can be effectively improved by containing a proper amount of salidroside in the low-temperature preservation solution, and meanwhile, the preservation effect of the cells can be influenced by the content of the salidroside in the preservation solution.
In order to study the influence of the content of salidroside in the preservation solution on the cell preservation effect, experimental groups 1-9 are set, and are specifically shown in table 2:
TABLE 2 concentration design of salidroside in preservation solution
Umbilical cord mesenchymal stem cells are prepared according to the method of the embodiment 1, and the umbilical cord mesenchymal stem cells are respectively added into the umbilical cord mesenchymal stem cell preservation solution of the experiment groups 1-9, and the concentration of the umbilical cord mesenchymal stem cells is adjusted to be 0.8 multiplied by 106Obtaining umbilical cord mesenchymal stem cell preparation; 2mL of the umbilical cord mesenchymal stem cell preparation is respectively taken from each experimental group and stored in a refrigerator at 4 ℃, and the storage effect is detected on the 5 th day after storage.
Cell viability was detected by trypan blue staining, with non-stained cells being live cells and blue stained cells being dead cells. The results are shown in table 3:
TABLE 3 test results of preservation effect of umbilical cord mesenchymal stem cell preservation solution of experimental groups 1-9
From the above results, it is understood that the preservation effect is good when the concentration of salidroside in the umbilical cord mesenchymal stem cell preservation solution is 10 to 30uM (experimental group 2 to 8), better when the concentration of salidroside is 15 to 25uM (experimental group 4 to 6), and particularly the best when the concentration of salidroside in the preservation solution is 20uM (experimental group 5). When the concentration of salidroside in the umbilical cord mesenchymal stem cell preservation solution is lower than 10uM or higher than 30uM, the preservation effect is reduced (experiment group 1 and experiment group 9). Other experiments on the concentration of salidroside were also performed in this example, and the detailed results are omitted.
Example 9
The umbilical cord mesenchymal stem cell preservation solution comprises the following components in concentration: the low-sugar DMEM culture medium comprises a low-sugar DMEM culture medium, human serum albumin, sodium chloride, low-molecular heparin sodium, glucose, vitamin C and salidroside, wherein the salidroside can be used for effectively preserving umbilical cord mesenchymal stem cells at 4 ℃ and maintaining the survival rate of the cells.
In order to study the influence of salidroside on the preservation effect of the preservation solution, experimental groups 10 and 11 were set, wherein the composition of the umbilical cord mesenchymal stem cell preservation solution in experimental group 10 was the same as that in example 5, and the other components of the umbilical cord mesenchymal stem cell preservation solution in experimental group 11 were the same as those in experimental group 10 except that salidroside was not contained.
Umbilical cord mesenchymal stem cells are prepared according to the method of the embodiment 1, and the umbilical cord mesenchymal stem cells preservation solution of the experiment groups 10 and 11 is respectively added to adjust the umbilical cordThe concentration of mesenchymal stem cells is 0.8 × 106Obtaining umbilical cord mesenchymal stem cell preparation; 2mL of the umbilical cord mesenchymal stem cell preparation is respectively taken from each experimental group and stored in a refrigerator at 4 ℃, and the storage effect is detected on the 5 th day after storage.
Cell viability was detected by trypan blue staining, with non-stained cells being live cells and blue stained cells being dead cells. The results are shown in table 4:
table 4 test results of preservation effect of umbilical cord mesenchymal stem cell preservation solution of experiment groups 10-11
| Group of | Cell survival rate (day 5) |
| Experimental group 10 | 90.8％ |
| Experimental group 11 | 63.7％ |
From the above results, when the umbilical cord mesenchymal stem cell preservation solution does not contain salidroside, the preservation effect of the umbilical cord mesenchymal stem cell preservation solution on umbilical cord mesenchymal stem cells is significantly reduced (experimental group 11), which indicates that salidroside can effectively improve the preservation effect of the umbilical cord mesenchymal stem cell preservation solution.
Example 10
When the umbilical cord mesenchymal stem cell preservation solution is used for umbilical cord mesenchymal stem cells, the concentration of the umbilical cord mesenchymal stem cells is adjusted to be 0.5 multiplied by 106～1×106Better preservation effect can be obtained by/mL.
In this embodiment, the umbilical cord mesenchymal stem cell preservation solution of embodiment 5 is used as an example to study the influence of umbilical cord mesenchymal stem cell concentration on the preservation effect in the umbilical cord mesenchymal stem cell preparation, and experimental groups 12 to 18 are provided, specifically as shown in table 5:
TABLE 5 umbilical cord mesenchymal stem cell concentration design
Preparing umbilical cord mesenchymal stem cells according to the method in the embodiment 1, respectively adding the umbilical cord mesenchymal stem cell preservation solution in the embodiment 5, and adjusting the concentration of the umbilical cord mesenchymal stem cells according to the experimental group 12-18 to obtain corresponding umbilical cord mesenchymal stem cell preparations; 2mL of the umbilical cord mesenchymal stem cell preparation is respectively taken from each experimental group and stored in a refrigerator at 4 ℃, and the storage effect is detected on the 5 th day after storage.
Cell viability was detected by trypan blue staining, with non-stained cells being live cells and blue stained cells being dead cells. The results are shown in Table 6:
TABLE 6 test results
| Group of | Cell survival rate (day 5) |
| Experimental group 12 | 83.8％ |
| Experimental group 13 | 87.4％ |
| Experimental group 14 | 88.2％ |
| Experimental group 15 | 90.7％ |
| Experimental group 16 | 86.0％ |
| Experimental group 17 | 85.2％ |
| Experimental group 18 | 81.9％ |
From the above results, it is understood that the umbilical cord mesenchymal stem cell preservation solution of the present invention, when used for umbilical cord mesenchymal stem cells, adjusts the concentration of umbilical cord mesenchymal stem cells to 0.5 × 106～1×106the/mL can obtain better preservation effect (experiment group 13-17), especially the concentration of the umbilical cord mesenchymal stem cells is 0.8 multiplied by 106At a concentration of/mL, the storage effect was better. And the concentration of umbilical cord mesenchymal stem cells is less than 0.5 x 106/mL or greater than 1X 106The storage effect was reduced at/mL (Experimental group 12 and Experimental group 18).
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. The umbilical cord mesenchymal stem cell preservation solution is characterized by comprising the following components in concentration: 5-10% (V/V) of low-sugar DMEM medium, 15-25 g/L of human serum albumin, 8-10 g/L of sodium chloride, 2000-molecular heparin sodium, 3000AXaIU/mL, 20-50 g/L of glucose, 100-150 mg/L of vitamin C and 10-30 uM of salidroside.
2. The umbilical cord mesenchymal stem cell preservation solution according to claim 1, wherein the concentration of salidroside is 15-25 uM.
3. The umbilical cord mesenchymal stem cell preservation fluid according to claim 1, wherein the concentration of salidroside is 20 uM.
4. The umbilical cord mesenchymal stem cell preservation solution according to claim 1, wherein the glucose concentration of the low-sugar DMEM medium is 1000 mg/L.
5. Use of umbilical cord mesenchymal stem cell preservation solution according to any one of claims 1 to 4 in preservation of umbilical cord mesenchymal stem cells.
6. A preservation method of umbilical cord mesenchymal stem cells is characterized by comprising the following steps: counting the prepared umbilical cord mesenchymal stem cells, adding the umbilical cord mesenchymal stem cell preservation solution of any one of claims 1 to 4 into the umbilical cord mesenchymal stem cells, and adjusting the concentration of the umbilical cord mesenchymal stem cells to 0.5 x 106～1×106/mL。
7. The method for preserving umbilical cord mesenchymal stem cells according to claim 6, wherein the umbilical cord mesenchymal stem cell concentration is 0.8 x 106/mL。
8. The preservation method of umbilical cord mesenchymal stem cells according to claim 6, wherein the preservation solution of umbilical cord mesenchymal stem cells is pre-cooled to 4 ℃ and then added into umbilical cord mesenchymal stem cells.
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