CN110050782B - Stem cell cryopreservation solution and preparation method and cryopreservation method thereof - Google Patents
Stem cell cryopreservation solution and preparation method and cryopreservation method thereof Download PDFInfo
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Abstract
The invention discloses a stem cell cryopreservation solution, a preparation method thereof and a cryopreservation method, wherein the stem cell cryopreservation solution comprises the following components in parts by weight: 3-10 parts of dimethyl sulfoxide, 2-7 parts of human serum albumin, 0.5-3 parts of trehalose, 400.2-2 parts of dextran and 2-6 parts of hydroxyethyl starch. The stem cell cryopreservation solution can be used for cryopreserving cells for a long time, can obviously reduce the freezing damage of the cells, enables the recovered cells to have higher survival rate and adherence, and improves the cryopreservation efficiency of the cells. Meanwhile, the components of the stem cell frozen stock solution are clear, all the components are medical pharmacopoeia injection grade auxiliary materials, no serum is contained, the risk of introducing pollution and allergen by using heterogeneous serum is effectively prevented, the DMSO content is low, the negative influence of DMSO on cells is reduced, the safety is high, the stability is good, the requirements of CFDA and FDA are met, and the stem cell frozen stock solution can be directly used for human infusion and is suitable for clinical research and treatment.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a stem cell cryopreservation solution, a preparation method thereof and a cryopreservation method thereof.
Background
Stem cells are a class of cells with self-renewal and multipotential differentiation potential that have been widely used in scientific research and clinical therapy. The stem cells have wide application prospects in cell therapy, regenerative medicine, drug discovery, toxicology, developmental biology and other aspects. At present, stem cell medicine products are on the market worldwide, and with the development of science and technology, stem cells can be more effectively and widely used for treating various diseases so as to make up the situation that the traditional medicine treatment has poor effect on certain diseases.
In the process of preparation or clinical application of the stem cells, the stem cells are stored in liquid nitrogen at low temperature and then recovered for continuous culture or infusion when needed. Cell cryopreservation is one of important links in biological research, normal cells can have the conditions of aging and function loss along with the increase of generation number during in vitro culture, and the cell cryopreservation plays an important role in preventing cell degeneration caused by cell subculture and bacterial pollution in the subculture and solving a series of problems in the process of maintaining the subculture.
Currently, in the field of cell cryopreservation, in order to protect cells from freezing damage, it is widely adopted to add a Cryoprotectant (CPA) to a cryopreservation solution. Dimethyl sulfoxide (DMSO) is the most commonly used cryoprotectant in cell cryopreservation, but excessive DMSO concentration has certain toxicity to cells, which can cause intracellular protein denaturation, so that improper DMSO concentration selection can have great influence on cell preservation. In addition, serum is generally added into the conventional cell freezing medium, so that on one hand, the serum can protect cells and reduce the freezing damage of the cells, and the damage of DMSO to the cells can also be reduced; on the other hand, serum can provide various growth factors and nutrients, and maintain the cell viability. However, the addition of serum may present the risk of introducing contamination and allergens, which may not meet the clinical needs of stem cell therapy. Therefore, there is a need to develop a safe and efficient frozen stem cell culture solution for clinical use.
Disclosure of Invention
In order to solve the defects and shortcomings in the prior art, the invention aims to provide a stem cell cryopreservation solution, a preparation method thereof and a cryopreservation method thereof. The stem cell cryopreservation solution disclosed by the invention is low in DMSO content, does not contain serum, can be used for cryopreserving stem cells for a long time, enables the recovered stem cells to have higher survival rate and adherence, and can be safely and stably used for clinical research and treatment.
In order to realize the purpose, the invention adopts the technical scheme that:
a stem cell cryopreservation liquid comprises the following components in parts by weight: 3-10 parts of dimethyl sulfoxide, 2-7 parts of human serum albumin, 0.5-3 parts of trehalose, 400.2-2 parts of dextran and 2-6 parts of hydroxyethyl starch.
Dimethyl sulfoxide (DMSO) can permeate into cells, increase intracellular osmotic pressure, reduce freezing point, delay the process of cryopreservation, ensure that part of water in the cells can permeate out of the cells before freezing, reduce the formation of ice crystals in the cells in the process of freezing and cooling, thereby protecting the cells, reducing the damage of freezing the cells and increasing the protective effect of the stem cell cryopreservation solution on the cells. The Human Serum Albumin (HSA) can protect cell membranes in the processes of cell cryopreservation and cell recovery and improve the cell activity in the process of cell cryopreservation and cell recovery. Trehalose (Trehalose) is natural micromolecular disaccharide, can interact with cell membranes, stabilizes the structure of cell membrane protein in the processes of cryopreservation and recovery, can form a glassy matrix, reduces the damage of ice crystals formed outside cells to the cells, and improves the activity of the stem cell cryopreservation solution in the process of cell freeze thawing. Dextran 40(Dextran 40) can increase the extracellular osmotic pressure, reduce the free water in the cells, and reduce the cell damage caused by the formation of ice crystals during the freezing storage process.
Preferably, the stem cell frozen stock solution comprises the following components in parts by weight: 3-10 parts of dimethyl sulfoxide, 2-7 parts of human serum albumin, 0.5-3 parts of trehalose, 400.2-2 parts of dextran, 2-6 parts of hydroxyethyl starch and 70-95 parts of ionic buffer solvent. Experiments show that the stem cell cryopreservation liquid of the formula can effectively perform cryopreservation on cells, has high use safety, and can replace the serum-containing cryopreservation liquid of the conventional formula.
More preferably, the stem cell frozen stock solution comprises the following components in parts by weight: 3-7 parts of dimethyl sulfoxide, 3-7 parts of human serum albumin, 1-2 parts of trehalose, 400.2-1 parts of dextran, 2-6 parts of hydroxyethyl starch and 70-95 parts of an ionic buffer solvent. The formula is the preferable formula further screened by experiments, has better cryopreservation effect on cells, has higher cell activity after the stem cells are cryopreserved and recovered, and has larger diameter, better adherence and production state.
Most preferably, the stem cell frozen stock solution comprises the following components in parts by weight: 5 parts of dimethyl sulfoxide, 7 parts of human serum albumin, 2 parts of trehalose, 400.2 parts of dextran, 4 parts of hydroxyethyl starch and 81.8 parts of ion buffer solvent. The stem cell cryopreservation liquid has the best cryopreservation effect on cells and the highest cryopreservation efficiency.
In particular, the components adopted by the invention are all in medical pharmacopoeia injection grade.
Preferably, the ionic buffer solvent comprises at least one of Boehmeria injection, PBS buffer, physiological saline, ringer's solution and Hanks solution. Preferably, the ionic buffer solvent is Bomaili A injection. Bo Mai Li A injection (Plasma-Lyte A injection) is a multiple electrolyte injection (pH7.4), and is also a sterile pyrogen-free isotonic solution for intravenous administration, and can well maintain the osmotic pressure inside and outside cells.
The Plasma-Lyte A injection used in the invention can be obtained commercially or prepared by self.
Preferably, the Plasma-Lyte A electrolyte injection is prepared by adopting the following method:
(1) accurately weighing 5.26g of USP-grade sodium chloride (NaCl), 0.37g of USP-grade potassium chloride (KCl) and USP-grade magnesium chloride hexahydrate (MgCl) by using an electronic balance in sequence2·6H2O)0.3g, pharmacopeia grade sodium gluconate (C)6H11NaO7)5.02g, pharmacopeia grade sodium acetate trihydrate (C)2H3NaO2·3H2O)3.68g；
(2) Sequentially adding the auxiliary materials weighed in the step (1) into a 1L beaker, adding 900mL of ultrapure water into the beaker, and fully dissolving to prepare an aqueous solution;
(3) measuring the pH value of the aqueous solution obtained in the step (2) by using a pH meter, adjusting the pH value of the aqueous solution to 7.2-7.4, and finally fixing the volume to 1L;
(4) and (4) subpackaging the aqueous solution obtained in the step (3) into a plurality of 500mL blue-covered reagent bottles, sterilizing at high temperature and high pressure (121 ℃, 30min), and storing at normal temperature for later use.
The invention also provides a preparation method of the stem cell frozen stock solution, which comprises the following steps:
(1) dissolving trehalose, dextran 40 and hydroxyethyl starch with appropriate amount of water to obtain sterile trehalose solution, dextran 40 solution and hydroxyethyl starch solution;
(2) and sequentially adding a trehalose solution, a dextran 40 solution, a hydroxyethyl starch solution and human serum albumin into an ion buffer solvent, uniformly mixing, then dropwise adding dimethyl sulfoxide, filtering, and refrigerating to obtain the stem cell frozen stock solution.
The whole process of the preparation method of the invention needs to be carried out in a dark place. In the step (1), when the trehalose, the dextran 40 and the hydroxyethyl starch are dissolved by water, the components can be blown by a liquid transfer gun to promote the dissolution of the components.
The invention avoids the problem by reasonably controlling the dosage of the dimethyl sulfoxide, and on the other hand, the dimethyl sulfoxide is added to the last step when the stem cell freezing solution is prepared, so that the problem can be further avoided.
Preferably, the concentrations of the trehalose solution, the dextran 40 solution and the hydroxyethyl starch solution are respectively more than or equal to 10%. When the stem cell frozen stock solution is prepared, the trehalose, the dextran 40 and the hydroxyethyl starch are dissolved and filtered by water, so that the sterility of the components can be ensured, and the frozen stock solution is convenient to prepare subsequently.
Preferably, the refrigerating temperature is 2-8 ℃. At this temperature, the components of the stem cell cryopreservation solution can be kept active.
The invention also provides an application of the stem cell cryopreservation liquid, and preferably the stem cell cryopreservation liquid is used for cryopreservation of umbilical cord mesenchymal stem cells.
The invention also provides a stem cell cryopreservation method, and particularly relates to a method for cryopreserving stem cells by using the stem cell cryopreservation liquid.
Preferably, the density of the stem cells is 1-20 x 106cells/mL.
Preferably, the stem cells are P1-P9 generation stem cells.
Preferably, the stem cell is an umbilical cord mesenchymal stem cell.
Compared with the prior art, the invention has the beneficial effects that: the stem cell cryopreservation solution can be used for cryopreserving cells for a long time, can obviously reduce the freezing damage of the cells, enables the recovered cells to have higher survival rate and adherence, and improves the cryopreservation efficiency of the cells. Meanwhile, the components of the stem cell frozen stock solution are clear, all the components are medical pharmacopoeia injection grade auxiliary materials, no serum is contained, the risk of introducing pollution and allergen by using heterogeneous serum is effectively prevented, the DMSO content is low, the negative influence of DMSO on cells is reduced, the safety is high, the stability is good, the requirements of CFDA and FDA are met, and the stem cell frozen stock solution can be directly used for human infusion and is suitable for clinical research and treatment.
Drawings
FIG. 1 shows the growth state of stem cells after cryopreservation recovery using the cryopreservation solutions of comparative example 1 and examples 1 to 3;
FIG. 2 is a graph showing the growth state of stem cells after cryopreservation recovery using the cryopreservation solutions of examples 4 to 7;
FIG. 3 shows the growth state of stem cells after cryopreservation recovery using the cryopreservation solutions of comparative examples 2 to 3.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention is further illustrated by the following examples. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. It should be understood that the embodiments of the present invention are only for illustrating the technical effects of the present invention, and are not intended to limit the scope of the present invention. In the examples, the methods used were all conventional methods unless otherwise specified.
The invention provides a stem cell cryopreservation solution which comprises the following components in parts by weight: 3-10 parts of dimethyl sulfoxide, 2-7 parts of human serum albumin, 0.5-3 parts of trehalose, 400.2-2 parts of dextran and 2-6 parts of hydroxyethyl starch.
The stem cell cryopreservation solution does not contain serum, has low DMSO content and good cell cryopreservation effect, and can be safely and stably used for clinical research and treatment.
In a preferred embodiment, the stem cell cryopreservation solution mainly comprises the following components in parts by weight: 3-10 parts of dimethyl sulfoxide, 2-7 parts of human serum albumin, 0.5-3 parts of trehalose, 400.2-2 parts of dextran, 2-6 parts of hydroxyethyl starch and 70-95 parts of ionic buffer solvent. The ionic buffer solvent comprises at least one of Bomaili A injection, PBS buffer solution, normal saline, ringer's solution and Hanks solution. Preferably, the ionic buffer solvent is Bomaili A injection. Bomaili A injection can be purchased commercially or prepared by oneself. Experiments show that the stem cell cryopreservation liquid of the formula can effectively perform cryopreservation on cells, has high use safety, and can replace the serum-containing cryopreservation liquid of the conventional formula.
In a more preferred embodiment, the stem cell cryopreservation solution consists essentially of the following components in parts by weight: 3-7 parts of dimethyl sulfoxide, 3-7 parts of human serum albumin, 1-2 parts of trehalose, 400.2-1 parts of dextran, 2-6 parts of hydroxyethyl starch and 70-95 parts of an ionic buffer solvent. The formula is the preferable formula further screened by experiments, has better cryopreservation effect on cells, has higher cell activity after the stem cells are cryopreserved and recovered, and has larger diameter, better adherence and production state.
In a most preferred embodiment, the stem cell cryopreservation solution mainly comprises the following components in parts by weight: 5 parts of dimethyl sulfoxide, 7 parts of human serum albumin, 2 parts of trehalose, 400.2 parts of dextran, 4 parts of hydroxyethyl starch and 81.8 parts of ion buffer solvent. Experiments show that the stem cell cryopreservation liquid has the best cryopreservation effect on cells and the highest cryopreservation efficiency.
The invention also provides a preparation method of the Plasma-Lyte A electrolyte injection, which comprises the following steps:
(1) accurately weighing 5.26g of USP-grade sodium chloride (NaCl), 0.37g of USP-grade potassium chloride (KCl) and USP-grade magnesium chloride hexahydrate (MgCl) by using an electronic balance in sequence2·6H2O)0.3g, pharmacopeia grade sodium gluconate (C)6H11NaO7)5.02g, pharmacopeia grade sodium acetate trihydrate (C)2H3NaO2·3H2O)3.68g；
(2) Sequentially adding the auxiliary materials weighed in the step (1) into a 1L beaker, adding 900mL of ultrapure water into the beaker, and fully dissolving to prepare an aqueous solution;
(3) measuring the pH value of the aqueous solution obtained in the step (2) by using a pH meter, adjusting the pH value of the aqueous solution to 7.2-7.4, and finally fixing the volume to 1L;
(4) and (4) subpackaging the aqueous solution obtained in the step (3) into a plurality of 500mL blue-covered reagent bottles, sterilizing at high temperature and high pressure (121 ℃, 30min), and storing at normal temperature for later use.
The invention also provides a preparation method of the stem cell frozen stock solution, which comprises the following steps:
(1) respectively dissolving trehalose, hydroxyethyl starch and dextran 40 by using a proper amount of ultrapure water, wherein the trehalose solution, the hydroxyethyl starch solution and the dextran 40 solution with the concentrations of 10% are obtained by blowing with a liquid transfer gun in the dissolving process;
(2) adding the Bomaili A injection into a centrifugal tube, then sequentially adding a trehalose solution, a dextran 40 solution, a hydroxyethyl starch solution and human serum albumin, uniformly mixing, then dropwise adding DMSO, filtering, and refrigerating in a refrigerator at 2-8 ℃ to finish the preparation.
The invention also provides a stem cell cryopreservation method, which comprises the following steps: the stem cells are frozen by using the stem cell freezing medium.
In a preferred embodiment, the density of the stem cells is 1 to 20X 106cells/mL.
In a preferred embodiment, the stem cells are P1-P9 generation stem cells.
In a preferred embodiment, the stem cell is an umbilical cord mesenchymal stem cell.
In order to understand the present invention more clearly, the technical solution of the present invention will be further described below with reference to examples and comparative examples.
Example 1
A stem cell cryopreservation liquid comprises the following components in parts by weight: 1 part of trehalose, 400.2 parts of dextran, 3 parts of human serum albumin, 3 parts of dimethyl sulfoxide, 5 parts of hydroxyethyl starch and 87.8 parts of Bomaili A injection.
Example 2
A stem cell cryopreservation liquid comprises the following components in parts by weight: 1 part of trehalose, 400.5 parts of dextran, 5 parts of human serum albumin, 5 parts of dimethyl sulfoxide, 6 parts of hydroxyethyl starch and 82.5 parts of Bomaili A injection.
Example 3
A stem cell cryopreservation liquid comprises the following components in parts by weight: 1 part of trehalose, 401 parts of dextran, 7 parts of human serum albumin, 7 parts of dimethyl sulfoxide, 2 parts of hydroxyethyl starch and 82 parts of Bomaili A injection.
Example 4
A stem cell cryopreservation liquid comprises the following components in parts by weight: 1.5 parts of trehalose, 400.2 parts of dextran, 5 parts of human serum albumin, 7 parts of dimethyl sulfoxide, 5 parts of hydroxyethyl starch and 81.3 parts of Bomaili A injection.
Example 5
A stem cell cryopreservation liquid comprises the following components in parts by weight: 1.5 parts of trehalose, 401 parts of dextran, 3 parts of human serum albumin, 5 parts of dimethyl sulfoxide, 6 parts of hydroxyethyl starch and 83.5 parts of Bomaili A injection.
Example 6
A stem cell cryopreservation liquid comprises the following components in parts by weight: 2 parts of trehalose, 400.2 parts of dextran, 7 parts of human serum albumin, 5 parts of dimethyl sulfoxide, 4 parts of hydroxyethyl starch and 81.8 parts of Bomaili A injection.
Example 7
A stem cell cryopreservation liquid comprises the following components in parts by weight: 2 parts of trehalose, 400.5 parts of dextran, 3 parts of human serum albumin, 7 parts of dimethyl sulfoxide, 5 parts of hydroxyethyl starch and 82.5 parts of Bomaili A injection.
The method for preparing the stem cell cryopreservation solution of the embodiment 1 to 7 includes the following steps:
(1) respectively dissolving trehalose, hydroxyethyl starch and dextran 40 by using a proper amount of ultrapure water, wherein the trehalose solution, the hydroxyethyl starch solution and the dextran 40 solution with the concentrations of 10% are obtained by blowing with a liquid transfer gun in the dissolving process;
(2) adding Bomaili A injection into a centrifuge tube, then sequentially adding trehalose solution, dextran 40 solution, hydroxyethyl starch solution and human serum albumin, uniformly mixing, then dropwise adding DMSO, filtering, and refrigerating in a refrigerator at 4 ℃ for later use to finish the preparation.
The boehmeria injection A adopted in the examples is self-made, and the specific preparation method of the invention is specifically given in the above description, and is not repeated herein.
Comparative example 1
A conventional cell cryopreservation liquid comprises the following components in percentage by mass: fetal bovine serum 90% and dimethylsulfoxide 10%.
Comparative example 2
A stem cell cryopreservation liquid comprises the following components in parts by weight: 1.5 parts of trehalose, 400.5 parts of dextran, 8 parts of human serum albumin, 2 parts of dimethyl sulfoxide, 8 parts of hydroxyethyl starch and 80 parts of Bomaili A injection.
Comparative example 3
A stem cell cryopreservation liquid comprises the following components in parts by weight: 4 parts of trehalose, 403 parts of dextran, 1 part of human serum albumin, 4 parts of dimethyl sulfoxide, 8 parts of hydroxyethyl starch and 80 parts of Bomaili A injection.
The preparation methods of the stem cell frozen stocks of comparative examples 2 to 3 refer to the preparation methods of the above examples.
Examples of the experiments
Firstly, preparing umbilical cord mesenchymal stem cells, comprising the following steps:
(1) separating and culturing umbilical cord mesenchymal stem cells:
after the consent of family members, the cord of the infant which is discarded in clinic is collected, and after the cord is disinfected, the cord is put into a bottle of preservation solution and preserved at 4 ℃ for standby.
And taking out the umbilical cord, transferring the umbilical cord to a culture dish filled with normal saline, removing blood stains and mucus on the surface of the umbilical cord and blood coagulation in blood vessels, and transferring the umbilical cord to a second culture dish filled with normal saline. The cord was cut into several small sections with scissors and the cord sections were washed in a third petri dish. The umbilical cord was transferred to a fourth petri dish, the vein was completely exposed and removed with forceps, and the artery was removed with two pairs of ophthalmic forceps. The Wal-gel was peeled from the amniotic membrane, placed in a fifth petri dish, and the tissue was cut into small pieces and resuspended in basal medium.
Centrifugation, discarding the supernatant, and resuspension of the medium. Subpackaging in culture flask, adding37℃，5％CO2Culturing in an incubator. Changing the solution every 3 days, observing cell climbing out under a microscope for about 7-14 days, digesting the cells by trypsin when the cells grow to 80-90% confluence, and carrying out subculture.
(2) Amplification culture and cryopreservation of umbilical cord mesenchymal stem cells:
when the primary cells grow to 80% -90% confluence, absorbing the culture solution, washing the inner wall of the culture bottle once with normal saline, adding trypsin for digestion, beating the culture bottle to promote the cells to be separated from the bottle wall when the cells begin to shrink, immediately adding stop solution for mixing, collecting the cell suspension in a centrifugal tube, sieving the cell suspension with a 200-mesh cell sieve to remove umbilical tissue blocks, centrifuging the filtered suspension, removing the supernatant, adding a serum-free culture medium for resuspension, and resuspending according to the proportion of 0.5-2 × 104Cells/cm2And (5) density passage. And when the cells are passaged to the P1 generation, taking 100 ten thousand cells for flow detection, measuring cell surface markers, and identifying the umbilical cord mesenchymal stem cells.
When the cells were passaged to P7 generation, the cells were subjected to cryopreservation. Cell number, viability, diameter, etc. were measured prior to cryopreservation. The cell freezing density is 1 × 107cells/mL, 1mL of the cryopreservation solution was added to each cryopreservation tube.
(3) Resuscitating and collecting umbilical cord mesenchymal stem cells:
the frozen P7 generation cells were revived, and immediately after reviving, the cells were counted to determine the number, viability, diameter, and the like of the cells. According to the ratio of 0.5-2 x 104Cells/cm2Inoculating into culture flask at a density of 5% CO at 37 deg.C2Culturing in an incubator, wherein the culture medium is a serum-free culture medium. After culturing for a certain time, removing the culture medium, washing the inner wall of the culture flask with PBS once, then adding trypsin for digestion, beating the culture flask to promote the cells to be separated from the flask wall when the cells begin to shrink, immediately adding a stop solution for uniformly mixing, collecting cell suspension in a centrifuge tube, centrifuging to remove supernatant, adding PBS for resuspension, and counting the cells to determine the number, activity, diameter and the like of the cells.
And secondly, verifying the cryopreservation effect of the cryopreservation liquid of the embodiment and the comparative example on the umbilical cord mesenchymal stem cells.
The frozen stock solutions of examples 1 to 7 and comparative examples 1 to 3 were prepared. Selecting P7 umbilical cord mesenchymal stem cells, sucking and discarding culture solution when the cells grow to fill 80-90% of the bottom of a culture bottle, adding PBS for rinsing once, then adding a proper amount of trypsin, beating the culture bottle to promote the cells to be separated from the bottle wall and immediately adding a stop solution when the cells shrink and become round, collecting the cells, centrifuging at 1300rpm for 4min, discarding supernatant, adding PBS for re-suspension, counting by using a cell counter, removing PBS after counting, using prepared freezing stock solution according to the proportion of 1 multiplied by 107Cells were frozen at a density of 1mL per tube, with each group set in triplicate.
And (4) putting the freezing tube into a programmed cooling box recovered to room temperature, putting the programmed cooling box into a refrigerator at the temperature of minus 80 ℃ for overnight, and transferring to a liquid nitrogen tank for storage the next day. Ten days later, taking out the cells in the liquid nitrogen tank for resuscitation, putting the cryopreservation tube into a 37 ℃ water bath kettle for dissolution, continuously shaking the cryopreservation tube in the dissolution process, adding a culture medium to dilute cell suspension after complete melting, washing the cryopreservation tube for 1-2 times by using the culture medium, centrifuging at 1300rpm for 4min, discarding supernatant, adding PBS (phosphate buffer solution) for resuspension, counting by using a cell counter, and finally counting the cells according to the weight of 0.5-2 multiplied by 104Cells/cm2Inoculating into culture flask at a density of 5% CO at 37 deg.C2Culturing in an incubator for 12 hours, photographing to observe the growth state of the cells, collecting the cells, counting by using a cell counter, and measuring the number, activity, diameter and the like of the cells.
1. The results of measuring the number, activity, diameter, etc. of cells are shown in Table 1.
TABLE 1
As can be seen from Table 1, in examples 1-7, compared with comparative example 1, the cryopreservation efficiency of the cryopreservation solution on cells is significantly higher than that of comparative example 1 after the cryopreservation recovery of examples 2, 4, 5 and 6; after the examples 1, 3 and 7 are subjected to cryopreservation recovery, the cryopreservation efficiency of the cryopreservation solution on the cells is not obviously different from that of the comparative example 1. The stem cell cryopreservation liquid of the formula can achieve the cell cryopreservation effect even superior to that of the existing cell cryopreservation liquid containing heterogeneous serum, can replace the existing serum-containing cryopreservation liquid of the conventional formula, and prevents the risks of pollution and allergen caused by the application of the heterogeneous serum. In addition, the cryopreservation efficiency of the cryopreservation solution formula in example 6 on umbilical cord mesenchymal stem cells is highest, and the ratio of the number of cells after recovery culture for 12 hours to the number of cells before cryopreservation is 118.93 +/-4.35%, which is higher than that of comparative example 1(104.27 +/-2.64%) by 14.66%. This demonstrates that the cell cryopreservation liquid formulation of example 6 has a good cryopreservation effect on umbilical cord mesenchymal stem cells. After the freezing recovery of the comparative examples 2 and 3, the freezing efficiency of the freezing solution to the cells is obviously lower than that of the comparative example 1. The different proportions of the components in the stem cell cryopreservation solution can also obviously influence the cryopreservation effect of the stem cell cryopreservation solution on cells.
2. The growth state of the cells is shown in figures 1-3, and it can be seen from the figures that compared with the comparative example, the growth state of the umbilical cord mesenchymal stem cells after the freeze storage recovery of examples 1-7 is not significantly different from that of the comparative example 1; comparative examples 2 and 3 after cryopreservation and recovery, the growth state of umbilical cord mesenchymal stem cells is significantly lower than that of comparative example 1.
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 (6)
1. The umbilical cord mesenchymal stem cell cryopreservation liquid is characterized by comprising the following components in parts by weight:
3-10 parts of dimethyl sulfoxide, 2-7 parts of human serum albumin, 0.5-3 parts of trehalose, 400.2-2 parts of dextran, 2-6 parts of hydroxyethyl starch and 70-95 parts of Bomaili A injection.
2. The umbilical cord mesenchymal stem cell cryopreservation liquid as claimed in claim 1, which consists of the following components in parts by weight: 3-7 parts of dimethyl sulfoxide, 3-7 parts of human serum albumin, 1-2 parts of trehalose, 400.2-1 parts of dextran, 2-6 parts of hydroxyethyl starch and 70-95 parts of Bo Mai injection.
3. The umbilical cord mesenchymal stem cell cryopreservation liquid as claimed in claim 1, which consists of the following components in parts by weight: 5 parts of dimethyl sulfoxide, 7 parts of human serum albumin, 2 parts of trehalose, 400.2 parts of dextran, 4 parts of hydroxyethyl starch and 81.8 parts of Bomaili A injection.
4. The method for preparing the umbilical cord mesenchymal stem cell cryopreservation liquid as claimed in any one of claims 1 to 3, comprising the following steps:
(1) dissolving trehalose, dextran 40 and hydroxyethyl starch with appropriate amount of water to obtain sterile trehalose solution, dextran 40 solution and hydroxyethyl starch solution;
(2) sequentially adding a trehalose solution, a dextran 40 solution, a hydroxyethyl starch solution and human serum albumin into an ion buffer solvent, uniformly mixing, then dropwise adding dimethyl sulfoxide, filtering and refrigerating to prepare the umbilical cord mesenchymal stem cell frozen stock solution;
the concentrations of the trehalose solution, the dextran 40 solution and the hydroxyethyl starch solution are respectively more than or equal to 10 percent;
the refrigerating temperature is 2-8 ℃.
5. A cryopreservation method of umbilical cord mesenchymal stem cells, which is characterized in that the stem cells are cryopreserved by using umbilical cord mesenchymal stem cell cryopreservation liquid as defined in any one of claims 1 to 3; the density of the stem cells is 1-20 multiplied by 106cells/mL.
6. The cryopreservation method of umbilical cord mesenchymal stem cells as claimed in claim 5, wherein the stem cells are generation P1-P9 stem cells.
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| CN107711823B (en) * | 2017-11-23 | 2021-05-04 | 深圳市北科生物科技有限公司 | Cell cryopreservation liquid stored at normal temperature and application thereof |
| CN110551684B (en) * | 2019-09-16 | 2021-09-03 | 福建省海西细胞生物工程有限公司 | Preparation method of human umbilical cord mesenchymal stem cells |
| CN110684722A (en) * | 2019-11-12 | 2020-01-14 | 广东唯泰生物科技有限公司 | Preparation method of mesenchymal stem cells derived from placenta chorion plate tissue |
| CN111838136A (en) * | 2020-08-24 | 2020-10-30 | 深圳普瑞金生物药业有限公司 | Mesenchymal stem cell low-temperature preservation solution and preparation method thereof |
| CN112021304A (en) * | 2020-09-17 | 2020-12-04 | 山东省齐鲁干细胞工程有限公司 | Cryopreservation method and recovery method of umbilical cord mesenchymal stem cells |
| CN112753694A (en) * | 2020-12-25 | 2021-05-07 | 江苏艾尔康生物医药科技有限公司 | Cell cryopreservation liquid, freezing method for mesenchymal stem cells and application of cell cryopreservation liquid |
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| CN112806355A (en) * | 2021-01-20 | 2021-05-18 | 山东科硕生物技术有限公司 | Cell preservation solution and preparation method and application thereof |
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| CN114391536B (en) * | 2022-02-15 | 2022-12-20 | 山东省齐鲁细胞治疗工程技术有限公司 | Mesenchymal stem cell cryopreservation liquid |
| CN114766471A (en) * | 2022-05-31 | 2022-07-22 | 宁波建顺生物科技有限公司 | Cryopreservation liquid and cryopreservation method for long-term storage of stem cells and products thereof |
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| CN115386549A (en) * | 2022-08-03 | 2022-11-25 | 河南省遗传资源细胞库有限公司 | Cryopreservation method for umbilical cord red blood cells |
| CN115443985A (en) * | 2022-08-18 | 2022-12-09 | 宁波希诺赛生物科技有限公司 | Stem cell cryopreservation liquid and cryopreservation method and recovery method thereof |
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