CN117796384A - High-concentration mesenchymal stem cell cryopreservation liquid, preparation and application thereof - Google Patents
High-concentration mesenchymal stem cell cryopreservation liquid, preparation and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 239000007788 liquid Substances 0.000 title claims abstract description 23
- 210000004027 cell Anatomy 0.000 claims abstract description 158
- 210000000130 stem cell Anatomy 0.000 claims abstract description 86
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Abstract
The invention discloses a high-concentration mesenchymal stem cell cryopreservation solution, a preparation and application thereof. The technical problem to be solved is to improve the survival rate of the frozen cells and maintain the high-efficiency biological performance of the frozen preparation with high concentration. The cell freezing solution comprises omnipotent nuclease, human serum albumin, dimethyl sulfoxide and compound electrolyte injection; the mass ratio of the omnipotent nuclease to the human serum albumin to the dimethyl sulfoxide to the compound electrolyte injection is 25kU:50g:55g:10.241g. Compared with the conventional cell cryopreservation liquid, the cell cryopreservation liquid can obviously improve the cell viability of cryopreservation when the high-concentration stem cell product is frozen, contributes to improving the cell viability of cryopreservation, and can maintain high biological performance of stem cells.
Description
Technical Field
The invention particularly relates to a high-concentration mesenchymal stem cell cryopreservation solution, a preparation and application thereof.
Background
The human umbilical cord mesenchymal stem cells (mesenchymal stem cells, MSC) are mainly obtained by separation from umbilical cords, are simple in material taking, wide in source, basically not limited by ethics, have the characteristics of low immunogenicity, strong immunoregulation capability, easier in-vitro expansion and the like, and gradually become seed cells with the most development prospect in tissue engineering and regenerative medicine.
MSC has been widely used clinically, and the concentration of domestic stem cell preparations is generally lower than 10 at present 7 The preparation specification is more than 10mL, the concentration of the preparation for treating related diseases is relatively low in the clinical research process, and the high-concentration frozen stem cell product is freshly reported. However, in view of the amount of administration in the clinical stage, the administration period and the specificity of certain indications, a large amount of administration and local administration treatment are required, and the limitation of the volume to be used is required, so that the need for a high-concentration lyophilized stem cell preparation is more urgent.
Disclosure of Invention
The invention solves the technical problems of improving the cell viability of high-concentration frozen stem cells and maintaining high biological performance, in particular to mesenchymal stem cells.
In order to solve the above problems, the present invention provides the following applications.
Use of a omnipotent nuclease in any one of the following:
a1 Improving the activity rate of the high-concentration frozen stem cells and/or preparing the product for improving the activity rate of the high-concentration frozen stem cells;
a2 Preparing cell freezing solution for improving the activity rate of high-concentration stem cells;
a3 Improving the total lymphocyte proliferation inhibition capacity of the high-concentration frozen stem cells and/or preparing a product for improving the total lymphocyte proliferation inhibition capacity of the high-concentration frozen stem cells;
a4 The application of the cell frozen stock solution for improving the proliferation inhibition capability of the high-concentration frozen stock cells to the total lymphocytes;
a5 Improving the secretion capacity of the high-concentration frozen stem cells for inhibiting the inflammatory factor TNF-alpha and/or preparing the product for improving the secretion capacity of the high-concentration frozen stem cells for inhibiting the inflammatory factor TNF-alpha;
a6 Preparing cell frozen stock solution for improving the capability of high-concentration frozen stem cells in inhibiting inflammatory factor TNF-alpha secretion;
a7 Improving the ability of the high-concentration frozen stem cells to promote the secretion of the growth factors TGF-beta and/or preparing the products for improving the ability of the high-concentration frozen stem cells to promote the secretion of the growth factors TGF-beta;
a8 Preparation of cell cryopreservation liquid for improving the capability of high-concentration cryopreservation stem cells for promoting the secretion of growth factors TGF-beta.
In order to solve the problems, the invention also provides a preparation method of the product for improving the activity rate of the high-concentration frozen stem cells.
The method comprises the step of taking omnipotent nuclease as an effective ingredient.
In the above, the substance may be a reagent, a kit or a composition.
Above, the substance contains the omnipotent nuclease.
In order to solve the problems, the invention also provides a product for improving the activity rate of the high-concentration frozen stem cells.
The product comprises omnipotent nuclease and also comprises any one of the following components:
k1 No;
k2 At least one of human serum albumin, dimethyl sulfoxide (DMSO) and a compound electrolyte.
In the product, the proportion of the omnipotent nuclease, the human serum albumin, the dimethyl sulfoxide and the compound electrolyte is 25kU:50g:55g:10.241g.
The compound electrolyte is composed of sodium chloride, sodium gluconate, sodium acetate trihydrate, potassium chloride and magnesium chloride hexahydrate, wherein the proportion of the sodium chloride, the sodium gluconate, the sodium acetate trihydrate, the potassium chloride and the magnesium chloride hexahydrate is 5.26g:5.02g:3.68g:0.37g:0.30g.
In order to solve the problems, the invention also provides a cell freezing solution.
The cell freezing solution contains the product and a solvent.
In order to solve the problems, the invention also provides the cell freezing solution.
The content of the omnipotent nuclease is 25kU/L.
In the above, the solvent may be water.
In the above, the water may be water for injection.
In the above, the cell cryopreservation solution may be composed of the following components: 5% DMSO, 70% compound electrolyte injection, 25% human serum albumin (20% mass-volume ratio of human serum albumin) and 1×10 -2 % of totipotent nuclease, wherein the percentage is the volume percentage.
The Human Serum Albumin (HSA) is available from jetty biologicals inc, switzerland under the designation S20170005. In the product, the mass volume ratio of human serum albumin is 20%.
Above, the omnipotent nuclease is available from offshore company under the trade designation GMP-1707. In this product, the concentration of the omnipotent nuclease was 250kU/mL.
The compound electrolyte injection can be purchased from Shanghai Baite medical supplies limited company, and the product number is national medicine standard H20000475. In the product, the compound electrolyte injection comprises: sodium chloride 5.26g/L; 5.02g/L of sodium gluconate; sodium acetate (C) 2 H 3 NaO 2 ·3H 2 O) 3.68g/L; potassium chloride 0.37g/L; chlorineMagnesium (MgCl) 2 ·6H 2 O)0.30g/L。
Above, the dimethyl sulfoxide (DMSO) was purchased from the company, nine classical pharmaceutical limited, hunan under the trade designation F2020999079. In this product, the purity of dimethyl sulfoxide is analytically pure.
In the present application, the stem cell density in the high-concentration cryopreserved stem cells may be 1×10 7 Up to 5X 10 7 Individual cells/mL.
In the present application, the stem cell density in the high-concentration cryopreserved stem cells may be 3×10 7 Individual cells/mL.
In the above, the stem cells have a cell density of 1×10 in the cell cryopreservation solution 7 Up to 5X 10 7 Individual cells/mL.
In the above, the stem cells have a cell density of 3×10 in the cell cryopreservation solution 7 Individual cells/mL.
In the above, the stem cells may be mesenchymal stem cells.
In order to solve the above problems, the present invention also provides the following applications.
The use of the above product in any of the following:
a1 Improving the activity rate of the high-concentration frozen stem cells and/or preparing the product for improving the activity rate of the high-concentration frozen stem cells;
a2 Preparing cell freezing solution for improving the activity rate of high-concentration stem cells;
a3 Improving the total lymphocyte proliferation inhibition capacity of the high-concentration frozen stem cells and/or preparing a product for improving the total lymphocyte proliferation inhibition capacity of the high-concentration frozen stem cells;
a4 The application of the cell frozen stock solution for improving the proliferation inhibition capability of the high-concentration frozen stock cells to the total lymphocytes.
A5 Improving the secretion capacity of the high-concentration frozen stem cells for inhibiting the inflammatory factor TNF-alpha and/or preparing the product for improving the secretion capacity of the high-concentration frozen stem cells for inhibiting the inflammatory factor TNF-alpha;
a6 Preparing cell frozen stock solution for improving the capability of high-concentration frozen stem cells in inhibiting inflammatory factor TNF-alpha secretion;
a7 Improving the ability of the high-concentration frozen stem cells to promote the secretion of the growth factors TGF-beta and/or preparing the products for improving the ability of the high-concentration frozen stem cells to promote the secretion of the growth factors TGF-beta;
a8 Preparation of cell cryopreservation liquid for improving the capability of high-concentration cryopreservation stem cells for promoting the secretion of growth factors TGF-beta.
The use of the cell cryopreservation solution described above in any of the following:
b1 Improving the viability of the high-concentration cryopreserved stem cells and/or preparing a high-concentration product for improving the viability of the cryopreserved stem cells;
b2 Improving the total lymphocyte proliferation inhibition capacity of the high-concentration frozen stem cells and/or preparing a product for improving the total lymphocyte proliferation inhibition capacity of the high-concentration frozen stem cells;
b3 Improving the secretion capacity of the high-concentration frozen stem cells for inhibiting the inflammatory factor TNF-alpha and/or preparing the product for improving the secretion capacity of the high-concentration frozen stem cells for inhibiting the inflammatory factor TNF-alpha;
b4 Improving the capability of the high-concentration frozen stem cells for promoting the secretion of the growth factors TGF-beta and/or preparing a product for improving the capability of the high-concentration frozen stem cells for promoting the secretion of the growth factors TGF-beta.
In the above, the high concentration stem cells may have a cell density of 1×10 7 Up to 5X 10 7 Individual cells/mL.
In the above, the high concentration stem cells may have a cell density of 3×10 7 Individual cells/mL.
In order to solve the problems, the invention also provides a preparation method of the frozen mesenchymal stem cells.
The method comprises the step of freezing and preserving the mesenchymal stem cells by using the cell freezing and preserving solution.
In the above, when the cell cryopreservation solution is used for cryopreservation, the density of the mesenchymal stem cells can be 1×10 7 Up to 5X 10 7 Individual cells/mL. The density of the mesenchymal stem cells may be 3×10 7 Individual cells/mL.
In the above, the stem cells may be mesenchymal stem cells. The mesenchymal stem cells may be one or more of umbilical cord mesenchymal stem cells, amniotic mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells and mesenchymal stem cells obtained by differentiation of pluripotent stem cells.
In the above, the mesenchymal stem cells have a cell density of 1×10 in the cell cryopreservation solution 7 Up to 5X 10 7 Individual cells/mL.
In the above, the mesenchymal stem cells have a cell density of 3×10 in the cell cryopreservation solution 7 Individual cells/mL.
In order to solve the above problems, the present invention also provides mesenchymal stem cells.
The mesenchymal stem cells are obtained by thawing the frozen mesenchymal stem cells prepared by the method.
In the above method or the above mesenchymal stem cells, the cell density of the mesenchymal stem cells is 1×10 7 Up to 5X 10 7 Individual cells/mL.
In the above, the mesenchymal stem cells have a cell density of 3×10 in the cell cryopreservation solution 7 Individual cells/mL.
Advantageous effects
The invention discloses a high-concentration mesenchymal stem cell cryopreservation solution, a preparation and application thereof. The technical problem to be solved is to improve the cryopreservation density of stem cells and maintain the viability and biological performance of stem cells at a very high level. The cell freezing solution comprises omnipotent nuclease, human serum albumin, dimethyl sulfoxide and compound electrolyte injection; the proportion of the omnipotent nuclease, the human serum albumin (the mass volume ratio of the human serum albumin is 20%), the dimethyl sulfoxide and the compound electrolyte is 25kU/L:25vol%:5vol%:70vol%, wherein the percentage is volume percentage.
The cell cryopreservation liquid of the invention is conventional cell cryopreservation liquid [ cryopreservation liquid 1:75% of compound electrolyte injection, 5% of dimethyl sulfoxide, 5% of dextran 40 sodium chloride injection and 15% of human serum albumin (the mass volume ratio of the human serum albumin is 20%), wherein the percentage is the volume percentage; frozen stock solution 2: compared with 70% of compound electrolyte injection, 5% of dimethyl sulfoxide and 25% of human serum albumin (the mass volume ratio of the human serum albumin is 20%), the high-concentration stem cells frozen by the frozen stock solution can obviously improve the activity of frozen stock cells, and the frozen stock stem cells of the frozen stock solution have good effects of inhibiting the proliferation capacity of total lymphocytes, inhibiting the secretion capacity of inflammatory factors TNF-alpha and promoting the secretion capacity of growth factors TGF-beta in terms of biological performance.
The above-mentioned omnipotent nuclease was purchased from a near-shore company under the trade designation GMP-1707 (in this product, the concentration of the omnipotent nuclease was 250 kU/mL). The above dimethyl sulfoxide was purchased from Hunan Jiujingzhu pharmaceutical Co., ltd, under the trade designation F2020999079 (in this product, the purity of dimethyl sulfoxide was analytically pure). The above mentioned human albumin, jetty berlin biologicals limited, switzerland, has a product number S20170005 (in this product, the mass-to-volume ratio of human albumin is 20%). The compound electrolyte injection is purchased from Shanghai Baite medical supplies Co., ltd, and the product number is national medicine standard H20000475 (in the product, the compound electrolyte injection contains 5.26g/L sodium chloride, 5.02g/L sodium gluconate, sodium acetate (C) 2 H 3 NaO 2 ·3H 2 O) 3.68g/L; potassium chloride 0.37g/L; magnesium chloride (MgCl) 2 ·6H 2 O)0.30g/L)。
The company researches and invents a high-concentration cryopreservation preparation of human umbilical mesenchymal stem cells, which has high cryopreservation cell activity rate and excellent biological performance, meets the requirement of mass administration and can be used as a stem cell product for clinical use.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In the following examples, mesenchymal stem cells serum-free complete medium: the culture medium is obtained by adding 5mL of mesenchymal stem cell serum-free culture medium additive (Youkang organism, product number: NC0103. S) into 500mL of mesenchymal stem cell serum-free basic culture medium (Youkang organism, product number: NC 0103).
In the examples below, trypLE is the product of GIBCO, accession number 12563-029.
In the following examples, 0.9% sodium chloride injection is a product of Shijia four-medicine, and the product number is national medicine standard H13023201. In this product, the sodium chloride concentration was 0.9g/100ml.
In the examples described below, dimethyl sulfoxide (DMSO) is a product of the pharmaceutical company, nine classics, hunan, under the trade designation F20209990795. In this product, the purity of dimethyl sulfoxide is analytically pure.
In the following examples, the compound electrolyte injection is a product of Shanghai Baite medical supplies Co., ltd, and the product number is national medicine standard H20000475. In the product, the compound electrolyte injection comprises: sodium chloride 5.26g/L; 5.02g/L of sodium gluconate; sodium acetate (C) 2 H 3 NaO 2 ·3H 2 O) 3.68g/L; potassium chloride 0.37g/L; magnesium chloride (MgCl) 2 ·6H 2 O)0.30g/L。
In the examples described below, human serum albumin is a product of jetty bellin biologicals, inc. In switzerland, product number S20170005. In the product, the mass volume ratio of human serum albumin is 20%.
In the examples below, the omnipotent nuclease is a near shore product, under the trade designation GMP-1707. In this product, the concentration of the omnipotent nuclease was 250kU/mL.
In the following examples, dextran 40 sodium chloride injection is a product of Sichuan Korea pharmaceutical Co., ltd, product number of national medicine standard H51020230.
In the following examples, the AO/PI dye liquor is Nexcelom company product with the product number CS2-0106-5ml.
In the following examples, CFSE is a BD company product, under the product number 565082.
In the following examples, 1640 complete medium: to 450mL of 1640 basal medium (GIBCO Co., ltd., product No. 11875-093) was added 50mL of fetal bovine serum (BI Co., product No. 04-001-1 ACS).
In the examples described below, phytohemagglutinin (PHA) is a product of Sigma company under the product number L8754-5mg.
Peripheral Blood Mononuclear Cells (PBMCs) referred to in the following examples were isolated from platelet-rich buffy coat (platelet-rich buffy coat derived from healthy adults in the blood center of the Tianjin city) and were designated PBMC20211123. The specific separation method is as follows: platelet-rich white membranes were slowly added to the surface of the human lymphocyte separation solution and allowed to separate by centrifugation at 300g for 20 min. The middle buffy coat layer was carefully aspirated with a pipette and washed twice with 300g of physiological saline for 5 minutes by centrifugation to obtain peripheral blood mononuclear cells.
In the examples described below, the TNF- α ELISA kit was purchased from NOVUS company under the product number VAL105.
In the examples below, TGF-beta ELISA kits were purchased from NOVUS company under the product number VAL127.
Example 1 preparation of frozen stock solution
The experimental group frozen stock solution consists of 4 components of omnipotent nuclease, human serum albumin, dimethyl sulfoxide and compound electrolyte injection. In the experimental group frozen stock solution, the ratio of the omnipotent nuclease to the human serum albumin (the mass volume ratio of the human serum albumin is 20 percent) to the dimethyl sulfoxide to the compound electrolyte injection is 25kU/L:25vol%:5vol%:70vol%; the percentage content is volume percentage content.
The preparation method comprises the following steps: prepare a 15mL centrifuge tube and add DMSO0.5mL of compound electrolyte injection (7 mL) and 1 mu L of total nuclease are sequentially added respectively, after uniform mixing, 2.5mL of human serum albumin is slowly added finally, and a light shaking centrifuge tube is added for full uniform mixing, so as to obtain experimental group frozen stock solution, and the experimental group frozen stock solution is preserved at 4 ℃ for standby (prepared at present and used in the same day).
The experimental group frozen stock solution is prepared from the following components: omnipotent nuclease, human serum albumin, dimethyl sulfoxide and compound electrolyte.
25kU/L of the omnipotent nuclease, 50g/L of the human serum albumin, 55g/L of the dimethyl sulfoxide and 10.241g/L of the compound electrolyte; the solvent is water for injection.
The compound electrolyte consists of sodium chloride, sodium gluconate, sodium acetate trihydrate, potassium chloride and magnesium chloride hexahydrate, wherein the proportion of the sodium chloride, the sodium gluconate, the sodium acetate trihydrate, the potassium chloride and the magnesium chloride hexahydrate is 5.26g:5.02g:3.68g:0.37g:0.30g.
Control group 1 frozen stock: consists of 4 components of dextran 40 sodium chloride injection, human serum albumin, dimethyl sulfoxide and compound electrolyte; the ratio of the dextran 40 sodium chloride injection to the human serum albumin (the mass volume ratio of the human serum albumin is 20 percent) to the dimethyl sulfoxide to the compound electrolyte injection is 5vol%:15vol%:5%:75vol%; the percentage content is volume percentage content.
Wherein, the freezing solution of the control group 1 is prepared from the following components: according to the volume ratio, 75% of compound electrolyte injection, 5% of DMSO,5% of dextran 40 sodium chloride injection and 15% of human serum albumin (the mass volume ratio of the human serum albumin is 20%), wherein the percentage content is the volume percentage content.
Control group 2 frozen stock:
consists of 3 components of human serum albumin, dimethyl sulfoxide and compound electrolyte; the ratio of the human serum albumin (the mass volume ratio of the human serum albumin is 20 percent) to the dimethyl sulfoxide to the compound electrolyte injection is 25vol%:5vol%:70vol%; the percentage content is volume percentage content.
Wherein, the frozen stock solution of the control group 2 is prepared from the following components: according to the volume ratio, 70% of compound electrolyte injection, 5% of DMSO and 25% of human serum albumin (the mass volume ratio of the human serum albumin is 20%), wherein the percentage is the volume percentage.
Example 2 preparation procedure of high concentration formulation
The preparation method of the frozen cells comprises the step of freezing the cells by using a freezing solution of an experimental group. The specific method comprises the following steps:
1. mesenchymal stem cell resuscitation
And taking mesenchymal stem cells P4 generation working library cells stored by liquid nitrogen, placing the cells in a freezing box, and placing the freezing tube into a water bath kettle at 37 ℃ as soon as possible, and starting to randomly check the thawing condition for 1-3 minutes until the frozen cells are completely thawed. In a biosafety cabinet, the cryopreservation tube was opened, the thawed cell suspension was aspirated, 1.5mL of the cell suspension was added to a centrifuge tube containing 10mL of complete medium (mesenchymal stem cells serum-free complete medium), and centrifuged at 300g for 5 minutes. After centrifugation, the supernatant was aspirated, and the cells were resuspended in serum-free complete medium of mesenchymal stem cells equilibrated to room temperature, according to 11000 cells/cm 2 Is inoculated in T175 culture flask, after inoculation, each flask is added with complete medium without serum of mesenchymal stem cells to 35mL, and after marking, the flask is placed in 5% CO 2 The culture of cells was continued in an incubator at 37℃under saturated humidity for about 3 days with a confluence of about 80%, and mesenchymal stem cells were harvested to prepare a preparation.
The specific preparation method of the mesenchymal stem cells MSC (P4 generation) comprises the following steps: taking an isolated umbilical cord (neonatal puerpera comes from a south-open hospital, has no infectious diseases such as hepatitis, syphilis and AIDS, and the puerpera and family members agree with each other when the umbilical cord is used for experimental study) for disinfection, and obtaining a disinfected umbilical cord; then cleaning the sterilized umbilical cord, and cutting the cleaned umbilical cord into 1-2cm sections to obtain umbilical cord sections; cleaning umbilical cord segment, and cutting into 1-2mm pieces 3 Tissue blocks, using fetal bovine serum complete medium (90% DMEM/F12 medium+10% FBS), were placed in 5% CO 2 Culturing the isolated umbilical cord tissue blocks under the conditions of saturated humidity and 37 ℃; and finally, after the mesenchymal stem cells climb out, subculturing until the P4 generation of cells are frozen, and obtaining the mesenchymal stem cells P4 generation of working library cells stored by liquid nitrogen.
2. Preparation of washing liquid
The formula of the washing liquid 1 is as follows: 250mL of 0.9% sodium chloride injection is added with 1.3mL of human serum albumin (the mass volume ratio of the human serum albumin is 20%), and the mixture is gently mixed to prepare a washing liquid 1; in washing solution 1, the concentration of sodium chloride was 0.009g/ml, and the concentration of human serum albumin was 0.001g/ml, to obtain washing solution 1.
The formula of the washing liquid 2 is as follows: 200mL of washing liquid 1 is taken, 20 mu L of omnipotent nuclease is added, and the washing liquid 2 is obtained after uniform mixing, wherein the concentration of sodium chloride in the washing liquid 2 is 0.009g/mL, the concentration of human serum albumin is 0.001g/mL, and the concentration of omnipotent nuclease is 25U/mL (25 kU/L).
3. Mesenchymal stem cell preparation
The experiments were divided into 3 groups, namely an experimental group, a control group 1 and a control group 2.
The experimental group performed the following operations: the culture solution in the mesenchymal stem cell culture bottles is sucked and removed, 30mL of 0.9% sodium chloride injection is added into each T175 culture bottle to wash out the residual culture medium, 5mL of TrypLE is added into the supernatant to digest for about 2 minutes, and 10mL of 0.9% sodium chloride injection is added to stop digestion after the cells are completely suspended.
Adding the cell suspension which is stopped to digest into a centrifuge tube, washing each culture bottle by using 0.9% sodium chloride injection, adding the washed cell suspension into the centrifuge tube again, balancing the centrifuge tubes pairwise, centrifuging for 8 minutes, and sucking all supernatant after centrifuging.
The cells were resuspended in 40mL of washing solution 1, mixed well, centrifuged for 8 min, and the whole supernatant was aspirated after centrifugation, and this washing step was repeated 1-2 times, with washing times no less than 2 times.
The washed cells were resuspended in 40mL of washing solution 2, homogenized, 300g was centrifuged for 8 minutes, centrifuged, resuspended in 40mL of washing solution 2, and about 200. Mu.L of cell suspension was used for cell counting in an EP tube after homogenized, the total number of cells was adjusted according to the counting result, 300g was centrifuged for 8 minutes, the whole supernatant was sucked after centrifugation, and the cells were resuspended in the experimental group frozen stock in example 1, and the cell concentration was adjusted to 3X 10 according to the counting result 7 And (3) obtaining cell/mL, namely obtaining cell liquid before freezing and preserving the experimental group, and subpackaging the cell liquid in liquid nitrogen after freezing and preserving the experimental group.
Control group 1 was subjected to the following operations:
the operation difference between the control group 1 and the experimental group is that the experimental group frozen stock solution is replaced by the control group 1 frozen stock solution, and the rest operation is the same as that of the experimental group, so as to obtain the control group 1 frozen stock cells.
Control group 2 was operated as follows:
the operation difference between the control group 2 and the experimental group is that the experimental group frozen stock solution is replaced by the control group 2 frozen stock solution, and the rest operation is the same as that of the experimental group, so as to obtain the control group 2 frozen stock cells.
EXAMPLE 3 cell viability of high concentration formulation
1. Cell resuscitation
Randomly extracting 3 freezing cells of each experimental group, 1 freezing cell of the control group and 2 freezing cells of the control group, recovering the freezing cells of the experimental group, 1 freezing cell of the control group and 2 freezing cell of the control group based on the step of recovering the mesenchymal stem cells in the embodiment 2 until the freezing cells are completely melted, and respectively obtaining recovered freezing cells 1-3 of the experimental group, 1-3 of the control group and 1-3 of the control group.
2. Cell viability assay
The cell viability statistics of the recovered experimental group frozen cells, the recovered control group 1 frozen cells and the recovered control group 2 frozen cells are carried out by using the AO/PI dye liquor, and the results show that the cell viability of the recovered experimental group frozen cells is over 95 percent, the cell viability of the recovered control group 1 frozen cells is only 78.65 percent, the cell viability of the recovered control group 2 frozen cells is only 74.37 percent, and the specific results are shown in Table 1.
The cell viability statistical method is carried out on the AO/PI dye liquor: taking 20 mu L of cell suspension from an EP tube, adding 20 mu L of AO/PI dye liquor, and fully and uniformly mixing; 20. Mu.L of the suspension was added to the well of the counting plate and detected on-machine using a double fluorescence cytometry.
TABLE 1 results of cell viability assay
EXAMPLE 4 biological Property detection of high concentration formulations
Biological performance assays include assays that inhibit the proliferation capacity of total lymphocytes, inhibit the secretion of inflammatory factor TNF- α, and promote the secretion of growth factor TGF- β.
1. Inhibition of Total lymphocyte proliferation
The immunoregulatory function was evaluated by the level of mesenchymal stem cells inhibiting proliferation of human total lymphocytes. The carboxyl fluorescein diacetate succinimidyl ester (CFSE) is used for carrying out fluorescent marking on human peripheral blood mononuclear cells, and the CFSE is a novel dye capable of carrying out fluorescent marking on living cells and can mark living cells. In the process of cell division and proliferation, the fluorescence intensity of the cell is gradually decreased along with the division of the cell, and the marker fluorescence can be evenly distributed into two sub-generation cells, so that the fluorescence intensity of the cell is half of that of a parent cell, the proliferation capacity of the cell is not affected, and the flow cytometry is used for detecting the fluorescence intensity of the cell, so that the proliferation capacity of the cell is detected.
3 experimental groups of frozen cells are randomly extracted, and based on the step of recovering the mesenchymal stem cells in the embodiment 2, the frozen cells in the experimental groups are recovered until the frozen cells are completely melted, and the recovered frozen cells 1-3 of the experimental groups are respectively obtained.
Peripheral Blood Mononuclear Cell (PBMC) staining: peripheral blood mononuclear cell suspension (cell concentration 1×10) 7 Per mL,1 mL) was added with 2. Mu.L of 5mM CFSE, mixed well, the CFSE reached a final concentration of 10. Mu.M, the cell suspension was cultured in a 37℃water bath for staining for 10-15min, 10mL of 1640 complete medium was added, centrifuged, the supernatant was decanted, and this step was repeated twice, and the cells were resuspended in 1640 complete medium to a concentration of 6X 10 6 cells/mL, stained peripheral blood mononuclear cells were obtained for cell culture.
Preparing PHA working solution: taking 40 mu L of 1mg/mL PHA solution (PHA concentration is 1mg/mL in 1mg/mL PHA solution), adding 960 mu L of 1640 complete medium, and obtaining PHA working solution; the concentration of PHA in the PHA working solution was 40. Mu.g/mL.
Experimental group: the thawed experiment groups were counted for 1-3 cells at a ratio of 6X 10 4 cells/well were seeded in 48-well plates at 37℃with 5% CO 2 Culturing overnight to adhere to wall. Absorbing and removing supernatant from the 48-well plate, inoculating the dyed peripheral blood mononuclear cells into the 48-well plate with mesenchymal stem cells adhered to the wall at a concentration of 100 mu L/well, adding125 mu L of PHA working solution and 275 mu L of 1640 complete medium, namely an experimental group system, under the condition of 37 ℃,5% CO 2 After culturing in an incubator for five days, the cells were collected, washed three times with 3mL of 1 XPBS, centrifuged, and 200. Mu.L of 1 XPBS was added to resuspend the cells to obtain experimental group cells 1-3.
Negative control was set: inoculating the stained peripheral blood mononuclear cells into a 48-well plate at a concentration of 100 μl/well, adding 400 μl of 1640 complete medium, and adding 5% CO at 37deg.C 2 After culturing in an incubator for five days, cells were collected, washed three times with 3mL of 1 XPBS, centrifuged, and 200. Mu.L of 1 XPBS was added to resuspend the cells to obtain negative control cells.
Setting a positive control: inoculating the above stained peripheral blood mononuclear cells into 48-well plate at a ratio of 100 μl/well, adding 125 μl PHA working solution and 275 μl 1640 complete medium, and adding 5% CO at 37deg.C 2 After culturing in an incubator for five days, cells were collected, washed three times with 3mL of 1 XPBS, centrifuged, and 200. Mu.L of 1 XPBS was added to resuspend the cells to obtain positive control cells.
The flow cytometer detects the fluorescence intensity of CFSE and calculates the primary cell proportion and proliferation inhibition rate.
And (3) calculating results: the data were obtained with CellQuest software, analyzed with CellQuest software and Modfit software, and exported. The inhibition of total lymphocyte proliferation by mesenchymal stem cells was calculated by analysis of the percentage of CFSE expression in different groups.
Inhibition = 1- ((negative control primary cell ratio-experimental group primary cell ratio)/(negative control primary cell ratio-positive control primary cell ratio) ×100%).
The results are shown in Table 2, 3 branches of randomly extracted high-concentration preparations of the frozen stock solution of the invention, compared with a positive control, the ratio of the primary cells of the total lymphocytes can be obviously improved by the 1-3 experimental group cells, and the primary cells are respectively improved from 22.80% to 81.63%, 78.21% and 80.74%; the inhibition rates of lymphocyte proliferation are respectively 76.43%, 71.99% and 75.28%, and the inhibition rates are all above 70%, which shows that the preparation has very high biological performance in immunoregulation.
TABLE 2 inhibition of Total lymphocyte proliferation
| Group of | Primary cell ratio | Inhibit proliferation rate |
| Negative control | 99.77％ | / |
| Positive control | 22.80％ | / |
| Cryopreserved cells 1 of experimental group | 81.63％ | 76.43％ |
| Cryopreserved cells of experimental group 2 | 78.21％ | 71.99％ |
| Cryopreserved cells of experimental group 3 | 80.74％ | 75.28％ |
2. Inhibition of inflammatory factor TNF-alpha secretion and promotion of growth factor TGF-beta secretion assays
3 experimental groups of frozen cells are randomly extracted, and based on the step of recovering the mesenchymal stem cells in the embodiment 2, the frozen cells in the experimental groups are recovered until the frozen cells are completely melted, and the recovered frozen cells 1-3 of the experimental groups are respectively obtained.
Cell density of 1.6X10 6 Preparation of peripheral blood mononuclear cells/mL: peripheral blood mononuclear cell density was adjusted to 1.6X10 using 1640 complete medium 6 Per mL, a cell density of 1.6X10 was obtained 6 Peripheral blood mononuclear cells per mL for later use.
Preparing PHA working solution: taking 40 mu L of 1mg/mL PHA solution (PHA concentration is 1mg/mL in 1mg/mL PHA solution), adding 960 mu L of 1640 complete medium, and obtaining PHA working solution; the concentration of PHA in the PHA working solution was 40. Mu.g/mL.
Experimental group: the thawed experiment groups were counted for 1-3 cells, respectively, at 8X 10 4 cells/well were seeded in 24-well plates at 37℃with 5% CO 2 Overnight culture, allowing to adhere, removing supernatant from 24-well plate, and concentrating to a cell density of 1.6X10 6 Peripheral blood mononuclear cells per mL are inoculated into a 24-well plate attached with mesenchymal stem cells at a concentration of 250 mu L/well, 250 mu L of PHA working solution is added, the temperature is 37 ℃ and the concentration of CO is 5% 2 After 72h incubation in an incubator, 300g was centrifuged for 10min and the supernatant was collected to obtain experimental group supernatants 1-3 for detection of TNF- α and TGF- β protein levels.
Negative control was set: the cell density was 1.6X10 6 Peripheral blood mononuclear cells per mL were inoculated into 24-well plates at 250. Mu.L/well, 250. Mu.L of 1640 complete medium was supplemented, at 37℃with 5% CO 2 After 72h incubation in incubator, 300g was centrifuged for 10min, and the supernatant was collected to obtain negative control supernatant, and the levels of TNF-. Alpha.and TGF-. Beta.proteins were detected.
Setting a positive control: inoculating the above stained peripheral blood mononuclear cells into 24-well plate at a concentration of 250 μL/well, adding 250 μLPHA working solution, and adding 5% CO at 37deg.C 2 After 72h incubation in incubator, 300g was centrifuged for 10min, and the supernatant was collected to obtain positive control supernatant, and the levels of TNF- α and TGF- β proteins were detected.
And the TNF- α secretion inhibition rate and TGF- β secretion promotion rate were calculated, TNF- α secretion inhibition rate= (positive control concentration-experimental group concentration)/positive control concentration×100%. TGF- β secretion promotion rate= (experimental group concentration-positive control concentration)/positive control concentration×100%.
TNF-alpha protein level detection: protein levels were detected using an ELISA kit for TNF- α, available from NOVUS company under the designation VAL105.
TGF-beta protein level detection: protein levels were detected using an ELISA kit for TGF-beta. The kit was purchased from NOVUS company under the designation VAL127.
As shown in Table 3, compared with the positive control, the experimental group can remarkably inhibit the secretion of the inflammatory factor TNF-alpha, the secretion amount is respectively reduced from 6150.212pg/mL to 865.719pg/mL, 864.088pg/mL and 844.052pg/mL, and the inhibition rates are 85.92%, 85.63% and 85.97%, which are all above 80%; meanwhile, the secretion of the growth factor TGF-beta can be promoted, the secretion amount of the TGF-beta is respectively increased from 113.050pg/mL to 4184.699pg/mL, 3810.143pg/mL and 3593.506pg/mL, and the promotion rates are 3601.64%, 3270.32% and 3078.69% respectively, which are all over 2000%. The index reflects that the frozen cells of the experimental group have high biological performance in inflammatory regulation and damage repair.
TABLE 3 influence on factor expression
In summary, the invention provides the high-concentration human umbilical mesenchymal stem cell cryopreservation solution with high cell activity and high biological performance, and the preparation prepared by adopting the cryopreservation solution can ensure that cells have high activity under the condition of higher stem cell preparation concentration, and has excellent performance on biological performance indexes such as immunoregulation, inflammatory regulation, tissue repair and the like.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (11)
1. Use of a omnipotent nuclease in any one of the following:
a1 Improving the activity rate of the high-concentration frozen stem cells and/or preparing the product for improving the activity rate of the high-concentration frozen stem cells;
a2 Preparing cell freezing solution for improving the activity rate of high-concentration stem cells;
a3 Improving the total lymphocyte proliferation inhibition capacity of the high-concentration frozen stem cells and/or preparing a product for improving the total lymphocyte proliferation inhibition capacity of the high-concentration frozen stem cells;
a4 The application of the cell frozen stock solution for improving the proliferation inhibition capability of the high-concentration frozen stock cells to the total lymphocytes;
a5 Improving the secretion capacity of the high-concentration frozen stem cells for inhibiting the inflammatory factor TNF-alpha and/or preparing the product for improving the secretion capacity of the high-concentration frozen stem cells for inhibiting the inflammatory factor TNF-alpha;
a6 Preparing cell frozen stock solution for improving the capability of high-concentration frozen stem cells in inhibiting inflammatory factor TNF-alpha secretion;
a7 Improving the ability of the high-concentration frozen stem cells to promote the secretion of the growth factors TGF-beta and/or preparing the products for improving the ability of the high-concentration frozen stem cells to promote the secretion of the growth factors TGF-beta;
a8 Preparation of cell cryopreservation liquid for improving the capability of high-concentration cryopreservation stem cells for promoting the secretion of growth factors TGF-beta.
2. The method for preparing the high-concentration freeze-stored stem cell viability product according to claim 1, wherein the method comprises the step of using omnipotent nuclease as an active ingredient.
3. The product for improving the activity rate of high-concentration frozen stem cells according to claim 1, which comprises totipotent nuclease and further comprises any one of the following:
k1 No;
k2 At least one of human serum albumin, dimethyl sulfoxide and compound electrolyte.
4. The product of claim 3, wherein the ratio of said omnipotent nuclease, said human serum albumin, said dimethyl sulfoxide and said compound electrolyte is 25kU:50g:55g:10.241g.
5. A cell cryopreservation solution comprising the product of claim 3 or 4 and a solvent.
6. The cell freezing medium according to claim 5, wherein the content of the totipotent nuclease is 25kU/L.
7. Use of a product according to any one of claims 1-4 in any one of the following:
a1 Improving the activity rate of the high-concentration frozen stem cells and/or preparing the product for improving the activity rate of the high-concentration frozen stem cells;
a2 Preparing cell freezing solution for improving the activity rate of high-concentration stem cells;
a3 Improving the total lymphocyte proliferation inhibition capacity of the high-concentration frozen stem cells and/or preparing a product for improving the total lymphocyte proliferation inhibition capacity of the high-concentration frozen stem cells;
a4 The application of the cell frozen stock solution for improving the proliferation inhibition capability of the high-concentration frozen stock cells to the total lymphocytes;
a5 Improving the secretion capacity of the high-concentration frozen stem cells for inhibiting the inflammatory factor TNF-alpha and/or preparing the product for improving the secretion capacity of the high-concentration frozen stem cells for inhibiting the inflammatory factor TNF-alpha;
a6 Preparing cell frozen stock solution for improving the capability of high-concentration frozen stem cells in inhibiting inflammatory factor TNF-alpha secretion;
a7 Improving the ability of the high-concentration frozen stem cells to promote the secretion of the growth factors TGF-beta and/or preparing the products for improving the ability of the high-concentration frozen stem cells to promote the secretion of the growth factors TGF-beta;
a8 Preparation of cell cryopreservation liquid for improving the capability of high-concentration cryopreservation stem cells for promoting the secretion of growth factors TGF-beta.
8. Use of the cell cryopreservation solution of claim 5 or 6 in any of the following:
b1 Improving the viability of the high-concentration cryopreserved stem cells and/or preparing a high-concentration product for improving the viability of the cryopreserved stem cells;
b2 Improving the total lymphocyte proliferation inhibition capacity of the high-concentration frozen stem cells and/or preparing a product for improving the total lymphocyte proliferation inhibition capacity of the high-concentration frozen stem cells;
b3 Improving the secretion capacity of the high-concentration frozen stem cells for inhibiting the inflammatory factor TNF-alpha and/or preparing the product for improving the secretion capacity of the high-concentration frozen stem cells for inhibiting the inflammatory factor TNF-alpha;
b4 Improving the capability of the high-concentration frozen stem cells for promoting the secretion of the growth factors TGF-beta and/or preparing a product for improving the capability of the high-concentration frozen stem cells for promoting the secretion of the growth factors TGF-beta.
9. A method for preparing a frozen mesenchymal stem cell, comprising freezing a mesenchymal stem cell using the cell freezing solution according to claim 5 or 6.
10. Mesenchymal stem cells, characterized in that the mesenchymal stem cells are obtained by thawing frozen mesenchymal stem cells prepared using the method of claim 9.
11. The method of claim 9 or the mesenchymal stem cell of claim 10, wherein the mesenchymal stem cell has a cell density of 1 x 10 7 Up to 5X 10 7 Individual cells/mL.
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