CN117617232A - Preparation and use method of stem cell storage liquid - Google Patents
Preparation and use method of stem cell storage liquid Download PDFInfo
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Abstract
The invention relates to a preparation and use method of stem cell storage liquid, which belongs to the technical field of cell biology, and each 100 parts of stem cell storage liquid comprises the following components in parts by weight: 5-10 parts of compound amino acid injection, 0.05-0.075 part of glutamine dipeptide injection, 0.05-0.075 part of vitamin C injection, 5 parts of human serum albumin solution, 1-2.5 parts of dextran 40 glucose injection, 77-89 parts of compound electrolyte injection and a proper amount of sodium bicarbonate injection. In the technical scheme of the invention, the dosage of human serum albumin is reduced, the components of compound amino acid, proglumide and vitamin C are increased, the in-vitro living environment of stem cells is improved, and the stem cell storage liquid of the optimized formula can maintain the activity rate of the stem cells to be not lower than 85% in 168 hours. The addition of the dextran 40 glucose injection increases the solution viscosity so as to reduce the agglomeration of the stem cells, and the rate of the agglomeration of the stem cells is lower than 10% within 168 hours.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a preparation and use method of stem cell storage liquid.
Background
In recent years, with the development of technical means such as stem cell therapy, immune cell therapy and gene editing, and clinical medical research, cell therapy has become an important development direction as a drug research.
The preparation of cell preparations from cells that can be used clinically is one of the keys to cell therapy. Among them, how to maintain the effectiveness of cells and how to ensure the clinical safety of the products are the main difficulties of the clinical transformation of the cell products at present. The cell preparation takes cell suspension as a main form, and besides the cell, an adjuvant solution, namely a cell storage solution, has the tasks of maintaining cell activity, guaranteeing high cell activity, total cell density, living cell density and low agglomeration rate. Therefore, the discovery of a stem cell stock solution to solve the above problems is an important research in the cell therapy industry.
The human serum albumin has the functions of maintaining the osmotic pressure and the pH value of blood colloid, transporting substances inside and outside cells, resisting oxidization, combining with Fc receptors on the surfaces of the cells, regulating in-vitro physiological environment and the like, so that the human serum albumin has a protective effect on the cells. In the prior art, normal saline containing human serum albumin is generally used as a cell protection liquid, and cells are easy to agglomerate in the transportation process due to the biological property of the human serum albumin, so that great potential safety hazard exists in intravenous infusion. The cell aggregation rate represents the degree of aggregation of cells and is an important index for evaluating the safety of cell therapy products. When the cell aggregation rate is high, it is necessary to strictly control the aggregation rate because it is injected into animals to cause fatal reactions such as pulmonary embolism and vascular embolism. Thus, attempts to reduce the use of albumin while exploring a range of compositions to protect cells are an important requirement for clinical cell preparations.
Disclosure of Invention
The invention aims to provide a preparation and use method of a stem cell storage liquid, which reduces the dosage of human serum albumin, increases the components of compound amino acid, prognostics dipeptide and vitamin C, improves the in-vitro living environment of stem cells, optimizes the preservation efficiency of the stem cell storage liquid, improves the stability and the oxidation resistance of the stem cell storage liquid, and optimizes the stem cell storage liquid of a formula, so that the stem cell storage liquid can maintain the stem cell activity to be not lower than 85% for 168 hours and has lower agglomeration rate. The stem cell storage liquid can ensure that cell products such as mesenchymal stem cell preparations and the like have higher cell activity rate and living cell density within a period of up to 5 days or even longer within a range of 2-8 ℃, and simultaneously maintain low agglomeration rate.
The invention aims to solve the technical problems: in the prior art, normal saline containing human serum albumin is generally used as a stem cell protection liquid, and due to the biological property of the human serum albumin, stem cells are easy to agglomerate in the transportation process, and great potential safety hazard exists in intravenous infusion.
The aim of the invention can be achieved by the following technical scheme:
the stem cell storage liquid comprises the following components in parts by weight per 100 parts of stem cell storage liquid: 5-10 parts of compound amino acid injection, 0.05-0.075 part of glutamine dipeptide injection, 0.05-0.075 part of vitamin C injection, 5 parts of human serum albumin solution, 1-2.5 parts of dextran 40 glucose injection, 77-89 parts of compound electrolyte injection and a proper amount of sodium bicarbonate injection.
A preparation method of a stem cell storage liquid, 100mL of which comprises the following steps: in a sterile environment, sequentially adding 5mL of human serum albumin solution, 0.05-0.075mL of a proglumide dipeptide injection, 1-2.5mL of a dextran 40 glucose injection, 5-10mL of a compound amino acid injection (14 AA-V-SF), 0.05-0.075mL of a vitamin C injection into 75mL of a compound electrolyte injection, and adjusting the pH of the sodium bicarbonate injection to 7.0-7.4, wherein 100mL of the compound electrolyte injection is used; filtering, sealing in sterile container, and refrigerating at 2-8deg.C.
Further, the osmotic pressure of the stem cell stock solution is 278-343mOsmol/kg.
Further, the compound amino acid injection is balanced fourteen compound amino acid injection 14AA-SF without sulfite antioxidants.
Further, the concentration of the balanced fourteen compound amino acid injection 14AA-SF without sulfite antioxidants is 4.2g/50mL.
The compound amino acid injection provided by the invention does not contain sulfite antioxidants, is not easy to cause adverse reactions of sensitive physique patients, has low clinical application safety risk, can provide various essential and non-essential amino acids for stem cells, and participates in synthesis and metabolism of stem cell substances to maintain cell functions.
Further, the original concentration of the human serum albumin solution is 10g/50mL, and the human serum albumin is an important substance for maintaining osmotic pressure and protecting the activity of stem cells.
The L-alanyl-L-glutamine contained in the glutamine injection provided by the invention has stable structure in an aqueous solution, can not be degraded spontaneously, but can slowly release glutamine for a long time under the condition that living cells exist, thereby providing important raw materials for synthesizing nucleic acid and protein by stem cells, and simultaneously being used as an antioxidant for protecting the stem cells.
The compound electrolyte injection is an important auxiliary material for maintaining osmotic pressure and ion balance and other multiple biological functions.
A method of using a stem cell stock solution, comprising the steps of:
selecting a good P5 generation mesenchymal stem cell, when the cell fusion degree is more than 80%, using recombinant human trypsin to digest, adding an equal volume of complete culture medium to stop the digestion, lightly blowing into single cell suspension, centrifuging for 8 minutes at 200G, washing cell sediment for 2-3 times by using physiological saline containing 1% of human serum albumin, measuring the cell density after the stem cell storage solution is resuspended, adjusting the cell density to be the required density by using the stem cell storage solution, filling and sealing in a cell reinfusion bag, and preserving at low temperature.
Further, the mesenchymal stem cell density is 0.5-1.2E+06/mL.
Further, the low temperature is in the range of 2-8 ℃.
Further, the complete medium was complete medium for passage of cells containing friends Kang Gan.
In the stem cell storage liquid provided by the invention, the dosage of human serum albumin is reduced, the components of compound amino acid, proglumide and vitamin C are increased, the in-vitro living environment of stem cells is improved, the storage efficiency of the stem cell storage liquid is optimized, and the stability and the antioxidation capability of the stem cell storage liquid are improved. The dextran 40 glucose injection can increase the solution viscosity so as to reduce the aggregation of stem cells, can provide energy for the stem cells, reduces the consumption of the glycogenic amino acid, and prolongs the action duration of the compound amino acid. The invention also optimizes the pH adjusting mode of the stem cell storage liquid, and uses sodium bicarbonate injection to replace solid sodium hydroxide pharmaceutic adjuvant, thereby avoiding the process of preparing and sterilizing sodium hydroxide. The stem cell preparation using the stem cell storage liquid can be directly applied to clinical infusion, and can provide a key preparation formula for the drug reporting registration of stem cell research and development enterprises.
The invention has the beneficial effects that:
(1) In the technical scheme of the invention, the dosage of human serum albumin is reduced, the components of compound amino acid, proglumide and vitamin C are increased, the in-vitro living environment of stem cells is improved, the preservation efficiency of stem cell storage liquid is optimized, the stability and the antioxidation capability of the stem cell storage liquid are improved, the stem cell storage liquid with optimized formula can maintain the activity rate of the stem cells to be not lower than 85% within 168 hours, and the mesenchymal stem cells have normal phenotype characteristics and no differentiation phenomenon after being preserved in the stem cell storage liquid for 168 hours. The technical scheme provided by the invention obviously prolongs the preservation time of the stem cell preparation and can realize clinical application in a great range.
(2) In the technical scheme of the invention, all applied components are clinical injection medicines, and can be directly prepared in proportion, so that the operation steps are obviously reduced, the operation risk is reduced, and the clinical application is safe and reliable.
(3) In the technical scheme of the invention, the stability of the stem cell storage solution designed by the invention is not lower than 180 days, and the stem cell storage solution can be prepared into a shelf product and has excellent commercialized potential.
(4) According to the technical scheme, the stem cell storage liquid can effectively maintain the density of living cells for a long time, the number of detected living cells after 168 hours is still more than 90% of the original density, the stem cells can be stored at the temperature of 2-8 ℃, and the stem cells can be directly infused by intravenous infusion after short rewarming, so that the cell damage in the reprocessing process of stem cell recovery and the like is avoided, the potential safety risk is reduced, and the safety and the cell utilization rate are improved.
(5) In the technical scheme of the invention, the dextran 40 glucose injection is added to increase the viscosity of the solution, so that the formula has good anti-agglomeration effect, the agglomeration rate of the stem cells in the embodiment is lower than 10% in 168h, and the consumption of the glycogenic amino acid is reduced.
Drawings
FIG. 1 shows the change of the stem cell viability of mesenchymal stem cells stored in different stem cell stock solutions with the storage time.
FIG. 2 shows the change of the stem cell clumping rate of mesenchymal stem cells stored in different stem cell stock solutions with the storage time.
FIG. 3 shows the number of viable cells in a storage solution of mesenchymal stem cells stored in different stem cell storage solutions as a function of the storage time.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The sources of each reagent in the following examples are as follows:
human serum albumin solution: the specification is 10g/50mL, purchased from Jie Te Balin biological products Co., ltd., U.S. and national medicine standard code number S20140078;
the proglutidine injection comprises: the specification is 20g/100mL, and the Chinese medicine standard is H20053409, which is purchased from Feisen You Sika to Huarui pharmaceutical Co., ltd;
compound amino acid injection (14 AA-V-SF): the specification is 4.2g/50mL, and the medicine is purchased from Hubei pharmaceutical Co., ltd, national medicine standard character size H20174069;
dextran 40 glucose injection: 30g/500mL, available from Tianjin Shanlan pharmaceutical industry Co., ltd, national drug standard number H20053212;
glucose injection: the specification is 50g/500mL, and the Chinese medicine standard is H37021824, which is purchased from Chenxin pharmaceutical industry Co., ltd;
vitamin C injection: the specification is 0.5g/2mL, and the product is purchased from Henan Koren pharmaceutical Co., ltd, and the national medicine standard is H20064634;
compound electrolyte injection: 500 mL/bottle, available from Shanxi Zhengtaisheng pharmaceutical Co., ltd, national medicine standard number H20113035;
sodium bicarbonate injection: the specification was 12.5g/250mL, purchased from Sichuan Korea pharmaceutical Co., ltd., national drug standard H20043739.
Examples
A preparation method of a stem cell storage liquid, 100mL of which comprises the following steps: in a sterile environment, sequentially adding 5mL of human serum albumin solution, 0.05mL of a glutamine dipeptide injection, 2.5mL of a dextran 40 glucose injection, 5mL of a compound amino acid injection (14 AA-V-SF), 0.05mL of a vitamin C injection into 75mL of a compound electrolyte injection, adjusting the pH of the sodium bicarbonate injection to 7.0, and supplementing 100mL of the compound electrolyte injection; filtering, sealing in sterile container, and refrigerating at 2-8deg.C.
Examples
A preparation method of a stem cell storage liquid, 100mL of which comprises the following steps: in a sterile environment, sequentially adding 5mL of human serum albumin solution, 0.05mL of a glutamine dipeptide injection, 2.5mL of a dextran 40 glucose injection, 10mL of a compound amino acid injection (14 AA-V-SF), 0.05mL of a vitamin C injection into 75mL of a compound electrolyte injection, adjusting the pH of the sodium bicarbonate injection to 7.0, and supplementing 100mL of the compound electrolyte injection; filtering, sealing in sterile container, and refrigerating at 2-8deg.C.
Examples
A preparation method of a stem cell storage liquid, 100mL of which comprises the following steps: in a sterile environment, sequentially adding 5mL of human serum albumin solution, 0.075mL of a glutamine dipeptide injection, 2.5mL of a dextran 40 glucose injection, 5mL of a compound amino acid injection (14 AA-V-SF), 0.075mL of a vitamin C injection into 75mL of a compound electrolyte injection, adjusting the pH of the sodium bicarbonate injection to 7.0, and supplementing 100mL of the compound electrolyte injection; filtering, sealing in sterile container, and refrigerating at 2-8deg.C.
Comparative example 1
In comparison with example 1, the vitamin C injection was not added in comparative example 1, and the other steps and raw materials were synchronized with example 1.
Comparative example 2
Compared with example 1, the dosage of the dextran 40 glucose injection in comparative example 2 is changed from 2.5mL to 0.5mL, and other steps and raw materials are synchronous with example 1.
Comparative example 3
Compared with example 3, the dosage of the compound amino acid injection in comparative example 3 is changed from 5mL to 1mL, and other steps and raw materials are synchronous in example 3.
Comparative example 4
In comparative example 4, the dextran 40 glucose injection was changed to glucose injection, and the other steps and raw materials were synchronized with comparative example 3, as compared with comparative example 3.
Comparative example 5
In comparative example 5, the glucose injection was changed from 2.5mL to 0.5mL, and the other steps and materials were synchronized with comparative example 4, as compared with comparative example 4.
Blank control group
The blank control group only contains 10mL of human serum albumin solution and 90mL of compound electrolyte injection.
Performance detection
Selecting a good-growth-state P5 generation mesenchymal stem cell, when the cell fusion degree is more than 80%, using recombinant human trypsin to digest, adding an equal volume of cells containing Kang Gan for passage, stopping with a complete culture medium, gently blowing to form single cell suspension, centrifuging for 8 minutes at 200G, washing cell sediment for 3 times with physiological saline containing 1% human albumin, then re-suspending the cells with stem cell storage solutions prepared in examples 1-3, comparative examples 1-5 and blank control groups respectively, measuring the cell density, regulating the cell density to 0.5E+06/mL with the stem cell storage solution, filling and sealing in a cell reinfusion bag, and preserving at a low temperature of 2-8 ℃. The stem cell viability, the clumping rate and the viable cell count were measured every 24 hours by staining with AO/PI dye and counting with a counter, and the results are shown in tables 1-3 and FIGS. 1-3.
TABLE 1
As can be seen from Table 1 and FIG. 1, after the mesenchymal stem cells were stored in the stem cell storage solutions prepared in examples 1 to 3 for 120 hours at a low temperature of 2 to 8 ℃, the stem cell viability was more than 77%, wherein the mesenchymal stem cells were maintained at 85.7% after being stored in the stem cell storage solution prepared in example 3 for 168 hours, indicating that the storage solution prepared in the present invention can effectively prolong the storage time of the stem cells and ensure a high viability.
As can be seen from the data of comparative example 1 and comparative example 1, the addition of vitamin C injection can increase the stem cell viability.
As can be seen from the data of comparative examples 1 and 2, the dosage of the dextran 40 glucose injection is changed from 2.5mL to 0.5mL, and the stem cell viability is obviously reduced due to the reduction of the dosage, which indicates that the dosage of the dextran 40 glucose injection has obvious influence on the stem cell viability.
The data of comparative example 3 and comparative example 3 show that the effect of the reduction of the dosage of the compound amino acid injection on the stem cell viability is very remarkable, and the stem cell viability is reduced from 90.9% to 69.6% after 96 hours.
As can be seen from the data of comparative examples 3 and 4, the stem cell stock solution added with dextran 40 glucose injection can improve the stem cell viability more than the glucose injection.
As can be seen from the data of comparative examples 4 and 5, the dosage of glucose injection is changed from 2.5mL to 0.5mL, and the stem cell viability is reduced slightly, which indicates that the effect of glucose injection on stem cell viability is small.
TABLE 2
As can be seen from Table 2 and FIG. 2, the mesenchymal stem cells were stored in the stem cell stock solutions prepared in examples 2 to 3 for 168 hours at a low temperature of 2 to 8℃and the stem cell agglomeration rate was lower than 10%; wherein the caking rates of the comparative example 2 and the comparative example 4 are higher than those of other groups, which shows that the addition of the dextran 40 glucose injection has obvious improvement effect on the caking rate.
TABLE 3 Table 3
As can be seen from Table 3 and FIG. 3, the number of viable cells was higher than 3.2E+05/mL after the mesenchymal stem cells were stored in the stem cell stock solutions prepared in examples 2 to 3 for 168 hours at a low temperature of 2 to 8℃and satisfied the quality control criteria.
In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Claims (9)
1. A preparation method of stem cell stock solution is characterized in that: the preparation method of 100mL stem cell stock solution comprises the following steps: in a sterile environment, sequentially adding 5mL of human serum albumin solution, 0.05-0.075mL of a proglumide dipeptide injection, 1-2.5mL of a dextran 40 glucose injection, 5-10mL of a compound amino acid injection and 0.05-0.075mL of a vitamin C injection into 75mL of a compound electrolyte injection, adjusting the pH of the sodium bicarbonate injection to 7.0-7.4, and supplementing 100mL of the compound electrolyte injection; filtering, sealing in sterile container, and refrigerating at 2-8deg.C.
2. The method for preparing a stem cell stock solution according to claim 1, wherein: the osmotic pressure of the stem cell stock solution is 278-343mOsmol/kg.
3. The method for preparing a stem cell stock solution according to claim 1, wherein: the compound amino acid injection is balanced fourteen compound amino acid injection 14AA-SF which does not contain sulfite antioxidants.
4. A method of preparing a stem cell stock solution according to claim 3, wherein: the concentration of the balanced fourteen compound amino acid injection 14AA-SF without sulfite antioxidants is 4.2g/50mL.
5. The method for preparing a stem cell stock solution according to claim 1, wherein: the original concentration of the human serum albumin solution is 10g/50mL.
6. A method of using the stem cell stock solution prepared by the preparation method of any one of claims 1 to 5, characterized in that: the method comprises the following steps:
selecting a good P5 generation mesenchymal stem cell, when the cell fusion degree is more than 80%, using recombinant human trypsin to digest, adding an equal volume of complete culture medium to stop the digestion, lightly blowing into single cell suspension, centrifuging for 8 minutes at 200G, washing cell sediment for 2-3 times by using physiological saline containing 1% of human serum albumin, measuring the cell density after the stem cell storage solution is resuspended, adjusting the cell density to be the required density by using the stem cell storage solution, filling and sealing in a cell reinfusion bag, and preserving at low temperature.
7. The method of claim 6, wherein the step of using the stem cell stock solution comprises the steps of: the mesenchymal stem cell density is 0.5-1.2E+06/mL.
8. The method of claim 6, wherein the step of using the stem cell stock solution comprises the steps of: the low temperature is in the range of 2-8 ℃.
9. The method of claim 6, wherein the step of using the stem cell stock solution comprises the steps of: the complete medium is complete medium for passage of cells containing friends Kang Gan.
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