CN112616831B - Inductive pluripotent stem cell preserving fluid and preparation method thereof - Google Patents

Inductive pluripotent stem cell preserving fluid and preparation method thereof Download PDF

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CN112616831B
CN112616831B CN202110026917.9A CN202110026917A CN112616831B CN 112616831 B CN112616831 B CN 112616831B CN 202110026917 A CN202110026917 A CN 202110026917A CN 112616831 B CN112616831 B CN 112616831B
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sodium hyaluronate
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宋文海
刘通
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Shanghai Yuantian Biotechnology Co.,Ltd.
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents

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Abstract

The invention discloses an induced pluripotent stem cell preserving fluid which is prepared from the following raw materials: DMEM medium and fullerene C90Sesamol, adenosine cyclic phosphate ester and ethanolAmine, linoleic acid, low molecular weight sodium hyaluronate, high molecular weight sodium hyaluronate and vitamin E. The DMEM medium in the preservation solution is helpful for maintaining the activity of the cells, and the added fullerene C90The sesamol and the adenosine cyclophosphate solve the problem that induced pluripotent stem cells are easy to aggregate in the preservation process to influence the activity of the cells, and the ethanolamine, the linoleic acid, the low molecular weight sodium hyaluronate and the high molecular weight sodium hyaluronate are beneficial to maintaining the osmotic pressure outside the cells, providing a stable external environment for the cells and improving the survival rate of the cells. The preservation solution effectively prolongs the preservation time, establishes a stable induced pluripotent stem cell preservation system, and provides guarantee for clinical application. The invention also provides a preparation method of the preserving fluid, which has the advantages of simple preparation process, easy operation and suitability for batch production and application.

Description

Inductive pluripotent stem cell preserving fluid and preparation method thereof
Technical Field
The invention relates to a cell preservation solution, in particular to an induced pluripotent stem cell preservation solution and a preparation method thereof.
Background
Induced Pluripotent Stem Cells (iPSCs) are pluripotent stem cells obtained by introducing four transcription factors, namely Oct4, Sox2, Klf4 and c-Myc, into mouse skin fibroblasts by using a retrovirus vector for the first time in japan scientist mountains in 2006 and reprogramming the fibroblasts. The iPSCs are very similar to embryonic stem cells in morphology, gene and protein expression, epigenetic modification state, cell multiplication capacity, embryoid body and teratoma generation capacity, differentiation capacity and the like.
The application value of the iPSCs as induced pluripotent stem cells in the aspect of clinical disease treatment is huge, because the iPSCs do not need human ova or human embryonic stem cells like the traditional stem cell obtaining modes such as nuclear transplantation or cell fusion, and the like, thereby avoiding various barriers such as ethics and laws and the like, and the iPSCs avoid immune rejection by utilizing the characteristic of reprogramming adult cells into stem cells so that autologous transplantation becomes more feasible; on the other hand, the iPSCs have great significance for the research of stem cell self-renewal mechanism, the research of stem cell signal regulation and the research of pathogenesis of a plurality of diseases and the search of treatment methods.
Clinically prepared iPSCs often need to be preserved in a preservation solution for later use. The conventional cell preservation solution contains DMSO (dimethyl sulfoxide) which is an osmotic protective agent and can reduce the freezing point of cells, reduce the formation of ice crystals and reduce the damage of free radicals to the cells. However, DMSO has a certain toxic effect, can react with protein hydrophobic groups, causes protein denaturation, and has vascular toxicity and hepatorenal toxicity. When the DMSO-containing preservation solution is used for preserving cells, ultralow temperature preservation is required, and the requirement on preservation conditions is high. The existing cell preservation solution without DMSO has various problems, such as short preservation time, strict preservation condition, easy occurrence of cell aggregation phenomenon, easy damage of cell activity, low survival rate and the like, and can not meet the requirements of clinical application. Therefore, improvement of the storage solution for iPSCs is required to improve the safety and storage efficiency thereof.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the induced pluripotent stem cell preserving fluid, which can effectively prolong the preservation time, maintain the survival rate of cells and avoid the aggregation of the cells without adding DMSO.
The second purpose of the invention is to provide a preparation method of the induced pluripotent stem cell preserving fluid.
One of the purposes of the invention is realized by adopting the following technical scheme:
an induced pluripotent stem cell preserving fluid is composed of the following raw materials: DMEM medium and fullerene C90Sesamol, adenosine cyclophosphate, ethanolamine, linoleic acid, low molecular weight sodium hyaluronate, high molecular weight sodium hyaluronate and vitamin E.
Further, the dosage of each component is as follows based on the final volume of the culture medium: fullerene C9040-50 mu g/mL, sesamol 35.5-39.5 mu g/mL, adenosine cyclophosphate 45-55 mu g/mL, ethanolamine 1-5mg/mL, linoleic acid 8-12mg/mL, low molecular weight sodium hyaluronate 1-5mg/mL, high molecular weight sodium hyaluronate 1-5mg/mL, and vitamin E5-10 mg/mL.
Further, the dosage of each component is as follows based on the final volume of the culture medium: fullerene C9045 mu g/mL, sesamol 37.5 mu g/mL, adenosine cyclophosphate 50 mu g/mL, ethanolamine 3mg/mL, linoleic acid 10mg/mL, low molecular weight sodium hyaluronate 2mg/mL, high molecular weight sodium hyaluronate 2mg/mL, vitamin E8 mg/mL.
Further, the average molecular weight of the low molecular weight sodium hyaluronate is less than or equal to 1 ten thousand daltons, and the average molecular weight of the high molecular weight sodium hyaluronate is 100-150 ten thousand daltons.
The second purpose of the invention is realized by adopting the following technical scheme:
the preparation method of the induced pluripotent stem cell preserving fluid comprises the following steps: adding fullerene C into DMEM medium90Sesamol, adenosine cyclophosphate, ethanolamine, linoleic acid, low molecular weight sodium hyaluronate, high molecular weight sodium hyaluronate and vitamin E, and uniformly stirring to obtain the composition.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides an induced pluripotent stem cell preserving fluid, wherein a DMEM (DMEM) culture medium in the preserving fluid is helpful for maintaining cell activity, and added fullerene C90The sesamol and the adenosine cyclic phosphate solve the problem that induced pluripotent stem cells are easy to aggregate in the preservation process to influence the activity of the cells, and the ethanolamine, the linoleic acid, the low molecular weight sodium hyaluronate and the high molecular weight sodium hyaluronate are beneficial to maintaining the osmotic pressure outside the cells, providing a stable external environment for the cells and improving the survival rate of the cells. According to the invention, through the synergistic effect of the components, aggregation and agglomeration of the induced pluripotent stem cells in the low-temperature (4-10 ℃) storage process are effectively avoided, the survival rate of the cells is improved, the storage time is prolonged, a stable induced pluripotent stem cell storage system is established, and a guarantee is provided for clinical application.
2. The invention also provides a preparation method of the induced pluripotent stem cell preservation solution, which is simple and convenient in preparation process, easy to operate and suitable for batch production and application.
Detailed Description
The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.
Example 1
An induced pluripotent stem cell preserving fluid is prepared fromThe following raw materials: DMEM medium and fullerene C90Sesamol, adenosine cyclophosphate, ethanolamine, linoleic acid, sodium hyaluronate with the average molecular weight of 1 ten thousand daltons, sodium hyaluronate with the average molecular weight of 100 ten thousand daltons and vitamin E;
based on the final volume of the culture medium, the dosage of each component is as follows: fullerene C9045 mu g/mL, sesamol 37.5 mu g/mL, adenosine cyclophosphate 50 mu g/mL, ethanolamine 3mg/mL, linoleic acid 10mg/mL, sodium hyaluronate 2mg/mL with an average molecular weight of 1 ten thousand daltons, sodium hyaluronate 2mg/mL with an average molecular weight of 100 ten thousand daltons, and vitamin E8 mg/mL.
The preparation method of the induced pluripotent stem cell preserving fluid comprises the following steps: adding fullerene C into DMEM medium90Sesamol, adenosine cyclophosphate, ethanolamine, linoleic acid, low molecular weight sodium hyaluronate, high molecular weight sodium hyaluronate and vitamin E, and uniformly stirring to obtain the composition.
Example 2
An induced pluripotent stem cell preserving fluid is composed of the following raw materials: DMEM medium and fullerene C90Sesamol, adenosine cyclophosphate, ethanolamine, linoleic acid, sodium hyaluronate with the average molecular weight of 8 kilodaltons, sodium hyaluronate with the average molecular weight of 120 kilodaltons and vitamin E;
based on the final volume of the culture medium, the dosage of each component is as follows: fullerene C9040 mu g/mL, sesamol 35.5 mu g/mL, adenosine cyclophosphate 45 mu g/mL, ethanolamine 1mg/mL, linoleic acid 8mg/mL, sodium hyaluronate 1mg/mL with an average molecular weight of 8 kilodaltons, sodium hyaluronate 1mg/mL with an average molecular weight of 120 kilodaltons, and vitamin E5 mg/mL.
The preparation method of the induced pluripotent stem cell preserving fluid is the same as the example.
Example 3
An induced pluripotent stem cell preserving fluid is composed of the following raw materials: DMEM medium and fullerene C90Sesamol, adenosine cyclophosphate, ethanolamine, linoleic acid, sodium hyaluronate with average molecular weight of 6 kilodaltons, sodium hyaluronate with average molecular weight of 150 kilodaltons, vitamin ABiotin (E);
based on the final volume of the culture medium, the dosage of each component is as follows: fullerene C9050 mu g/mL, sesamol 39.5 mu g/mL, adenosine cyclophosphate 55 mu g/mL, ethanolamine 5mg/mL, linoleic acid 12mg/mL, sodium hyaluronate 5mg/mL with an average molecular weight of 6 kilodaltons, sodium hyaluronate 5mg/mL with an average molecular weight of 150 kilodaltons, and vitamin E10 mg/mL.
The preparation method of the induced pluripotent stem cell preserving fluid is the same as the example.
Comparative example 1
Comparative example 1 provides an induced pluripotent stem cell preserving fluid, which is different from example 1 in that: omitting fullerene C90Otherwise, the same as in example 1 was repeated.
Comparative example 2
Comparative example 2 provides an induced pluripotent stem cell preserving fluid, which is different from example 1 in that: reacting fullerene C90Adjusted to fullerene C60Otherwise, the same as in example 1 was repeated.
Comparative example 3
Comparative example 3 provides an induced pluripotent stem cell preserving fluid, which is different from example 1 in that: sesamol was omitted and the procedure was as in example 1.
Comparative example 4
Comparative example 4 provides an induced pluripotent stem cell preserving fluid, which is different from example 1 in that: the adenosine cyclic phosphate was omitted and the rest was the same as in example 1.
Comparative example 5
Comparative example 5 provides an induced pluripotent stem cell preserving fluid, which is different from example 1 in that: the adenosine cyclic phosphate was replaced with adenosine, and the rest was the same as in example 1.
Comparative example 6
Comparative example 6 provides an induced pluripotent stem cell preserving fluid, which is different from example 1 in that: the amount of sodium hyaluronate having an average molecular weight of 1 kilodalton was adjusted to 4mg/mL without using sodium hyaluronate having an average molecular weight of 100 kilodalton, and the rest was the same as in example 1.
Comparative example 7
Comparative example 7 provides an induced pluripotent stem cell preserving fluid, which is different from example 1 in that: the amount of sodium hyaluronate having an average molecular weight of 100 kilodaltons was adjusted to 4mg/mL by omitting sodium hyaluronate having an average molecular weight of 1 kilodaltons, and the rest was the same as in example 1.
Induced pluripotent stem cells were resuspended in the storage solutions of examples 1 to 3 and comparative examples 1 to 5, respectively, at a cell concentration of 1X 106Each cell/mL was stored at 4 ℃ for 24 hours, 48 hours, 72 hours, 96 hours, and 120 hours, and then sampled, and the cell aggregation rate was measured by a Countstar cytometer using the trypan blue staining method, and the results are shown in Table 1.
TABLE 1
Figure GDA0003257863210000061
It can be seen from table 1 that the cell clumping rate in the preservation solutions of examples 1 to 3 is lower, and the cell clumps automatically disperse under the shaking of external force to form cell suspension again. Comparative examples 1 to 5 in which fullerene C was omitted90One of sesamol and adenosine cyclic phosphate or fullerene C90Substitution to Fullerene C60After the adenosine cyclic phosphate is replaced by adenosine, the cell aggregation rate is obviously increased along with the prolonging of the preservation time, and the cell aggregation rate exceeds 2% after 120h preservation, which indicates that the fullerene C is added into the preservation solution of the invention90Sesamol and adenosine cyclic phosphate reduce the aggregation rate of cells and prolong the storage time of induced pluripotent stem cells.
Induced pluripotent stem cells were resuspended in the storage solutions of examples 1 to 3 and comparative examples 1 to 7, respectively, at a cell concentration of 1X 106One cell/mL, which was rapidly recovered in a water bath at 37 ℃ after being stored at 10 ℃ for 0h and 120h, respectively, and then stained with trypan blue, followed by cell counting (three-time counting), the cell viability was calculated, and the results are shown in Table 2.
TABLE 2
Sample (I) 0h cell survival (%) 120h cell survival (%)
Example 1 98.79 91.69
Example 2 99.04 90.54
Example 3 98.83 91.20
Comparative example 1 99.16 63.79
Comparative example 2 98.35 72.03
Comparative example 3 97.91 67.61
Comparative example 4 98.57 58.74
Comparative example 5 99.33 75.95
Comparative example 6 98.54 79.36
Comparative example 7 97.44 77.25
It can be seen from table 2 that the cells in the preservation solutions of examples 1 to 3 still had higher survival rates after 120h of preservation than the preservation solutions of comparative examples 1 to 7. The preservation solutions of comparative examples 1 to 7 adjusted the composition of the raw materials and reduced the survival rate of the cells to various degrees, indicating that fullerene C was contained in the preservation solution of the present invention90Sesamol, adenosine cyclophosphate, ethanolamine, linoleic acid, low molecular weight sodium hyaluronate and high molecular weight sodium hyaluronate cooperate to establish a stable induced pluripotent stem cell preservation system, prolong the preservation time and provide guarantee for clinical application.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.

Claims (3)

1. An induced pluripotent stem cell preserving fluid is characterized by comprising the following raw materials: DMEM medium and fullerene C90Sesamol, adenosine cyclophosphate, ethanolamine, linoleic acid, low molecular weight sodium hyaluronate, high molecular weight sodium hyaluronate and vitamin E;
based on the final volume of the culture medium, the dosage of each component is as follows: fullerene C9040-50 mug/mL, sesamol 35.5-39.5 mug/mL, adenosine cyclophosphate 45-55 mug/mL, ethanolamine 1-5mg/mL, linoleic acid 8-12mg/mL, low molecular weight sodium hyaluronate 1-5mgPer mL, 1-5mg/mL of high molecular weight sodium hyaluronate and 5-10mg/mL of vitamin E;
the average molecular weight of the low molecular weight sodium hyaluronate is 6-1 ten thousand daltons, and the average molecular weight of the high molecular weight sodium hyaluronate is 100-150 ten thousand daltons.
2. The induced pluripotent stem cell preserving fluid according to claim 1, wherein the components are used in the following amounts by volume: fullerene C9045 mu g/mL, sesamol 37.5 mu g/mL, adenosine cyclophosphate 50 mu g/mL, ethanolamine 3mg/mL, linoleic acid 10mg/mL, low molecular weight sodium hyaluronate 2mg/mL, high molecular weight sodium hyaluronate 2mg/mL, vitamin E8 mg/mL.
3. The method for preparing the induced pluripotent stem cell preserving fluid according to any one of claims 1 to 2, comprising the steps of: adding fullerene C into DMEM medium90Sesamol, adenosine cyclophosphate, ethanolamine, linoleic acid, low molecular weight sodium hyaluronate, high molecular weight sodium hyaluronate and vitamin E, and uniformly stirring to obtain the composition.
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