CN117502435A - Method suitable for preserving cells under normal temperature condition - Google Patents
Method suitable for preserving cells under normal temperature condition Download PDFInfo
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- CN117502435A CN117502435A CN202410022994.0A CN202410022994A CN117502435A CN 117502435 A CN117502435 A CN 117502435A CN 202410022994 A CN202410022994 A CN 202410022994A CN 117502435 A CN117502435 A CN 117502435A
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- 238000000034 method Methods 0.000 title claims abstract description 44
- 229930182555 Penicillin Natural products 0.000 claims abstract description 35
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims abstract description 35
- 229940049954 penicillin Drugs 0.000 claims abstract description 35
- 239000006285 cell suspension Substances 0.000 claims abstract description 26
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000001816 cooling Methods 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 18
- 238000007710 freezing Methods 0.000 claims abstract description 15
- 230000008014 freezing Effects 0.000 claims abstract description 15
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract description 15
- 238000005070 sampling Methods 0.000 claims abstract description 9
- 239000011259 mixed solution Substances 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 15
- 229920002307 Dextran Polymers 0.000 claims description 11
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 10
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 10
- 229930195725 Mannitol Natural products 0.000 claims description 10
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 10
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 10
- 235000010355 mannitol Nutrition 0.000 claims description 10
- 239000000594 mannitol Substances 0.000 claims description 10
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 claims description 9
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 4
- 238000004321 preservation Methods 0.000 abstract description 8
- 210000004027 cell Anatomy 0.000 description 92
- 238000012795 verification Methods 0.000 description 11
- 239000000843 powder Substances 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 210000003954 umbilical cord Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000001120 cytoprotective effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
-
- 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
-
- 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
- A01N1/0278—Physical preservation processes
-
- 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
- A01N1/0278—Physical preservation processes
- A01N1/0289—Pressure processes, i.e. using a designated change in pressure over time
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method suitable for preserving cells under normal temperature conditions, which relates to the technical field of cell preservation and comprises the following steps: s1, cell pretreatment; s2, preparing a cell protection liquid: adding Dmso with the mass fraction of 1% -20% into the pretreated cell suspension; sampling and counting; s3, performing programmed cooling on the split penicillin bottles; the final temperature of program cooling is-70 ℃ to-100 ℃; s4, vacuum freeze-drying: placing the penicillin bottle subjected to program cooling in a cold trap of a freeze dryer precooled to the temperature ranging from-70 ℃ to-100 ℃; freezing for 1-24 h; turning on a vacuum pump of a vacuum freeze dryer to keep the cells under the condition of 1Pa-20Pa for 12-48h; by adopting the method suitable for preserving cells under normal temperature conditions, the cells can be preserved under normal temperature conditions, the transportation of the cells is convenient, and the transportation and preservation cost is reduced; the method provided by the application is simple to operate and high in practicability.
Description
Technical Field
The invention relates to the technology in the field of cell cryopreservation, in particular to a method suitable for preserving cells under normal temperature.
Background
The umbilical cord mesenchymal stem cells refer to multifunctional stem cells existing in umbilical cord tissues of newborns, have higher differentiation potential, and can differentiate towards multiple directions. The preparation method has the advantages of sufficient sources, easy acquisition, low immunogenicity, no social theoretical disputes and the like, and has better clinical application prospect; the invention of application number 202111258581.5 discloses a method for preserving a cell preparation; in the prior art, when the cells are stored, the cells are mostly stored under a deep low temperature condition, the storage condition is severe, and the transportation is inconvenient; therefore, in view of the current situation, there is an urgent need to develop a method suitable for preserving cells at normal temperature to meet the demands of practical use.
Disclosure of Invention
In view of the above, the present invention aims at the defects existing in the prior art, and its main objective is to provide a method suitable for preserving cells at normal temperature, which can preserve cells at normal temperature by adopting the method suitable for preserving cells at normal temperature provided by the present application, thereby facilitating transportation of cells and reducing transportation and preservation costs; the method provided by the application is simple to operate and high in practicability.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for preserving cells at room temperature comprising the steps of:
s1, cell pretreatment: uniformly mixing 30% -90% of dextran, 1% -10% of trehalose and 1% -10% of mannitol by mass with DPBS to form a mixed solution; adding 1 x 10 to the mixed solution 6 individual/mL-6 x 10 7 Cell suspension at a concentration of individual/mL; fully and uniformly mixing, and standing for 12h at 2-8 ℃;
s2, preparing a cell protection liquid: adding Dmso with the mass fraction of 1% -20% into the pretreated cell suspension; sampling and counting; subpackaging the prepared cell protection liquid into penicillin bottles to ensure that the cell concentration is 1 x 10 6 individual/mL-6 x 10 7 individual/mL;
s3, performing programmed cooling on the split penicillin bottles; the final temperature of program cooling is-70 ℃ to-100 ℃;
s4, vacuum freeze-drying:
placing the penicillin bottle subjected to program cooling in a cold trap of a freeze dryer precooled to the temperature ranging from-70 ℃ to-100 ℃; freezing for 1-24 h; turning on a vacuum pump of a vacuum freeze dryer to keep the cells under the condition of 1Pa-20Pa for 12-48h; the freeze-dried cells can be sealed and stored under normal temperature.
As a preferred embodiment: the mass fraction of the dextran in the step S1 is 40% -60%.
As a preferred embodiment: the mass fraction of trehalose in the step S1 is 2% -8%.
As a preferred embodiment: and in the step S1, the mass fraction of mannitol is 2% -8%.
As a preferred embodiment: the concentration of the cell suspension added to the mixed solution in the step S1 is 1 x 10 6 individual/mL-6 x 10 7 And each mL.
As a preferred embodiment: preparing a cell protection solution in the step S2: dmso with a mass fraction of 1% -10% is added to the pretreated cell suspension.
As a preferred embodiment: in the step S3, the procedure cooling is carried out on the split penicillin bottles; the final temperature of the program cooling is-80 ℃ to-90 ℃.
As a preferred embodiment: vacuum freeze-drying in the step S4: and placing the penicillin bottle subjected to the programmed cooling in a cold trap of a freeze dryer precooled to the temperature ranging from-80 ℃ to-90 ℃.
As a preferred embodiment: vacuum freeze-drying in the step S4: and placing the penicillin bottle subjected to the programmed cooling into a cold trap of a freeze dryer precooled to the temperature ranging from-80 ℃ to-90 ℃, and freezing for 1-12 h.
As a preferred embodiment: in the step S4, a vacuum pump of the vacuum freeze dryer is started, so that the cells are kept under the condition of 5Pa-10Pa for 12-24 hours.
Compared with the prior art, the method has obvious advantages and beneficial effects, and particularly, the technical scheme shows that the method suitable for preserving cells under normal temperature can preserve cells under normal temperature, is convenient for transporting the cells, and reduces transportation and preservation cost; the method provided by the application is simple to operate and high in practicability.
In order to more clearly illustrate the structural features and efficacy of the present invention, a detailed description thereof will be given below with reference to the accompanying drawings and examples.
Drawings
FIG. 1 is a first-order flow chart of verification example 3 of the present invention;
FIG. 2 is a second-order flow chart of verification example 3 of the present invention;
FIG. 3 is a third flow chart of verification example 3 of the present invention;
FIG. 4 is a fourth flow chart of verification example 3 of the present invention;
FIG. 5 is a fifth flow chart of verification example 3 of the present invention.
Detailed Description
The invention is a method for preserving cells under normal temperature conditions, as shown in fig. 1 to 5, comprising the following steps:
s1, cell pretreatment: uniformly mixing 30% -90% of dextran, 1% -10% of trehalose and 1% -10% of mannitol by mass with DPBS to form a mixed solution; adding 1 x 10 to the mixed solution 6 individual/mL-6 x 10 7 Cell suspension at a concentration of individual/mL; fully and uniformly mixing, and standing for 12h at 2-8 ℃;
s2, preparing a cell protection liquid: adding Dmso with the mass fraction of 1% -20% into the pretreated cell suspension; sampling and counting; subpackaging the prepared cell protection liquid into penicillin bottles to ensure that the cell concentration is 1 x 10 6 individual/mL-6 x 10 7 individual/mL;
s3, performing programmed cooling on the split penicillin bottles; the final temperature of program cooling is-70 ℃ to-100 ℃;
s4, vacuum freeze-drying:
placing the penicillin bottle subjected to program cooling in a cold trap of a freeze dryer precooled to the temperature ranging from-70 ℃ to-100 ℃; freezing for 1-24 h; turning on a vacuum pump of a vacuum freeze dryer to keep the cells under the condition of 1Pa-20Pa for 12-48h; the freeze-dried cells can be sealed and stored under normal temperature.
The mass fraction of the dextran in the step S1 is 40% -60%.
The mass fraction of trehalose in the step S1 is 2% -8%.
The mass fraction of mannitol in the step S1 is 2% -8%.
The concentration of the cell suspension added to the mixed solution in step S1 was 1×10 6 individual/mL-6 x 10 7 And each mL.
In this step S2, a cytoprotective solution is prepared: dmso with a mass fraction of 1% -10% is added to the pretreated cell suspension.
In the step S3, the procedure cooling is carried out on the split-packaged penicillin bottles; the final temperature of the program cooling is-80 ℃ to-90 ℃.
Vacuum freeze-drying in this step S4: and placing the penicillin bottle subjected to the programmed cooling in a cold trap of a freeze dryer precooled to the temperature ranging from-80 ℃ to-90 ℃.
Vacuum freeze-drying in this step S4: and placing the penicillin bottle subjected to the programmed cooling into a cold trap of a freeze dryer precooled to the temperature ranging from-80 ℃ to-90 ℃, and freezing for 1-12 h.
In the step S4, a vacuum pump of the vacuum freeze dryer is turned on, so that the cells are kept under the condition of 5Pa-10Pa for 12-24 hours.
Example 1: method suitable for preserving cells under normal temperature condition
S1, cell pretreatment;
s11, mixing 48% of dextran and 3% of mannitol uniformly by using DPBS to form a mixed solution;
s12, adding 2 x 10 7 Cell suspension at a concentration of individual/mL;
s13, fully and uniformly mixing, and standing for 12h at 4 ℃.
S2, preparing a cell protection liquid;
s21, adding 5% DMSO into the pretreated cell suspension;
s22, sampling and counting;
s23, subpackaging the prepared cell protection liquid into penicillin bottles to ensure that the cell concentration is 2 x 10 6 individual/mL;
s3, performing programmed cooling on the split charging penicillin bottles;
s31, the final temperature of program cooling is-80 ℃;
s4, vacuum freeze-drying:
s41, placing the penicillin bottle after the program is cooled in a cold trap of a freeze dryer precooled to-80 ℃;
s42, freezing for 2-12 h;
s43, turning on a vacuum pump of a vacuum freeze dryer to keep the cells under the condition of 10Pa for 24 hours;
s44, sealing the freeze-dried cells, and storing the cells under normal temperature.
Example 2: method suitable for preserving cells under normal temperature condition
S1, cell pretreatment;
s11, mixing 48% of dextran and 5% of trehalose uniformly by using DPBS to form a mixed solution;
s12, adding 2 x 10 7 Cell suspension at a concentration of individual/mL;
s13, fully and uniformly mixing, and standing for 12h at 4 ℃.
S2, preparing a cell protection liquid;
s21, adding 5% DMSO into the pretreated cell suspension;
s22, sampling and counting;
s23, subpackaging the prepared cell protection liquid into penicillin bottles to ensure that the cell concentration is 2 x 10 6 individual/mL;
s3, performing programmed cooling on the split charging penicillin bottles;
s31, the final temperature of program cooling is-80 ℃;
s4, vacuum freeze-drying:
s41, placing the penicillin bottle after the program is cooled in a cold trap of a freeze dryer precooled to-80 ℃;
s42, freezing for 2-12 h;
s43, turning on a vacuum pump of a vacuum freeze dryer to keep the cells under the condition of 10Pa for 24 hours;
s44, sealing the freeze-dried cells, and storing the cells under normal temperature.
Example 3; method suitable for preserving cells under normal temperature condition
S1, cell pretreatment;
s11, 5% of trehalose and 3% of mannitol are uniformly mixed with dextran to form a mixed solution;
s12, adding 2 x 10 7 Cell suspension at a concentration of individual/mL;
s13, fully and uniformly mixing, and standing for 12h at 4 ℃.
S2, preparing a cell protection liquid;
s21, adding 5% DMSO into the pretreated cell suspension;
s22, sampling and counting;
s23, subpackaging the prepared cell protection liquid into penicillin bottles to ensure that the cell concentration is 2 x 10 6 individual/mL;
s3, performing programmed cooling on the split charging penicillin bottles;
s31, the final temperature of program cooling is-80 ℃;
s4, vacuum freeze-drying:
s41, placing the penicillin bottle after the program is cooled in a cold trap of a freeze dryer precooled to-80 ℃;
s42, freezing for 2-12 h;
s43, turning on a vacuum pump of a vacuum freeze dryer to keep the cells under the condition of 10Pa for 24 hours;
s44, sealing the freeze-dried cells, and storing the cells under normal temperature.
Verification example 1: method suitable for preserving cells under normal temperature condition and method for measuring cell viability
S1, cell pretreatment;
s11, mixing 48% of dextran, 5% of trehalose and 3% of mannitol uniformly by using DPBS to form a mixed solution;
s12, adding 2 x 10 7 Cell suspension at a concentration of individual/mL;
s13, fully and uniformly mixing, and standing for 12h at 4 ℃.
S2, preparing a cell protection liquid;
s21, adding 5% DMSO into the pretreated cell suspension;
s22, sampling and counting;
s23, subpackaging the prepared cell protection liquid into penicillin bottles to ensure that the cell concentration is 2 x 10 6 individual/mL;
s3, performing programmed cooling on the split charging penicillin bottles;
s31, the final temperature of program cooling is-80 ℃;
s4, vacuum freeze-drying:
s41, placing the penicillin bottle after the program is cooled in a cold trap of a freeze dryer precooled to-80 ℃;
s42, freezing for 2-12 h;
s43, turning on a vacuum pump of a vacuum freeze dryer to keep the cells under the condition of 1Pa for 24 hours;
s5, storing the freeze-dried cells at 0-10 ℃, 10-20 ℃, 20-30 ℃ and 30-40 ℃ respectively.
S51, storing the freeze-dried cells at different temperatures for 7d, 14d, 21d, 28d and 35d.
S6, observing whether the appearance of the freeze-dried cells is changed.
S7, resuscitating the freeze-dried powder with unchanged appearance form;
s71, 5mL, DPBS dissolved freeze-dried powder
S72, forming a mixed solution and centrifuging;
s8, discarding the supernatant;
s81, resuspension is carried out by using normal saline, and cell resuscitating is completed.
S9, counting and measuring the cell viability.
Table 1: appearance form change results stored under different conditions
Table 2: resuscitating and counting the freeze-dried powder with unchanged appearance form to obtain cell activity
Verification example 2: method suitable for preserving cells under normal temperature condition and method for measuring cell viability
S1, cell pretreatment;
s11, mixing 48% of dextran, 5% of trehalose and 3% of mannitol uniformly by using DPBS to form a mixed solution;
s12, adding 2 x 10 7 Cell suspension at a concentration of individual/mL;
s13, fully and uniformly mixing, and standing for 12h at 4 ℃.
S2, preparing a cell protection liquid;
s21, adding 5% DMSO into the pretreated cell suspension;
s22, sampling and counting;
s23, subpackaging the prepared cell protection liquid into penicillin bottles to ensure that the cell concentration is 2 x 10 6 individual/mL;
s3, performing programmed cooling on the split charging penicillin bottles;
s31, the final temperature of program cooling is-80 ℃;
s4, vacuum freeze-drying:
s41, placing the penicillin bottle after the program is cooled in a cold trap of a freeze dryer precooled to-80 ℃;
s42, freezing for 2-12 h;
s43, turning on a vacuum pump of a vacuum freeze dryer to keep the cells under the condition of 1Pa for 24 hours;
s44, sealing the freeze-dried cells, and storing the cells under normal temperature.
S5, resuscitating the cells;
s51, respectively taking freeze-dried powder stored under normal temperature conditions at 7d, 14d, 21d, 28d and 35d after freeze-drying.
S52, 5mL, DPBS dissolved freeze-dried powder
S53, forming a mixed solution and centrifuging;
s54, discarding the supernatant;
s55, resuspension is carried out by using normal saline, and cell resuscitating is completed.
S56, counting the measured cell activity rate, and calculating the cell recovery rate.
Table 3: post-cell resuscitation data results
Verification example 3: flow detection of cells after 5 times of resuscitation
Table 4: delivering detection results of flow detection after resuscitation
Comparative example 1: the method for preserving cells by adopting the traditional method for adding the frozen stock solution comprises the following steps:
s1, preparing frozen stock solution:
s11, adding DMSO into a complete culture medium;
s2, adding the cell suspension into the prepared frozen stock solution;
s3, split charging cell suspension:
s31, sub-packaging the cell suspension into a freezing tube to ensure that the cell concentration is 2 x 10 6 individual/mL;
s4, program cooling;
s5, resuscitating the cells;
s51, taking the cells frozen in liquid nitrogen at 7d, 14d, 21d, 28d and 35d after freezing.
S52, melting in a water bath;
s53, washing the frozen stock solution with normal saline;
s54, discarding the supernatant;
s55, resuspension is carried out by using normal saline, and cell resuscitating is completed.
Table 5: post-resuscitation data outcome
Comparative example 2: flow detection of cells after 5 times of resuscitation
Table 6: delivering detection results of flow detection after resuscitation:
the experimental results of the table show that the cells can be prepared into the freeze-dried powder by adopting the verification example 1, and the freeze-dried powder can be verified to be stored under the normal temperature condition. The flow detection results after resuscitation in verification example 2 and verification example 3 normally prove that the cells stored in the freeze-dried powder are mesenchymal stem cells. The comparative example 1 is a traditional deep low temperature preservation method, and the comparison proves that the method has slightly lower recovery rate compared with the traditional deep low temperature freezing preservation method, but can completely realize cell preservation under normal temperature; from the above description, the beneficial effects of the invention are as follows: the cells can be preserved under normal temperature.
The design key point of the invention is that by adopting the method suitable for preserving cells under normal temperature conditions, the cells can be preserved under normal temperature conditions, the transportation of the cells is convenient, and the transportation and preservation cost is reduced; the method provided by the application is simple to operate and high in practicability.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the technical scope of the present invention, so any minor modifications, equivalent changes and modifications made to the above embodiments according to the technical principles of the present invention still fall within the scope of the technical solutions of the present invention.
Claims (10)
1. A method for preserving cells at room temperature, characterized by: the method comprises the following steps:
s1, cell pretreatment: uniformly mixing 30% -90% of dextran, 1% -10% of trehalose and 1% -10% of mannitol by mass with DPBS to form a mixed solution; adding 1 x 10 to the mixed solution 6 individual/mL-6 x 10 7 Cell suspension at a concentration of individual/mL; fully and uniformly mixing, and standing for 12h at 2-8 ℃;
s2, preparing a cell protection liquid: adding Dmso with the mass fraction of 1% -20% into the pretreated cell suspension; sampling and counting; subpackaging the prepared cell protection liquid into penicillin bottles to ensure that the cell concentration is 1 x 10 6 individual/mL-6 x 10 7 individual/mL;
s3, performing programmed cooling on the split penicillin bottles; the final temperature of program cooling is-70 ℃ to-100 ℃;
s4, vacuum freeze-drying: placing the penicillin bottle subjected to program cooling in a cold trap of a freeze dryer precooled to the temperature ranging from-70 ℃ to-100 ℃; freezing for 1-24 h; turning on a vacuum pump of a vacuum freeze dryer to keep the cells under the condition of 1Pa-20Pa for 12-48h; the freeze-dried cells can be sealed and stored under normal temperature.
2. The method for preserving cells under normal temperature conditions according to claim 1, wherein: the mass fraction of the dextran in the step S1 is 40% -60%.
3. The method for preserving cells under normal temperature conditions according to claim 1, wherein: the mass fraction of trehalose in the step S1 is 2% -8%.
4. The method for preserving cells under normal temperature conditions according to claim 1, wherein: and in the step S1, the mass fraction of mannitol is 2% -8%.
5. The method for preserving cells under normal temperature conditions according to claim 1, wherein: the concentration of the cell suspension added to the mixed solution in the step S1 is 1 x 10 6 individual/mL-6 x 10 7 And each mL.
6. The method for preserving cells under normal temperature conditions according to claim 1, wherein: preparing a cell protection solution in the step S2: dmso with a mass fraction of 1% -10% is added to the pretreated cell suspension.
7. The method for preserving cells under normal temperature conditions according to claim 1, wherein: in the step S3, the procedure cooling is carried out on the split penicillin bottles; the final temperature of the program cooling is-80 ℃ to-90 ℃.
8. The method for preserving cells under normal temperature conditions according to claim 1, wherein: vacuum freeze-drying in the step S4: and placing the penicillin bottle subjected to the programmed cooling in a cold trap of a freeze dryer precooled to the temperature ranging from-80 ℃ to-90 ℃.
9. The method for preserving cells under normal temperature conditions according to claim 8, wherein: vacuum freeze-drying in the step S4: and placing the penicillin bottle subjected to the programmed cooling into a cold trap of a freeze dryer precooled to the temperature ranging from-80 ℃ to-90 ℃, and freezing for 1-12 h.
10. The method for preserving cells under normal temperature conditions according to claim 1, wherein: in the step S4, a vacuum pump of the vacuum freeze dryer is started, so that the cells are kept under the condition of 5Pa-10Pa for 12-24 hours.
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