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Stem cell freezing solution and stem cell freezing method thereof

A freezing method and technology of stem cells, which are applied in the field of freezing liquid and freezing of stem cells, and can solve the problems of low heating rate and resurrection rate

Inactive Publication Date: 2020-02-28
ANHUI HUIEN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the deficiencies of the prior art, the object of the present invention is to provide a freezing liquid for stem cells and a freezing method thereof, which solves the problem of low heating and resurrection rate of stem cells in the prior art after freezing

Method used

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  • Stem cell freezing solution and stem cell freezing method thereof
  • Stem cell freezing solution and stem cell freezing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] S1. Preparation solution: select 1ml of dimethyl sulfoxide with a concentration of 10%, 2ml of human serum with a concentration of 10%, 1ml of trehalose with a concentration of 3(w / v), 1ml with a concentration of 3(w / v) % dextran and 5ml of 0.85% saline are mixed and stored in an incubator at 37°C;

[0040] S2. Cleaning: Select 0.5ml of stem cells in the logarithmic growth phase and wash them three times with PBS, then wash them with an appropriate amount of normal saline, and put them in a petri dish;

[0041] S3. Digestion: drop a few drops of trypsin with a concentration of 0.08% on the culture dish to eliminate the stem cells in the upper layer;

[0042] S4. Centrifugation: place the digested stem cells in a centrifuge, control the speed at 800 rpm, and centrifuge for 8 minutes;

[0043] S5. Filtration: take the supernatant from the centrifuged stem cells and filter to remove suspended impurities;

[0044] S6. Cultivation: transfer the filtered stem cells to the p...

Embodiment 2

[0050] S1. Preparation solution: choose 1ml of dimethyl sulfoxide with a concentration of 8%, 2ml of human serum with a concentration of 15%, 1ml of trehalose with a concentration of 4(w / v), 1ml with a concentration of 5(w / v) % dextran and 5ml of 0.9% saline are mixed and stored in an incubator at 37°C;

[0051] S2. Cleaning: Select 0.5ml of stem cells in the logarithmic growth phase and wash them three times with PBS, then wash them with an appropriate amount of normal saline, and put them in a petri dish;

[0052] S3. Digestion: drop a few drops of trypsin with a concentration of 0.08% on the culture dish to eliminate the stem cells in the upper layer;

[0053] S4. Centrifugation: place the digested stem cells in a centrifuge, control the speed at 1000 rpm, and centrifuge for 5 minutes;

[0054] S5. Filtration: take the supernatant from the centrifuged stem cells and filter to remove suspended impurities;

[0055] S6. Cultivation: transfer the filtered stem cells to the pr...

Embodiment 3

[0061] S1. Preparation solution: select 1ml of dimethyl sulfoxide with a concentration of 9%, 2ml of human serum with a concentration of 20%, 1ml of trehalose with a concentration of 6(w / v), 1ml with a concentration of 7(w / v) % dextran and 5ml of 0.9% saline are mixed and stored in an incubator at 37°C;

[0062] S2. Cleaning: Select 0.5ml of stem cells in the logarithmic growth phase and wash them three times with PBS, then wash them with an appropriate amount of normal saline, and put them in a petri dish;

[0063] S3. Digestion: drop a few drops of trypsin with a concentration of 0.08% on the culture dish to eliminate the stem cells in the upper layer;

[0064] S4. Centrifugation: place the digested stem cells in a centrifuge, control the speed at 900 rpm, and centrifuge for 10 minutes;

[0065] S5. Filtration: take the supernatant from the centrifuged stem cells and filter to remove suspended impurities;

[0066] S6. Cultivation: transfer the filtered stem cells to the prep...

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Abstract

The invention discloses a stem cell freezing solution and a stem cell freezing method thereof, and the stem cell freezing solution comprises the following raw material components in percentage by concentration: 8-10% of dimethyl sulfoxide, 10-20% of human serum, 2-6 (w / v)% of trehalose, 3-7 (w / v)% of glucan and 0.85-0.9% of normal saline. The stem cell freezing solution comprises the following components in parts by weight: 1 part of the dimethyl sulfoxide, 2 parts of the human serum, 1 part of the trehalose, 1 part of the glucan and 5 parts of the normal saline. The stem cell freezing methodcomprises the steps of solution preparation, stem cell cleaning, digestion, centrifugation, filtration, culture, tubing, primary cooling, secondary cooling and cryopreservation. Stepped cooling and freezing is adopted, so that cytoplasm damage of stem cells due to fast temperature change is prevented, and the influence on the temperature rise reactivation rate is avoided; the prepared solution isstored in a 37 DEG C environment, so that the stem cells are transferred into the solution to simulate a human body environment, and the influence of different external environments on the stem cellsis avoided. The problem that in the prior art, after the stem cells are frozen, the temperature rise reactivation rate is low is solved.

Description

technical field [0001] The invention belongs to the field of cell freezing, and in particular relates to a freezing solution of stem cells and a freezing method thereof. Background technique [0002] Cell storage is indispensable in cell culture, and cell freezing is a very effective way of cell storage today. Cell cryopreservation is a technology that places cells in a low temperature environment to reduce cell metabolism for long-term storage. [0003] The same is true for the storage of stem cells, but stem cells are very susceptible to freezing, thawing, and changes in osmotic pressure of the cell solution during the process of cooling, freezing, and resurrection, resulting in changes in the properties of the solution and intracellular damage, which in turn revives stem cells after freezing low rate. In order to ensure a high rate of cryopreservation of stem cells, it is necessary to ensure that the cell membrane structure of stem cells is stable and not easy to change...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0284
Inventor 王冬
Owner ANHUI HUIEN BIOTECH
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