Method for quickly cryopreserving and thawing cells

A cell and cryopreservation technology, applied in the field of cell culture, can solve the problems of failure to fully consider the impact of freezing rate and recovery rate, failure to consider the impact of recovery survival rate, and damage to cells, so as to ensure the genetic stability of cells and reduce Human operation differences to ensure the effect of high vitality

Active Publication Date: 2018-03-06
SINOVAC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The characteristics of the cell resuscitation technology in the prior art are (1) resuscitating cells at low temperature (37°C water bath), the recovery time is too long, and the cells are easily damaged during the rewarming process
(2) Resuscitate cells at high temperature (46-67°C water bath). Although the recovery time is shortened, the cells are easily damaged during the rewarming process.
(3) The influence of other solution temperatures on the recovery survival rate was not considered during the cell recovery process
(4) After the cells are resuscitated, t...

Method used

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  • Method for quickly cryopreserving and thawing cells

Examples

Experimental program
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Embodiment 1

[0028] Example 1 Rapid cryopreservation and recovery of human diploid cells (2BS, MRC-5, KMB17, WI-38)

[0029] 1. Digestion of human diploid cells: Add 250ml of 0.25% trypsin solution to a CF10 that has formed a dense monolayer of cells. Digest continuously for 5-10 minutes in a constant temperature room at 36.5±1°C.

[0030] Preparation of cell suspension: Add 500ml of growth medium containing 10% newborn calf serum to the digested CF10 to make human diploid cell suspension, and distribute it to 50ml centrifuge tubes. Centrifuge at 800-1000 r / min for 5 minutes, discard the supernatant;

[0031] 2. Dilute human diploid cells before freezing:

[0032] According to the second step of cell counting, add 10ml of cell freezing solution to ten 50ml centrifuge tubes, blow gently until the precipitated cells are evenly dispersed, collect the dispersed cells into a sterile saline bottle, take 0.1ml of cell suspension and count, Continue to add cell cryopreservation solution to adju...

Embodiment 2

[0042] Example 2 Quick Freezing and Resuscitating Vero Cells

[0043] 1. Digestion of African green monkey kidney cells:

[0044] to 175 cm of 10 dense monolayer cells 2 Add 7ml of 0.25% trypsin solution to the culture bottle, and when the intercellular substance appears pinhole-like, discard the digestion solution in the culture bottle and place it in a constant temperature room at 36.5±1°C for 5-10 minutes.

[0045] Preparation of cell suspension: 175cm of digested 2 Add 15ml of growth solution containing 10% newborn bovine serum to the culture bottle to make vero cell suspension, which is divided into 50ml centrifuge tubes. Centrifuge at 800-1000r / min for 5min, discard the supernatant;

[0046] 2. Dilute African green monkey kidney cells before cryopreservation:

[0047] According to the second step of cell counting, add 10ml of cell freezing solution to three 50ml centrifuge tubes, blow gently until the precipitated cells are evenly dispersed, collect the dispersed cel...

Embodiment 3

[0056] Example 3 Rapid Freezing and Resuscitation of Kidney Hamster Kidney Cells

[0057] 1. Digestion of Kidney Hamster Kidney Cells:

[0058] to 175 cm of 10 dense monolayer cells 2 Add 7ml of 0.25% trypsin solution to the culture bottle, and when the intercellular substance appears pinhole-like, discard the digestion solution in the culture bottle, and place it in a constant temperature room at 36.5±1°C for 10 minutes for continuous digestion.

[0059] Preparation of cell suspension: 175cm of digested 2Add 15ml of growth solution containing 10% newborn bovine serum to the culture bottle to make a suspension of suckling hamster kidney cells, which is divided into 50ml centrifuge tubes. Centrifuge at 800-1000r / min for 5min, discard the supernatant;

[0060] 2. Dilute the baby hamster kidney cells before cryopreservation:

[0061] According to the second step of cell counting, add 10ml of cell freezing solution to ten 50ml centrifuge tubes, blow gently until the precipitat...

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Abstract

The invention provides a method for quickly cryopreserving and thawing cells, which comprises a cryopreservation step and a thawing step. The cryopreservation step comprises: (1) cells are dissociated, and cell suspension is prepared; (2) the cells are diluted and separately packaged; (3) temperature is gradiently decreased, and is increased, temperature is then gradiently decreased to negative 120 DEG C to negative 180 DEG C, and the cells are stored in liquid nitrogen. The thawing step comprises: the cryopreserved cells are taken out of the liquid nitrogen, quickly immersed in thawing waterwhich is 38 DEG C to 40 DEG C and rapidly shaken until the cells are completely thawed, and the cells can be directly used without removing the cryopreserving liquid. The method disclosed by the invention can guarantee the quality consistency between batches of cryopreserved cells and the stability of cell genetics, ensure that the survival rate of thawed cells can be stably increased to 98 percent or more, and shorten the cell thawing and passage period by 50 to 60 percent.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for rapidly freezing and resuscitating cells. Background technique [0002] With the development of cell biology application technology, in vitro cell culture technology has been widely used in various biological research and industrial production fields. Therefore, cell cryopreservation and recovery technology is one of the key areas of cell culture technology. How to establish an efficient cell cryopreservation method to ensure long-term biological activity of cryopreserved cells and improve cell survival rate during cell recovery is a technical difficulty often encountered in current cell biology experiments and some industrial production. [0003] A large number of studies have shown that storing cells in liquid nitrogen at -196°C can preserve their characteristics for a long time and make the cells temporarily out of the growth state. Afterwards, the cells can be revived...

Claims

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Application Information

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IPC IPC(8): A01N1/02C12N5/07C12N5/09
CPCA01N1/0221C12N5/0602C12N5/0686C12N5/0693
Inventor 李进军张颖马敏华万金娥王云鹏张健姜德玉吴君兰李静尹卫东
Owner SINOVAC BIOTECH
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