CN102140433A

CN102140433A - Cell freezing method

Info

Publication number: CN102140433A
Application number: CN 201010622260
Authority: CN
Inventors: 林登宇; 徐希丽
Original assignee: BEIJING MIN HAI BIO-SCIENTIFIC Inc
Current assignee: BEIJING MIN HAI BIO-SCIENTIFIC Inc
Priority date: 2010-12-28
Filing date: 2010-12-28
Publication date: 2011-08-03
Anticipated expiration: 2030-12-28
Also published as: CN102140433B

Abstract

The invention provides a cell freezing method. The method comprises the following steps: digesting culture cells in a single layer with pancreatic enzyme for 1-8min, discarding pancreatic enzyme, adding 0.08-0.1ml/cm<2> of cell freezing medium according to the culture area of a culture bottle, blowing and mixing the cell suspension, filling in clean freezing tubes respectively; and standing the freezing tubes at 2-8 DEG C for 10-40min, then standing the freezing tubes at -80 DEG C for 10-20h, and soaking the freezing tubes in liquid nitrogen to store for long term. By adopting the method provided by the invention, the steps and operation process of cell freezing can be simplified, the operation cost can be reduced and the operating efficiency can be increased. Compared with the conventional cell freezing method, the method has far higher cell survival rate which is up to more than 90%.

Description

A kind of cell cryopreservation method

Technical field

The present invention relates to histocyte culture technique field, be specifically related to a kind of cell cryopreservation method.

Background technology

In today of biotechnology develop rapidly, we are in the research of biotechnics and related science thereof, increasing utilization histocyte is cultivated breeding technology, Research Significance will be arranged or have the histocyte of application prospect to adopt low temperature to carry out secular preservation, in case needs are arranged, can be at any time to cell recover, recover its complete morphology structure and biological property, use for life science, the frozen method of the cell particularly important that just seems, the quality of its frozen success or not, quality is the key factor that directly influences the cell proliferation result.

Frozen and the recovery of cell is one of importance of RESEARCH ON CELL-BIOLOGY, and its method is had nothing in common with each other but all followed and freezes fast molten principle slowly.For a long time, the relevant books of a large amount of cytobiologies both at home and abroad, the long-term employed cell cryopreservation method of the conventional cell cryopreservation method of introduction and people is:

Choose one bottle in the VERO, the BHK-21 that grow into individual layer, MRC-5,2BS or KMB17 cell, its cell density about 6 * 10 ⁴/ cm ², culture area is 225cm ², the trysinization 2min with 0.25% mass volume ratio discards pancreatin, adds nutrient solution 20ml, piping and druming mixing cell suspension, and the centrifugal 10min of 1000rpm abandons supernatant, adds conventional cells frozen storing liquid 20ml mixing, divides the clean frozen pipe of the 2ml/ pipe of packing into; Frozen pipe is placed 2～8 ℃ of 2h ,-20 ℃ of 2h ,-80 ℃ are spent the night, and immerse the medium-term and long-term preservation of liquid nitrogen then.

In secular biological experiment, be extensive use of this method freeze-stored cell.A large amount of actual biocytology experiment and statistic datas show that survival rate is generally 50～80% after recovery to adopt the frozen cell of this method, and surviving rate is lower; Freeze-stored cell after recovery is cultivated 12 hours is observed under ordinary optical microscope, and survival rate is generally about 65%; When survival rate less than 50% the time, the cell growth and breeding is slow, form is irregular, cell is more tiny, growing way is poor, can not survive basically; When cell survival rate during in 70% left and right sides, cell distribution is more even, and viable cell is many, and form is more normal, and covering with individual layer needs 4～5 days.

Summary of the invention

The object of the present invention is to provide a kind of cell cryopreservation method, in order to solve the lower problem of existing freeze-stored cell survival rate.

For achieving the above object, cell cryopreservation method provided by the invention comprises the steps:

With growing into the culturing cell trysinization 1～8min of individual layer, discard pancreatin, add 0.08～0.1ml/cm according to the culturing bottle culture area ²Cells frozen storing liquid, piping and druming mixing cell suspension divides the clean frozen pipe of packing into;

Frozen pipe in 2～8 ℃ of placement 10～40min, is placed 10～20h to frozen Guan Yu-80 ℃ then, immerse the medium-term and long-term preservation of liquid nitrogen.

Wherein, the described preferred cell density of culturing cell that grows into individual layer is 5.5 * 10 ⁴～6.5 * 10 ⁴/ cm ²

Wherein, the described mass volume ratio that is used for the pancreatin of peptic cell is preferably 0.15～0.40%, most preferably is 0.25%.

Wherein, the described trysinization time is preferably 2min.

Wherein, cells frozen storing liquid can be any cells frozen storing liquid well known in the art, as by volume percent being the cells frozen storing liquid that the dimethyl sulfoxide (DMSO) of 70% MEM cell culture fluid, 20% calf serum and 10% is formed.

Cell cryopreservation method provided by the invention can be used for frozen any culturing cell well known in the art, and preferred culturing cell can be VERO, BHK-21, MRC-5,2BS or KMB17 cell.

The inventive method has following advantage:

The inventive method has been simplified the step and the operating process of cell cryopreservation, in the process of peptic cell, has removed centrifugal this step in the conventional frozen method, and because of centrifugal pair cell has certain damaging action, the while is easy contamination of cells in the centrifugal process; In the process of freeze-stored cell, saved in the conventional frozen method 2 hours these steps of low temperature of-20 ℃, save this cyrogenic equipment.

The inventive method has reduced the pollution in the frozen process and the damage of pair cell, has reduced job costs, has improved working efficiency, has improved the surviving rate after the freeze-stored cell recovery is cultivated, and can reach more than 90%.Compare with the cell cryopreservation method of routine, the frozen cell survival rate of the inventive method is significantly improved, and quality and the biological characteristics of cell growth have been guaranteed, shortened the proliferating cycle of cell recovery, made the histocyte after the recovery can in time, offer biological experiment research with guaranteeing both quality and quantity.

Embodiment

Following embodiment further specifies content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.

Embodiment 1

Choose grow into individual layer VERO, BHK-21, MRC-5,2BS and KMB17 cell each one bottle, its cell density about 6 * 10 ⁴/ cm ², culture area is 225cm ²With mass volume ratio 0.25% trysinization 2min, discard pancreatin, adding is the conventional cells frozen storing liquid 20ml that the dimethyl sulfoxide (DMSO) of 70% MEM cell culture fluid, 20% calf serum and 10% is formed by volume percent, blow and beat the mixing cell suspension, divide the clean frozen pipe of the 2ml/ pipe of packing into; Frozen pipe is placed 2 ℃ of 20min, then frozen pipe is put into the previously prepd foam box and spend the night in-80 ℃, immerse in the liquid nitrogen and carry out prolonged preservation.

Embodiment 2

Choose grow into individual layer VERO, BHK-21, MRC-5,2BS and KMB17 cell each one bottle, its cell density about 6 * 10 ⁴/ cm ², culture area is 225cm ²With mass volume ratio 0.40% trysinization 1min, discard pancreatin, adding is the conventional cells frozen storing liquid 20ml that the dimethyl sulfoxide (DMSO) of 70% MEM cell culture fluid, 20% calf serum and 10% is formed by volume percent, blow and beat the mixing cell suspension, divide the clean frozen pipe of the 2ml/ pipe of packing into; Frozen pipe is placed 4 ℃ of 10min, then frozen pipe is put into the previously prepd foam box and spend the night in-80 ℃, immerse in the liquid nitrogen and carry out prolonged preservation.

Embodiment 3

Choose grow into individual layer VERO, BHK-21, MRC-5,2BS and KMB17 cell each one bottle, its cell density about 6 * 10 ⁴/ cm ², culture area is 225cm ²With mass volume ratio 0.15% trysinization 8min, discard pancreatin, adding is the conventional cells frozen storing liquid 20ml that the dimethyl sulfoxide (DMSO) of 70% MEM cell culture fluid, 20% calf serum and 10% is formed by volume percent, blow and beat the mixing cell suspension, divide the clean frozen pipe of the 2ml/ pipe of packing into; Frozen pipe is placed 8 ℃ of 40min, then frozen pipe is put into the previously prepd foam box and spend the night in-80 ℃, immerse in the liquid nitrogen and carry out prolonged preservation.

Comparative example 1

Choose grow into individual layer VERO, BHK-21, MRC-5,2BS and KMB17 cell each one bottle, its cell density about 6 * 10 ⁴/ cm ², culture area is 225cm ²Trysinization 2min with 0.25%, discard pancreatin, add MEM nutrient solution 10ml, piping and druming mixing cell suspension, the centrifugal 10min of 1500rpm, abandon supernatant, adding is the conventional cells frozen storing liquid 20ml that 70%MEM cell culture fluid, 20% calf serum and 10% dimethyl sulfoxide (DMSO) are formed by volume percent, and mixing divides the clean frozen pipe of the 2ml/ pipe of packing into; Frozen pipe is placed 4 ℃ of 2h ,-20 ℃ of 2h ,-80 ℃ are spent the night, and frozen pipe is immersed in the liquid nitrogen carry out prolonged preservation then.

Experimental example

Taking-up embodiment 1～3 and comparative example 1 frozen VERO, BHK-21, MRC-5,2BS and KMB17 cell respectively one are managed (2ml/ pipe) from liquid nitrogen, insert 37 ℃ of water-baths dissolvings, add 20% calf serum nutrient solution 10ml, at culture area 25cm ²Tissue Culture Flask in, put 37 ℃ of cultivations.

The freeze-stored cell viability rate of VERO, BHK-21, MRC-5,2BS and KMB17 that embodiment 1～3 and comparative example 1 are frozen is as shown in table 1.

Find out from the result of table 1, use survival rate after the freeze-stored cell recovery of cell cryopreservation method of the present invention institute is cultivated among the embodiment 1～3 apparently higher than the survival rate of using after the freeze-stored cell recovery of conventional cell cryopreservation method institute is cultivated in the comparative example 1.

Use the embodiment of the invention 1～3 the cell cryopreservation method frozen cell recovery cultivate 6 as a child the back observe, as seen the cell attachment about 90% is arranged, cellular form is good, cell is more plentiful, viable cell is a lot, is evenly distributed, and is adherent good; Change 10% calf serum nutrient solution and continue to cultivate, covered with monolayer cell in 2～3 days.

Use in the comparative example 1 conventional cell cryopreservation method frozen cell recovery cultivate 12 as a child the back observe, as seen form is irregular, cell is more tiny, growing way is poor, cell attachment about 60%, and adherent insecure, change 10% calf serum nutrient solution and continue to cultivate, covered with monolayer cell in 6～8 days.

Table 1: the survival rate after the freeze-stored cell recovery is cultivated relatively

	The VER cell	The BHK-2 cell	The MRC-5 cell	The 2BS cell	The KMB1 cell
						Embodiment 1	96％	92％	96％	92％	92％
Embodiment 2	94％	90％	95％	90％	90％
						Embodiment 3	94％	91％	93％	91％	91％
Comparative example 1	65％	63％	66％	58％	55％

Claims

1. a cell cryopreservation method is characterized in that, this method comprises the steps:

Frozen pipe in 2～8 ℃ of placement 10～40min, is placed 10～20h to frozen Guan Yu-80 ℃ then, immerse in the liquid nitrogen and carry out prolonged preservation.

2. method according to claim 1 is characterized in that, the described cell density that grows into the culturing cell of individual layer is 5.5 * 10 ⁴～6.5 * 10 ⁴/ cm ²

3. method according to claim 1, the described mass volume ratio that is used for the pancreatin of peptic cell is 0.15～0.40%.

4. method according to claim 3, the described mass volume ratio that is used for the pancreatin of peptic cell is 0.25%.

5. according to the described method of claim 1, the described trysinization time is 2min.

6. method according to claim 1, the described frozen pipe that cell suspension is housed is placed 20min in 2～8 ℃.

7. method according to claim 1, described culturing cell are VERO, BHK-21, MRC-5,2BS or KMB17 cell.