In general, the transfer cells, the best strategy is to kept at low temperature (70 ℃ ~ 196 ℃), the cell is the basic principle of deep cryogenics: under - 70 ℃, the activity of enzyme in cells have been stopped, the metabolism in full stop state, it can be long-term preservation.
The key cell cryopreservation, is processing through the stage of 0 ~ 20 ℃, in this temperature range, ice crystals are needle, easy to cause cells to serious damage.
After previous long-term experiments, the basic principle of cryopreservation and recovery of cells is to slow freezing and thaw, so that the scientific research can preserve the cell vitality.
Material preparation
1.Instrument, clean bench, centrifuge, constant temperature bath box, refrigerator (4 ℃, 20 ℃, 70 ℃), inverted phase contrast microscope, incubator, liquid nitrogen freezer.
2.Glassware: straw (elbow, straight head), culture bottle, glass bottle (250ml, 100ml), waste water cylinder.
3.Plastic utensils: suction head, gun head, rubber plug, pipette (10ml), 15ml centrifuge tube, frozen storage tube (1 ~ 2ml).
4.Other items: small sample, red blood cell counting board, marker pen, medical adhesive plaster, liquid gun.
5.PBS, fetal bovine serum, DMSO, medium, double resistance, trypsin (0.25%)
Cell freeze
At present, a lot of cells cryopreserved using glycerol or dimethyl sulfoxide as protective agent, the two substances can increase the cell membrane permeability to water, coupled with slow freezing can make the water seepage, cells in the cell to reduce the formation of ice crystals in the cell, thus reduce the cell damage formed by ice crystals.
1.10% of DMSO + 90% fetal calf serum.
Glycerol is generally used in the preservation of bacteria and is rarely used in cell cryopreservation.
2.To take logarithmic growth cells, the cells of the single layer were digested by trypsin, and the cells in suspension were transferred directly to the 15ml centrifuge tube.
3.Centrifuge 1000 RPM, 5 min.
4.Removal of trypsin and old culture, and adding suitable amount of nutrient solution prepared good cryopreserved, gently blowing through a straw even, the cell count, the final density of the cells in freeze-stored liquid is 5 x10 ^ 6 / ml ~ 1 x10 ^ 7 / ml.
5.Mark cell name, freeze time and operator on frozen storage tube.
6.Cryopreserved: standard procedures for the cooling rate of cryopreserved - 1 ~ 2 ℃ / min;
When the temperature reached 25 ℃, can be increased to 5 ℃ ~ - 10 ℃ / min;
When to - 100 ℃, it can rapidly immersed in liquid nitrogen.
Also can be cryopreserved tubes containing cells into - 20 ℃ refrigerator 2 h, then in the 70 ℃ refrigerator overnight, remove the cryopreserved tubes, in liquid nitrogen container.
Cell recovery
1.Pipe from the liquid nitrogen container cryopreserved, directly into 37 ℃ warm water, do not shake to the melt as soon as possible.
2.Frozen out of the 37 ℃ water bath tube, open the lid and out of the cell suspension through a straw, added to the centrifuge tube and drops, more than 10 times the culture blending.
3.Centrifuge, 1000 RPM, 5 min.
4.Abandon to supernatant liquid, add heavy suspension cells containing 10% calf serum broth, count, adjust the cell density, inoculation culture bottle, 37 ℃ incubator static culture.
5.Change the culture medium once the next day, continue to cultivate.
Cell count and survival test
1.Principle:
(1) counting the number of cells in the cell or the Coultercounter particle counter automatically counts.
(2) there are usually two Chambers in the blood count plate. In each chamber, there are nine 1mm2 squares, of which there are 16 small squares in four corners, and the depth is 0.1mm.
When the glass is covered over the chamber, the volume of each large square is 1mm2 * 0.1mm = 1.0x10-4ml.
When used, count the number of cells in each large square, multiplied by dilution, and multiplied by 104, which is the number of cells per ml.
(3) the survival test procedure is dyeexclusion, and the dye will infiltrate into the dead cells, and the living cells will not penetrate the cell membrane and the dye will not penetrate.
Usually use the blue trypan blue dye. If the cell is not easy to absorb trypan blue, then use the red erythrosine bluish.
Cell survival rate: number of living cells/(number of live cells + dead cells) x 100%.
The counting should be completed within minutes after the staining of the trypan orchid, and as time goes on, some living cells also start to ingest dye.
Because Taiwan hope orchid has a strong affinity to protein, use the diluent that does not contain serum, can make dye count more accurate.
2、Material:
0.4% w/v trypan blue (GibcoBRL15250-061);
Erythosin bluish stain;
0.1 gram erythrosine bluish (SigmaE - 9259) and 0.05 gram preservative (sigmah-3647) were dissolved in 100mlCa + + / mg + + freesaline.
Blood count plate and cover glass (Hemocytometerandcoverslip);
Counter (counter);
Low-power inverted microscope;
Particle counter (Coultercounter CoulterElectronics).
Leucocyte diluent (4% acetic acid solution).
3.Steps:
(1) take 50 mu l cell suspension and 50 mu l trypan blue (orErythrosinbluish) in a mixture of 1.5 ml small centrifugal tube.
(2)Take a little mixture (about 15 mu l) from the blood corpuscle count plate chamber to join the above groove, cover glass, in 100 times were observed under inverted microscope, living cells don't dye, dead cells is blue (or red - Erythrosin bluish).
(3)Count the total number of cells of four large squares, in addition to the four again, multiplied by the dilution ratio (at least 2 times, with the trypanblue volume mixing), finally multiplied by 104, the number of cells per ml in the cell suspension.
If the cell is located on the line, only the cells (or the cell line with the left line) of the right line will be on-line.
Note: the total number of cells of the four large cells is x 104/4 = the number of cells/ml;
The volume of each large lattice is 2.5 px x 2.5 px x 0.25 px = 10-4ml
When the counting plate count, the optimum concentration is 5 ~ 10 x 105 cells/ml, which has a large margin error.
High concentration cell suspension, which can be counted as dilution or continuous dilution.
Pay attention to the point
1.The slower the freeze the better, the faster the recovery
2.Operate with liquid nitrogen, and wear protective glasses and gloves.
When cells recover, shaking small bottles in a water bath explodes easily.
DMSO is a toxic carcinogen, with good gloves in contact.
3.Cryopreserved cells should be enough, general minimum to achieve 5 x10 ^ 6 / ml.
Concentration depends on the situation.
4.Mark the bottle: mark the bottle with markers and write cell name, algebra and freeze time.
5.Gently move the newly recovered cells away from the cell.
6.DMSO is not sensitive to most cells, so it is not necessary to remove DMSO after defrosting.
Some DMSO sensitive cells need to be removed DMSO.
7.The thawed cells use the same medium and serum as before, and suddenly different cultures and serums can cause cell growth and even death.