Cell freezing experiment
Cell freezing experiment
Cell freezing refers to the use of a protective agent added to the cryopreservation solution to freeze the cells in -175 ℃ liquid nitrogen. At present, the commonly used protective agents are dimethyl sulfoxide (DMSO) and glycerol, the protective agent is required to be non-toxic to the cells, small molecular weight, solubility, easy to penetrate the cells, the use of the concentration range of 5% -15%, the commonly used concentration of 10%. DMSO itself is toxic, no need for filtration or autoclave sterilization, while cryopreservation solution can be filtered to remove bacteria.
Principle
The basic principle of cell freezing experiment is that the cells are frozen in liquid nitrogen at -175℃. If the cells are frozen directly without any conditions, the water in the environment inside and outside the cells will form ice crystals, which will lead to a series of changes in the cells, such as mechanical damage, electrolyte elevation, osmotic pressure change, dehydration, pH change and protein denaturation, and finally lead to cell death.
If the cryopreservation solution is added to the protective agent, the freezing point can be lowered, and under the condition of slow freezing, it can make the water inside the cell seep out of the cell before freezing. -130 ℃ below the low temperature storage can reduce the formation of ice crystals.
Operation method
Cell cryopreservation
Principle
Traditionally, cell freezing (glycerol/dimethyl sulfoxide as a protective agent) is about: slow freezing and quick dissolution. Glycerol or dimethyl sulfoxide (DMSO) added to the culture medium during cell freezing, these two substances can increase the permeability of the cell membrane to water, coupled with the slow freezing can make the water inside the cell to seep out of the cell, reducing the formation of intracellular ice crystals, thus reducing the cellular damage due to the formation of ice crystals.
Materials and Instruments
[Experimental Materials Move 1. Prepare the freezing solution: Prepare the freezing solution according to the ratio of normal medium:DMSO = 9:1; 2. 2. Take the logarithmic growth cells, digest the monolayer growth cells with trypsin, centrifuge at 1000 rpm for 5 min; 3. Remove the trypsin and the old culture medium, add appropriate amount of prepared frozen culture medium, gently blow with a pipette to make the cells homogeneous, counting, adjust the final density of cells in the frozen culture medium to 5×106/mL~1×107/mL. 4; 4. Pack the cells into cryopreservation tubes, 1~1.5 ml per tube. 5; 5. Label the cryopreservation tubes with the name of the cells, the time of freezing, the number of cell generations, and the operator; 6. Add isopropyl alcohol to the cryopreservation box (it can be decreased by 1 ℃/min in negative 80 refrigerator) and put it into -80 ℃ refrigerator, and it is recommended to keep it for more than 12 hours. Generally, it can be stored in -80℃ for half a year to one year, and it should be transferred to liquid nitrogen for long-term storage; commercial freezing solution can be put into -80℃ refrigerator directly. Caveat 1. liquid nitrogen freezing is not recommended to use domestic freezing tube, easy to burst the tube when resuscitation;2. When digesting the cells, do not over-digest them;3. DMSO dissolved in DMEM medium will be exothermic, the freezing solution should be prepared 5-10 minutes in advance. Avoid wiping off the markers with isopropyl alcohol when using the cryopreservation kit. 4;4. DMSO penetrates many synthetic and natural membranes, including skin and rubber gloves. Therefore, it should be handled with care. Protective gloves and face masks must be used when placing cryopreservation tubes into liquid nitrogen. Common Problems 1. excessive cell digestion, resulting in poor cell status during recovery; 2. long term cell preservation in -80 ℃ freezer is not good, cells should be transferred to liquid nitrogen in time; 3. the domestic freezing tube in liquid nitrogen is easy to leak liquid nitrogen, resulting in the exposure of the tube during resuscitation, so put in liquid nitrogen try to use better quality freezing tube. For more product details, please visit Aladdin Scientific website.
Cells, DMSO, 0.25% Trypsin-EDTA (or cell scraper), 100X Double Antibody, PBS, Fetal Bovine Serum, DMEM Complete Medium, 75% Ethanol, Trypsin, Isopropanol;
[Instruments and Supplies
Freezing tube, 37 ℃ constant temperature water bath, Petri dish (6 cm), cell freezing box (with added isopropanol) or freezing foam box, marker, -80 ℃ refrigerator, inverted phase contrast microscope, liquid nitrogen tank, centrifuge, ultra-clean table for cell room, pipette gun and pipette tip (sterile or sterilized and then dried), disposable cell counting plate, cell autocounter, culture incubator, pipette.