normal liver cells of mice 

NCTC clone 1469

BNCC number: 102019

Growth properties: Semi-suspended semi-adherent growth

culture conditions : 37 ℃,5% CO₂

complete medium : 90% DMEM-H + 10% HS

frozen storage conditions: 50% DMEM-H + 40% FBS + 10% DMSO

Receiving notice: if any abnormality is found on the day of receipt, please contact the customer service within 24 hours. If it is overdue, it is deemed that the cells are well. For the frozen cells,  it shall be stored in the refrigerator at -80 ℃ upon arrival. If they are not used for a long time, they shall be transferred to liquid nitrogen for storage overnight.  Recovered cells in T25 culture flask, upon receipt, put the culture flask in the incubator for 2-3 hours, and then carry out handling procedure. Under sterile conditions, centrifuge the cell solution at 1000-1200rpm/min for 5-10 minutes, resuspend the cells, transfer the suspended cells in the T25 flask, add 10ml of complete medium, then put it in incubator. Please operate in strict accordance with this instruction, otherwise the replacement of cells are not be available in case of loss of cell viability.

Recovery steps: 
(1)    The frozen vial is taken out of liquid nitrogen or -80 ℃ refrigerator and put into PE gloves, quickly submersed into a 37 ℃ water bath, shaken the frozen vial to accelerate dissolution, and it is advisable to dissolve all within 1min; 
(2)    Put the dissolved cell solution into a centrifuge tube containing 9ml of complete culture medium in an ultra-clean table, centrifuge at 1000-1200rpm for 5min, discard the supernatant,  resuspend the cells with 1-2ml of complete media. 
(3)    The cell suspension was added to T25 flask containing 5-6ml complete medium and cultured in an incubator. 

cell subculture:

①Aspirate the culture medium (including suspended cells) into a centrifuge tube, centrifuge at 1000rpm for 5 minutes, and collect the cells;

②After gently rinsing the T25 bottle twice with PBS, add 1-2 mL of trypsin (0.25% Trypsin+0.02% EDTA); observe the digestion situation under the microscope. When the cell edge shrinks and the adherent is loose (a pasteur pipette can be used to suck up some pancreatin and gently blow somewhere in the cell layer, and the cell layer can be seen to detach with naked eyes, I .e. digestion is completed, otherwise digestion is continued), directly suck out pancreatin, add 5-6mL of complete medium, gently blow the cell layer off.

③ Mix the cells obtained in the above two steps evenly and distribute them into new T25 flasks at a ratio of 1:2, add appropriate complete medium, mix the cell suspension evenly, and culture in an incubator.

④ Pay attention to the pH value of the medium and the cell density, renew the medium regularly (2-3 times a week), and repeat the passage or cryopreservation when the cell density reaches 80%-90%.

Notes:
(1)    The cells are density dependent. Initiate the subcultivation in T25 flask when the cell density reaches 80%; A subcultivation ratio of 1:2 is recommended. 
(2)    If the cells in sealed culture bottle, put it into the incubator for cultivation after handling, with the cap unscrewed.

Recovery record:  According to the recovery instructions, the results of the cell recovery are reported as follows:
 

item	quality standard	Recovery record
Viability	adherence is observed in 18 hours, the cell adherence rate ≥ 80.0% in 172 hours	adherence is observed in 18 hours, the cell adherence rate ≥ 80.0% in 168 hours
cell morphology:	Epithelial cell-like, round	Epithelial cell-like, polygonal, round
attached figure:
Conclusion:	good viability, no abnormal cell morphology, qualified