BeNa Culture Collection
|Culture medium||DMEM-H complete medium: 90% DMEM-H + 10% FBS|
|Subculture procedure||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 pan, shaken The frozen vial to accelerate dissolution, and it is advisable to dissolve all within 1min; (2) Add 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: ① remove the medium, rinse twice with PBS, and add 1-2mL pancreatin (0.25% Trypsin + 0.02% EDTA); (2) Observe the digestion situation under the microscope. When the cell edge shrinks and the adherent is loose (a 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) dispense the cell suspension into a fresh T25 flask as a ratio of 1:2, add appropriate complete culture medium, mix the cell suspension evenly, and culture in an incubator.; ④ Pay attention to the change of pH of medium and cell density, renew the medium regularly (2-3 times a week), and repeat the subculture or cryopreservation when the cell density reaches 80%-90%.
|Growth conditions||Culture temperature 37 ℃; Atmosphere 5% CO2 + 95% air;|
|Growth characteristics||Adherent growth|
|Storage conditions||Liquid nitrogen|
|morphology||Fibroblast-like, polygonal, irregular margin, monolayer growth, clean background, no pigment, no vacuoles|
|Sharing mode||Public welfare sharing|
CEF chicken embryo fibroblasts
Adherence, epithelial cell-like
Product format: 2ml frozen vial x 2, or T25 flask x 1
Biosafety level: 1 , handle in ultra-clean table or safety cabinet
Culture:37 ℃,5% CO2 CM1-1 culture solution
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 4h, and then carry out handling procedure. During recovery, each vial shall be used up once and shall not be retained. After recovery, the cells can be passed on to the next generation and can be used normally. Please operate in strict accordance with this instruction, otherwise the replacement of cells are not be available in case of lose of cell viability.
Growth conditions :37 ℃,5% CO2,CM1-1 culture solution. CM1-1 culture solution: 90% DMEM-H + 10% FBS. DMEM-H:DMEM high sugar culture solution, containing glutamine and sodium pyruvate.
(1)Prepare a new 100mm culture dish containing 12ml of the above culture medium;
(2)Remove the frozen vial from liquid nitrogen or refrigerator at -80℃, thaw the vial in 37°C water bath for 1-2 minutes. Transfer the vial to biosafety cabinet for culture as soon as the contents are completely thawed.
(3)Draw the solution with a sterile pipette, drip into a new culture dish, and mix it evenly by shaking clockwise;
(4)Put it into incubator (37℃，5%CO2）, change the media overnight, and it will grow up in 2-4 days.
Subculture/ cryopreservation: remove old medium, and rinse twice with PBS, add 6ml of 0.25%Trypsin+0.02%EDTA, do not shake the culture dish during the period. Observed under the microscope, remove most of the trypsin(with 0.5ml left) when the cells detach. transfer it to the incubator for digestion about 3minute, and take it out.
(1) Subculture: terminate digestion with 12ml of CM1-1 culture media, aspirate and dispense into 3~6 culture dishes.
(2) Cryopreservation: terminate digestion with 6ml of cryopreservation solution （90%FBS+10%DMSO), aspirate and dispense into 6 frozen vials, and cryopreserve at -80 ℃ with a programmed cooling box.
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 12hours, the cell adherence rate ≥ 80.0% in 70hours||adherence is observed in 18 hours, the cell adherence rate ≥ 80.0% in 66hours|
|cell morphology||adherent, epithelial cell-like||in CM1-1 culture solution, adherent, epithelial cell-like, polygonal|
|conclusion||good viability, and no abnormal cell morphology, qualified|