Immortalized human brain microvascular endothelial cells|BNCC337728 |BNCC

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Immortalized human brain microvascular endothelial cells

Culture medium	Special culture medium for endothelial cells: 500mlECM + 25mlFBS + 5mlECGS + 5mlP/S
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 digesion 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 value of media and cell density, renew the media regularly (2-3 times a week), and repeat the subculture or cryopreservation when the cell density reaches 80%-90%.
Growth conditions	37 ℃;5% CO2 + 95% air;
Growth characteristics	Adherent growth
Storage conditions	Liquid nitrogen
Safety level	1
morphology	Epithelial-like, polygonal, irregular margin, monolayer adherent growth
Sharing mode	Public welfare sharing

hCMEC/D3 immortalized human brain microvascular endothelial cells

Adherence, epithelial cell-like

No. 337728

culture:37 ℃,5% CO₂ ECM liquid medium

product format: 2ml frozen vial x 2, or T25 flask x 1

Biosafety level: 1, handle in ultra-clean table or safety cabinet

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 loss of cell viability.

Culture conditions :37 ℃,5% CO₂,ECM endothelial cell special medium. ECM culture solution: 500mlECM + 25mlFBS + 5mlECGS + 5mlP/S

Recovery steps:
(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）, renew the media overnight, and it will grow up in 3-5 days.

Passage / cryopreservation: remove old medium, and rinse twice with PBS, add 2ml 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 0.5minute, and take it out.

(1) Passage: terminate digestion with 6ml of CM1-1 culture media, aspirate and dispense into 3~6 culture dishes.

(2) Cryopreservation: terminate digestion with 3ml of cryopreservation solution （90%FBS+10%DMSO), aspirate and dispense into 3 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 18 hours, the cell adherence rate ≥ 80.0% in 168 hours	adherence is observed in 18 hours, the cell adherence rate ≥ 80.0% in 108 hours
cell morphology	adherent, epithelial cell-like	ECM culture medium, adherent, epithelial cell-like, polygonal, fusiform
attached figure
Conclusion:	good viability, no abnormal cell morphology, qualified