Human rheumatoid arthritis fibroblasts|BNCC337864 |BNCC

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Human rheumatoid arthritis fibroblasts

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 according to 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
Type	fibroblast
Safety level	1
morphology	Fibroblast-like, irregularly shaped, margin irregular, monolayer adherent growth, clean background, no pigment, no vacuoles
Separation substrate	Joint
Sharing mode	Public welfare sharing

MH7A human rheumatoid arthritis fibroblasts

adherent, fibroblast-like

No. 337864

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

Biosafety level: 1, handle in ultra-clear 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 2-3h, 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.

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.

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）, change the media overnight, and it will grow up in 2-5 days.

Subculture/cryopreservation: suck out the old culture solution. after PBS cleaning twice, add 6mL(/100mm dish) of pancreatic enzyme and observe it under a microscope. during this period, it is forbidden to shake the culture dish. when the cells just fall off, most of the pancreatic enzyme is sucked out, leaving about 0.5mL, then move to the incubator for digestion and take out for about 5min. Subculture with 12mL CM1-1 culture solution to terminate digestion, gently blow uniform cells, and then can be divided into 3~6 dishes for culture; For cryopreservation, 6mL of cryopreservation solution (90% FBS + 10% DMSO) is used to terminate digestion, blow evenly, divide into 6 frozen storage tubes, and use a program cooling box to freeze storage at -80 ℃.

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 12 hours, the cell adherence rate ≥ 80.0% in 90 hours	adherence is observed in 18 hours, cell adherence rate ≥ 80.0% in 90 hours
cell morphology:	Adherent, fibroblast-like	CM1-1 culture medium, adherent, fibroblast-like
attached：
Conclusion:	good viability, and no abnormal cell morphology, qualified

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