ATT00029E

Culture of human liver-derived cell lines

Eriko Ave and Masayoshi Nanba
Okayama University, School of Medicine

(This section was kindly translated to English by Dr.Satoh, and Dr.Yoshida of the HSRRB.)
General caution to the JCRB04xx series cell lines.

    *In the passage procedure of liver cell lines, the step of removal of trypsin by centrifugation usually give the most damage to the cells and this is desirable to be avoided.

    *The schedule of passage at 7-10 day interval with split ratio 1:4 and medium change once in this period is adequate.

    *In the passage of JCRB0401 HuH-6, rinse the culture 3 times with trypsin-EDTA in PBS and incubate for 10-20 min. For T-25 flask, incubation if 0.5 ml trypsin-EDTA solution is recommended.

    *At the time of JCRB cell bank started, RPMI1640 with 0.2% lactalbumin hydrolysate and 5% fetal bovine serum was used for JCRB04** human liver cell lines. However, we recently changed the medium to Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Enzyme solution at passage is 0.2% trypsin and 0.02% EDTA in PBS.

    *The use of heat-inactivated serum is recommended.(treat the FBS 30 min. at 56 C prior to use)

    *Medium change at 3-4 day-interval will produce good result. This may be due to the accumulation of autocrine growth factors.

    *Passage interval is 7 days for HuH-7 (split ratio 1:4) and 10 days for HuH-6 (split ratio 1:4).

General procedure for passaging liver-derived cell lines

    In the case of using T-25 flask

    1. Remove culture medium.

    2. Add 2 ml trypsin-EDTA solution and quickly rinse. Discard immediately 1.5 ml trypsin-EDTA with 0.5 ml left behind in the flask.

    3. Incubate for 5 min at 37 C.

    4. Add 1.5 ml fresh culture medium and pipetting to suspend the cells.

    5. If at the split ratio 1:4, dilute the 0.5 ml cell suspension with 4.5 ml culture medium.

    6. Place the flask in CO2 incubator and culture.

    (The point of above method is avoiding the cells from centrifugation.The trypsin is not removed and its action is stopped by FBS included in the culture medium.However,the inclusion of centrifugation step that widely used passage procedure is also possible.)

    Methods for the passage of HuH-6 cells

    (relatively difficult to cultivate)

    1. Remove culture medium

    2. Rinse quickly twice with trypsin-EDTA.

    3. Add 2 ml trypsin-EDTA and quickly rinse. Discard immediately 1.5 ml trypsin-EDTA with 0.5 ml left behind in the flask.

    4. Incubate for 10 min at 37 C.

    5. Add 1.5 ml fresh culture medium and pipetting to suspend the cells.

    6. If at the split ratio 1:4, dilute the 0.5 ml cell suspension with 4.5 ml culture medium. (Centrifugation is "not" required.)

    7. Place the flask in CO2 incubator and culture.

Comments

    * Avoid collagen-coated vessels since it makes difficult to detach the cells at passage.

    * The cells tend not to attach the vessels unless completely dissociate into single cells.

    Therefore, thoroughly pipetting at step [5] is recommended.

    * Centrifugation is not required to remove trypsin.

    * If you afraid the activity remaining in culture, change culture medium at 1 day after step [6].