国立研究開発法人 医薬基盤・健康・栄養研究所 JCRB細胞バンク

Notes for Cultivation of MG5 Cells

[Culture Medium]

Mixture of 30% Dulbecco's modified Eagle's medium(*a) containing 10% fetal bovine serum (FBS) and 70% A1 conditioned medium(*b) (or 100% conditioned medium of astrocytes).

*a) Dulbecco's modified Eagle's medium with 4.5 g/L glucose. For example, GBCO BRL #12100-038.

*b) Preparation of A1-conditioned medium.
1. Grow A1 cells (IFO50519) in 10% FBS-DMEM (high glucose) to sub-confluency on T-150 flasks.

2. Replace the medium with 60 mL fresh 10% FBS-DMEM and culture the cells overnihgt.

3. Collect the culture medium and filtrate it using a filter unit (pore size, 0.22 um). Store the filtered medium (A1-conditioned medium) at -80 C.

4. Please take care not to contaminate the A1 cells to MG5 cells. Avoid sharing the media, enzymes, buffers, or other equipments between A1 and MG5.

[Culture Substrata]

Bacterial grade dish (such as Falcon #1001, #1016) or flask (such as Falcon #3009, #3012).

[Thawing Frozen Cells and Cultivation of MG5 cells]

1. Thaw the frozen vial quickly by vigorously shaking the vial in 37 C water bath.

2. Strilize the surface of vials with 70% ethanol. Surround the vial with sterile quaze and snap it at the neck.

3. Transfer the vial content to 10 mL culture medium in 15 mL tube, and centrifuge at 1000 rpm for 5 min,

4. Resuspend the cells in 5 mL culture medium and plated to 25-cm2 culture flask or dish.

[Passage]

1. Wash the cells twice with PBS and add adequate amount of PBS.

2. Cool the culture for 15-30 min at 4 C.

3. Detach the cells by pipetting in PBS with a glass Pasteur pipette.

4. Collect the cells by centrifugation at 1000 rpm for 5 min.

5. Resuspend the cells in fresh medium and seed onto bacterial grade dishes/flasks.

*MG5 cells stack to the culture substratum very tightly, and this attachment is trypsin-resistant. Even according to the above procedure, only (up to) 70% recovery is expected. In addition, the attachment will become increasingly hard if the cells are cultured for long period in a same dish/flask. We recommend passage interval should not exceed 1 week.

(The cell bank tested the above passage method, but like-macrophages, MG5 adhere very tightly and this passage method did not completely work. The cell bank also tested the pronase digestion, 25 ug/ml pronase in Hanks' BSS for 10 min. and pipetting, but it also appeared not completely to recover the cells.)

[Alternative method for passage.]

Use RepCell (Cell Seed Inc.). Repcell is temperature-responsive cell cutureware, and at 4 C, the cell culture surface lose to support cell attachment. At passage, place the RepCell dishes at 4 C for 30 min to detach the cells.
 



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• 02232011
• 02122009
• 12222008
• 1159
• 07172017

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