Primary bone marrow stromal cultures

(Note: This protocol begins after obtaining the bone marrow aspirate from an AML patient.)

1.    After MNCs have been collected from Ficoll-separated bone marrow, aspirate the remaining supernatant and Ficoll, leaving only the red blood cell pellet.

2.    Resuspend the red blood cell pellet in 12mL ACK lysis buffer.

3.    Mix by pipetting and incubate on ice for 20 min.

4.    After incubation, centrifuge at 1500 rpm for 5 min.

5.    Aspirate supernatant and resuspend cell pellet in 10 mL stromal growth media (MEM-alpha with 20% fetal bovine serum, 2% L-Glutamine, 1% Pen-Strep, and 0.1% Fungizone).

6.    Plate cells in 10cm dish. This stage is “Passage 0”.

7.    Incubate cells for 48 hr. After incubation, aspirate culture media to remove any non-adherent cells.

8.    Following initial media replacement, allow cells to incubate for 2 wks, changing culture media every 7 days. By day 14, adherent cells should adopt a fibroblast-like morphology and form small colonies in the dish. If this does not occur, discard the sample.

9.    Allow cells to culture until reaching ~80% confluency, then passage the sample into one 15cm dish as follows:

a.    Wash 10cm dish with 3 mL sterile PBS and aspirate.

b.    Add 2mL trypsin to dish and incubate at 37C until cells have lifted off of the dish (this time can vary widely between samples. Check using microscope every 5 min).

c.     Wash cells off the dish with 3mL of media, then centrifuge at 1500 rpm for 5 min.

d.    Resuspend cells in 15mL culture media and plate in 15cm dish.

After plating in one 15cm dish, the sample is then at “Passage 1”.

Further passages similar to above until desired cell number obtained.

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