Generation of bone marrow derived-cultured mast cells (BMCMC) from mouse bone marrow (PDF)
Donor mice
Generally, mice that are 4 – 6 weeks of age yield best cells for engraftment of mast cell-deficient mice. Male donor cells are not suitable for engraftment of female mice. Depending on your technique and culture maintenance, 5 – 10 x 107 cells may be generated after 5 weeks of culture per donor mouse.
Materials Needed
- Ice bucket
- Petri dishes: 35 x 10 mm and 100 x 20 mm
- 6-well culture plate
- Sterile scissors, forceps, scalpel blades
- Sterile syringes: 3, 5 and/or 10 ml
- 50 ml polypropylene (Falcon) tubes
- Sterile filter tissue culture flasks (T-25, T-75, and T-150)
Media
| Flushing Medium* (for retrieval of bone marrow) | |
| To DMEM add: | glutamine (1x) antibiotic/antimycotic (2x) ß-mercaptoethanol (1x) *Filter all through a 0.2 mm filter to sterilize |
| Culture Medium* (we use WEHI-3 conditioned medium as a source of IL-3; alternatively, use rmIL-3 (Peprotech) at 10 ng/ml) |
|
| To DMEM add: | 20% WEHI-3 conditioned medium antibiotic/antimycotic (1x) ß-mercaptoethanol (1x) glutamine (1x) 10% FBS, heat-inactivated **Filter all through a 0.2 µm filter to sterilize |
Bone marrow retrieval
In tissue culture hood:
1- Pour flushing medium into 6-well culture plate.
2- Place plate on ice and take to animal procedure room.
In animal procedure room:
3- Sacrifice animal and remove skin and feet from legs using sterile instruments.
4- Dislocate femur from hip, remove whole leg from animal, and reserve on ice until ready to remove bones.
5- Scrape away all tissue from bones using sterile scalpel blades, discard fibula.
6- Separate tibias from femurs and place bones in the flushing medium on ice until all bones are collected.
In tissue culture hood:
7- Aliquot 50 ml flushing medium into polypropylene tube on ice. Set aside empty 50 ml tube for collection of flushed marrow on ice.
8- Using sterile instruments, pick up a bone with forceps and cut off epiphyses (bone ends) with scissors to expose medullary cavity.
9- Holding bone over the empty 50 ml tube, use 25 gauge needle on 3 or 5 ml syringe filled with flushing medium to flush marrow out of the bone into 50 ml receiving tube.
10- When all bones have been flushed, you can break up any major marrow clumps with a 10 ml pipette.
11- Centrifuge marrow at 20°C, 1000 rpm, 5 minutes.
12- Pour off supernatant and resuspend pellet in culture medium. Place in T-25 flasks.1 mouse: 6-7 ml
3 mice: 14-15 ml
5 mice: 40-50 ml
Culture procedures
- After 2 days, remove medium (leaving debris to stick to bottom of flask) and double volume with fresh culture medium into new T-75 flask.
- After 2 more days, transfer half of medium to centrifuge and pellet cells at 1000 rpm, 5 minutes. Resuspend pellet in equal volume of medium and return to flask.
- In subsequent weeks, feed every 3-4 days. To increase yield of BMCMC, alternate:
- Centrifuge 1/3 to 1/2 of culture and replacing medium with equal volume (no total volume change)
- Adding 10 – 15 ml of fresh medium to culture to increase total volume
- With each feeding/volume change, use new flasks to remove fibroblast-like cells from culture (these cells adhere to the flask bottom).
- By week 4 of culture, if cells look heterogeneous (e.g., smaller cells amongst the larger round granular cells), you may need to supplement medium with IL-3.
- Complete differentiation will take 4-6 weeks. Assess differentiation by making a cytocentrifuge prep and stain with May-Grunwald Giemsa stain (Sigma GS-1L). If more than 90% of cells are mast cells, the culture is suitable for use in engraftment or in vitro studies. If not, continue to culture cells and use supplemental IL-3 as needed.
