Long-term culture of bone marrow hematopoietic cells
Long-term culture of bone marrow hematopoietic cells
Bone marrow is aspirated into the culture medium. The bone marrow is maintained as an attached multilayer of cells for at least 12 weeks and up to 30 weeks. Stem cells, maturing myeloid cells and mature myeloid cells are released from the attached cell layers into the culture medium. Progenitor cells of granulocytes or macrophages can be detected by soft gel culture.
Operation method
Long-term culture of bone marrow hematopoietic cells
Principle
Bone marrow is aspirated into the culture medium. The bone marrow is maintained as an attached multilayer of cells for at least 12 weeks and up to 30 weeks. Stem cells, maturing myeloid cells and mature myeloid cells are released from the attached cell layers into the culture medium. Progenitor cells of granulocytes or macrophages can be detected by soft gel culture.
Materials and Instruments
Mouse Move 1. Neck-breaking execution of mice (5 mice: (C57BI/6xDBA/2)F1 bone marrow is good for long term culture, but some strains of bone marrow are poorly cultured, e.g., CBA [ Greenberger, 1980]). Caveat All reagents must be pre-tested for their ability to support cell growth. For more product details, please visit Aladdin Scientific website.
Fischer's medium Growth medium
Syringe Gauze Swab Dissecting scissors Dissecting forceps Culture flasks
2. Wet the hair with 70 % ethanol, and excise the femur on both sides. Ten femurs were placed in Petri dishes containing Fischer's culture medium (16 mmol/L (1.32 g/L) NaHCO3 with 50 U/ml penicillin and 50 ug/ml streptomycin) on ice. 1 femur contained 1. 5x107-2. 0x107 nucleated cells.
3. Operate on a laminar-flow ultra-clean bench:
( a) Remove the remaining muscle tissue with gauze and swab.
(b) Fix the femur with anatomical forceps and cut the knee joint end. 21G needle should be able to be inserted tightly into the bone marrow cavity.
(c) Cut the other end of the femur as close to the end as possible.
(d) Insert the end of the femur into a 100 ml bottle of growth medium, withdraw and push the syringe plunger several times until the bone marrow is flushed out of the femur.
(e) Repeat steps (a)-(d) to remove the bone marrow from the other 9 femurs.
4. Blow large clumps of bone marrow with a 10 ml pipette to disperse the bone marrow into a cell suspension.
5. Dispense 10 ml aliquots of the cell suspension into 25 cm2 culture flasks. Shake the cell suspension continuously to ensure that the cells are evenly distributed into 10 culture flasks.
6. Fill the culture flasks with 5 % CO2 and screw the lids on tightly.
7. Place the flasks horizontally at 33°C and incubate the cells.
8. Change the fluid once a week.
(a) Gently shake the culture flask to suspend cells that are not firmly attached.
(b) Pipette off 5 ml of culture medium containing suspended cells. Take care that the pipette does not touch the adherent cell layer.
(c) Add 5 ml of growth medium (Growth medium: dispensed as 100 ml. Fischer's medium with 1x10-6 mol/L hydrocortisone and sodium butanedioate and 20 % horse serum) to each culture bottle. (Hydrocortisone and Sodium Succinate are prepared in Fischer Culture Solution as a storage solution and stored at -20°C). Do not add the culture solution directly to the adherent cell layer to avoid damaging the cells.
(d) After filling the culture flask with gas, put it into the incubator.