Source: Human Stem Cell Culture

Operation method

Discontinuous density gradient centrifugation of human bone marrow for MSCs production

Materials and Instruments

Bone marrow matrix
Complete Culture Medium (CCM) Phosphate buffer without calcium or magnesium Hank's Balanced Salt Solution without calcium or magnesium Ficoll-Paque Lymphocyte Separation Solution
Polypropylene centrifuge tubes 15 mL and 50 mL Plastic tissue culture dishes 15 cm diameter Plastic micropipette tips 10 μL

Move

(a) Remove the cap from the anticoagulation tube and transfer the bone marrow fluid to a 50 mL centrifuge tube. Add room temperature HBSS, making sure the volume reaches 25 mL.

(b) Take another 50 mL centrifuge tube and add 20 mL of Ficoll-Paque Lymphocyte Separation Solution. gently add 25 mL of cell suspension to the upper layer of Ficoll. the interface between HBSS and Ficoll must not be disrupted.

(c) Close the gate and centrifuge at 1800 g for 30 min at room temperature.

(d) After centrifugation, the white cell layer was collected at the interface between Ficoll and HBSS and transferred to a new 50 mL centrifuge tube.

(e) The volume of the collected cell suspension was adjusted to 3 times with HBSS and centrifuged at 1000 g for 10 min at room temperature. the washing was repeated.

(f) Resuspend cells with 30 mL of CCM preheated to 37°C

(g) Take 10 μL of cell suspension and add it to 10 μL of Taipan blue, and the viability of the cells was evaluated by a blood cell counter, and the viability needed to be higher than 80%.

(h)Transfer 30mL of cell suspension to a 15 cm diameter tissue culture dish and incubate in a 5% _C02 incubator for at least 15h.

(i)Remove the dish from the incubator and aspirate the culture medium.

(j) Add 20 mL of preheated PBSA, rinse the monolayer of cells and discard.

(k) Repeat the rinsing process 3 times.

(l) Add 30 mL of pre-warmed fresh CCM.

(m) Repeat this rinsing and medium change operation every other day for a total of 6 days.

(n) After 6 days, monolayers of cells were observed under an inverted microscope, and clones of adherent, fibroblast-like MSCs were clearly visible in the culture dish. Sometimes there may be signs of hematopoietic cell contamination, but these cells can be removed at passaging. Cultures are processed for expansion and cryopreservation when they reach 50% to 60% confluence.

Common Problems

Media Formulation.

Complete Culture Medium (CCM): α-MEM: α Low Limit Basal Medium Contains glutamine with no caustic or deoxyribonucleotides; add: additional L-glutamine 2 mmol/L (final concentration includes glutamine in the fractions for a total of 4 mmol/L), 20% purified by FBS hybridoma Non-heat inactivated, Penicillin 100 U/mL, Streptomycin 100 μg/mL. filtered to remove bacteria Storage. at 4 ℃ for no more than 2 weeks.

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