hBM-MSCs were isolated and cultured as previously described [17 (link),21 (link),42 (link),43 (link)]. Briefly, hBM-MSCs were isolated from the waste samples from surgery consisting of bone marrow extracted throughout washouts of the femurs’ medullary cavities of adult donor subjects undergoing primary total hip replacement. The procedure of isolation of stem cells is occasional and is not part of a specific project. All procedures were done after the consent of donors (60-year-old) and were in accordance with the Declaration of Helsinki.
Donor subjects gave informed consent and the institutional ethical committee approved the procedures. After density gradient on Lympholyte (Cedarlane Laboratories Limited, Hornby, ON, Canada), mononuclear cells were isolated from the bone marrow and were seeded in culture flasks in growth medium consisting of RPMI-1640 (Euroclone, Milano, Italy) medium containing FBS 10%, 2 mM L-glutamine, and 1% penicillin–streptomycin (Euroclone) in a humidified atmosphere and 5% CO2 at 37 °C. After 5 to 7 days, the non-adherent cells were removed, and fresh medium was added to the flasks. After 15 days, a fibroblast-like colony started to grow. The medium was changed every three days [17 (link),21 (link),42 (link),43 (link)]. The mesenchymal phenotype of hBM-MSCs was analyzed by flow cytometry FACScan (BD Biosciences, San Jose, CA, USA ) as previously described [21 (link)]. The human anti-CD44, -CD45, -CD73, -CD90, -CD105, human leukocyte antigen (HLA)-ABC (BD Biosciences) were used. Data analyses were performed with the FlowJo software (Tree Star, Ashland, OR, USA). Cells were electronically gated according to light-scattering properties to discriminate cell debris. Isotype-matched nonspecific antibodies were used as the negative control.
Donor subjects gave informed consent and the institutional ethical committee approved the procedures. After density gradient on Lympholyte (Cedarlane Laboratories Limited, Hornby, ON, Canada), mononuclear cells were isolated from the bone marrow and were seeded in culture flasks in growth medium consisting of RPMI-1640 (Euroclone, Milano, Italy) medium containing FBS 10%, 2 mM L-glutamine, and 1% penicillin–streptomycin (Euroclone) in a humidified atmosphere and 5% CO2 at 37 °C. After 5 to 7 days, the non-adherent cells were removed, and fresh medium was added to the flasks. After 15 days, a fibroblast-like colony started to grow. The medium was changed every three days [17 (link),21 (link),42 (link),43 (link)]. The mesenchymal phenotype of hBM-MSCs was analyzed by flow cytometry FACScan (BD Biosciences, San Jose, CA, USA ) as previously described [21 (link)]. The human anti-CD44, -CD45, -CD73, -CD90, -CD105, human leukocyte antigen (HLA)-ABC (BD Biosciences) were used. Data analyses were performed with the FlowJo software (Tree Star, Ashland, OR, USA). Cells were electronically gated according to light-scattering properties to discriminate cell debris. Isotype-matched nonspecific antibodies were used as the negative control.
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