Human bone marrow contains mesenchymal progenitors (mesenchymal stem cells, MSCs). MSCs produce adventitial cells in the human bone marrow microenvironment.
MSCs are multipotent stem cells that can differentiate into a variety of cell types, including: osteoblasts (bone cells), chondrocytes (cartilage cells) and adipocytes (fat cells).
These cells provide support to hematopoiesis by producing membrane-bound and soluble signals and cytokines. These stromal progenitors can be readily isolated from bone marrow and demonstrate extensive proliferative capacity in vitro. Purified and culture-expanded human MSCs differentiate along the osteogenic, chondrogenic, and adipogenic lineages both in vitro and in vivo.
Materials and Methods
1. On enrollment, approximately 35 days before scheduled peripheral-blood progenitor-cell (PBPC) infusion, 20 to 25 ml of bone marrow aspirate was obtained.
2. Aspirates were obtained 2 to 48 hours before high-dose cyclophosphamide mobilization chemotherapy.
3. The aspirate was taken to the class 10,000 quality clean production suite.
4. Aspirate was mixed with two volumes of Dulbecco’s phosphate-buffered saline (DPBS) in a sterile class II biologic safety cabinet and centrifuged at 900 x g for 10 minutes at 20°C in a Beckman GS-6R centrifuge.
5. Pellets were layered onto 25 mL of Percoll (density, 1.073 g/ml) at a density of 1 to 2 x 107 cells/ml.
6. Gradients were centrifuged at 900 x g for 30 minutes at 20°C, and recovered mononuclear cells were resuspended in DPBS and centrifuged at 460 x g for 10 minutes at 20°C.
7. Cells were resuspended at 1 x 106 cells/ml in Dulbecco’s modified Eagle medium, low glucose (DMEM-LG) with 10% fetal bovine serum and 30 ml of cell suspension was plated in a 175 cm2 flask.
8. The serum lot used was selected on the basis of optimal MSC growth with maximal retention of osteogenic differentiation as assessed with in vitro and in vivo assays.
9. MSCs were cultured in humidified incubators with 5% CO2 and initially allowed to adhere for 72 hours, followed by media change every 3 to 4 days.
10. When cultures reached more than 90% confluence, adherent cells were detached with 0.05% trypsin-EDTA and replated or passaged at a density of 1 x 106 per 175 cm2 flask until processing for infusion.
11. Cell cultures were tested for sterility weekly and for the presence of cells by immunocytochemical method, endotoxin by limulus amebocyte lysate test, and Mycoplasma by DNA-fluorochrome stain and PCR before infusion.
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