Organ: bone marrow Strain: (C57BL/6 x C3H)F2 -op/op Tissue: stroma
Permits/Forms:
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Applications:
supports hematopoietic differention
Age:
newborn newborn
Comments:
The OP9 cell line was established from newborn op/op mouse calvaria. The cells do not produce functional macrophage colony-stimulating factor (M-CSF) due to an osteopetrotic mutation in the gene encoding M-CSF. The presence of M-CSF had inhibitory effects on the differentiation of embryonic stem (ES) cells to blood cells other than macrophages. OP9 cells can be used to coculture mouse embryonic stem cells (ES cells) to induce the differentiation of embryonic stem (ES) cells into blood cells of erythroid, myeloid, and B cell lineages. Cocultivation with OP9 does not require exogenous growth factors or complex embryoid structures. This system will facilitate the study of molecular mechanisms involved in development and differentiation of hematopoietic cells.
Propagation:
ATCC complete growth medium: The base medium for this cell line is Alpha Minimum Essential medium without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate . To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 20% Atmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37.0°C
Subculturing:
Protocol: Note: Cell density is important. If the subculture ratio is too low, the culture will not reach confluence. However, do not overgrow. Very large cells tend to appear after overgrowth and these cells are a warning sign that the OP9 cells will not support the maintenance of hematopoietic cells. Subculture just before confluence.
Remove and discard culture medium.
Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
Transfer cell suspension to a centrifuge tube and spin at approximately 125 X g for 5 to 10 minutes. Discard supernatant.
Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels.
Incubate cultures at 37°C. Interval: Maintain cultures at a cell concentration between 4 X 10(3) and 1 X 10(4) cells/cm2. Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:5 is recommended Medium Renewal: Every 2 to 3 days
Preservation:
Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO Storage temperature: liquid nitrogen vapor phase
Doubling Time:
26 hrs
Related Products:
recommended serum:ATCC 30-2020
References:
61302: Nakano T, et al. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265: 1098-1101, 1994. PubMed: 8066449
64482: Nakano T, et al. In vitro development of primitive and definitive erythrocytes from different precursors. Science 272: 722-724, 1996. PubMed: 8614833
64484: Nakano T. Lymphohematopoietic development from embryonic stem cells in vitro. Semin. Immunol. 7: 197-203, 1995. PubMed: 7579206
64485: Motoyama N, et al. bcl-x prevents apoptotic cell death of both primitive and definitive erythrocytes at the end of maturation. J. Exp. Med. 189: 1691-1698, 1999. PubMed: 10359572
64486: Nakano T. In vitro development of hematopoietic system from mouse embryonic stem cells: a new approach for embryonic hematopoiesis. Int. J. Hematol. 65: 1-8, 1996. PubMed: 8990620
64487: Nakano T, et al. Development of erythroid cells from mouse embryonic stem cells in culture: potential use for erythroid transcription factor study. Leukemia 3: 496-500, 1997. PubMed: 9209437
64488: Suwabe N, et al. GATA-1 regulates growth and differentiation of definitive erythroid lineage cells during in vitro ES cell differentiation. Blood 92: 4108-4118, 1998. PubMed: 9834216
64489: Suzuki A, Nakano T. Development of hematopoietic cells from embryonic stem cells. Int. J. Hematol. 73: 1-5, 2001. PubMed: 11372743
64490: Eto K, et al. Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling. Proc. Natl. Acad. Sci. USA 99: 12819-12824, 2002. PubMed: 12239348
