OP9 (ATCC® CRL-2749)

Organism: Mus musculus, mouse  /  Cell Type: Embryonic Stem Cells, Macrophage  /  Tissue: bone marrow/stroma  / 

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Organism Mus musculus, mouse
Tissue bone marrow/stroma
Cell Type Embryonic Stem Cells, Macrophage
Product Format frozen
Morphology fibroblast-like
Culture Properties adherent
Biosafety Level 1
Age embryo
Strain (C57BL/6 x C3H)F2 -op/op
Applications
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.
Storage Conditions liquid nitrogen vapor phase
Derivation
The OP9 cell line was established from newborn op/op mouse calvaria.
Comments

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. 

Complete Growth Medium The base medium for this cell line is Alpha Minimum Essential Medium without ribonucleosides and deoxyribonucleosides and with 2.2 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%
Subculturing Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

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.

  1. Remove and discard culture medium.
  2. 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.
  3. 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.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Transfer cell suspension to a centrifuge tube and spin at approximately 125 X g for 5 to 10 minutes. Discard supernatant.
  6. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels.
  7. Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 4 X 103 and 1 X 10cells/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:5 is recommended
Medium Renewal: Every 2 to 3 days
Cryopreservation
Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Population Doubling Time 26 hrs
Name of Depositor T Nakano
References

Nakano T, et al. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265: 1098-1101, 1994. PubMed: 8066449

Nakano T, et al. In vitro development of primitive and definitive erythrocytes from different precursors. Science 272: 722-724, 1996. PubMed: 8614833

Nakano T. Lymphohematopoietic development from embryonic stem cells in vitro. Semin. Immunol. 7: 197-203, 1995. PubMed: 7579206

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

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

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

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

Suzuki A, Nakano T. Development of hematopoietic cells from embryonic stem cells. Int. J. Hematol. 73: 1-5, 2001. PubMed: 11372743

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

Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
References

Nakano T, et al. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265: 1098-1101, 1994. PubMed: 8066449

Nakano T, et al. In vitro development of primitive and definitive erythrocytes from different precursors. Science 272: 722-724, 1996. PubMed: 8614833

Nakano T. Lymphohematopoietic development from embryonic stem cells in vitro. Semin. Immunol. 7: 197-203, 1995. PubMed: 7579206

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

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

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

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

Suzuki A, Nakano T. Development of hematopoietic cells from embryonic stem cells. Int. J. Hematol. 73: 1-5, 2001. PubMed: 11372743

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

Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.