IDG-SW3 represent a non-homogenous population progressing from early osteoblasts to late osteocytic. These cells express functional SV40 large T antigen that is induced in the presence of IFN γ under permissive temperature (33°C) and express GFP under control of the dentin matrix (Dmp1) promoter. Initial culture shows osteoblastic phenotype, however when under mineralizing conditions, the cells start to express early osteocyte markers such as E11/podoplanin, followed by Dmp1 and mature markers SOST and FGF23 by 21-28 days of culture.
Similar to osteocytes in vivo, these cells respond to hormonal signals such as PTH and 1,25-dihydroxyvitamin-D3. IDG-SW3 cells can be maintained in both 2D and 3D cultures and are useful for study of osteocytes at various differentiation stages. It should be noted that these cultures may contain a mixture of osteoblasts and osteocytes at different stages of differentiation and for studies requiring enriched osteocytes, the Dmp1-GFP cells should be isolated by flow cytometry (FACS).Immortalization Method: Isolated from long bones of transgenic mice (in which the Dmp1 promoter drives the expression of GFP). BioSafety Level: IISpecies: MouseSource Organ: Long bonesGrowth Properties: AdherentMorphology: PolygonalPopulation Doubling: 40 - 50 hoursSeeding Density: 25,000 - 30,000 cells/cm²Markers: ALP (osteoblast), E11/gp38, Dmp1 and Phex (osteocyte), SOST/sclerostin and FGF23 (late osteocyte)Donor Age: N/ADonor Gender: N/ADonor Ethnicity: N/APropagation: The basal medium for this cell line is Prigrow III medium available in abm, Cat. No. TM003. To make the complete growth medium, add the following components to the basal medium: fetal bovine serum (TM999)* to a final concentration of 10%, 50U/ml IFN γ and Penicillin/Streptomycin Solution (G255) to a final concentration of 1%.Carbon dioxide (CO2): 5%, Temperature: 33.0°C.
To induce osteogenesis, replace the culture medium with the following osteogenic media: Prigrow III medium (TM003) with fetal bovine serum (TM999)* to a final concentration of 10%, 50 µg/ml ascorbic acid, 4 mM β-glycerophosphate and Penicillin/Streptomycin Solution (G255) in the absence of IFN γ at +37.0°C. Change the media every three days. GFP expression should be observed by day 4 under differentiation conditions, and become strong by day 10-15 of culture.
* abm does not recommend to use heat-inactivated FBS for cell culture unless specified otherwise.Quality Control: (1) Presence and absence of SV40 large T-antigen, under permissive and non-permissive conditions, were confirmed using western blot. (2) Fluorescent imaging was used to measure Dmp1 promoter driven GFP expression in osteogenic conditions. (3) Von Kossa staining was used to show focal nodular mineralization and Alizarin red S staining was used to detect calcium deposition of the differentiated cells. Shipping Condition: Dry IceStorage Condition: liquid nitrogen or -180°C