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HCEC-B4G12 is a clonal subpopulation started in 2005 from the parental cell line HCEC-12 (ACC 646) established from normal cells of the posterior epithelium of the cornea of a 91-year-old woman transformed with the early region of the SV40 genome including genes encoding large T-antigen and small t-antigen; the subclone is described in the literature to represent differentiated cells of the so-called corneal endothelium
Human-Endothelial-SFM + 10 ng/ml FGF-2 (observe the shelf-life of the medium; do not apply h.i. FBS in order to keep differentiation status)
seed out at ca. 1 x 106 cells/80 cm2 flask; flasks must be coated with 10 µg/ml laminin and 10 mg/ml chondroitin sulfate; split confluent culture ca. 1:3 every 3-4 days using 2-3 ml trypsin/EDTA per 80 cm2 flask for 2-5 min at 37 °C (wash cells twice with PBS before trypsination, avoid trypsination longer than 5 min and check cell dissociation by microscopy); stop trypsination by using medium containing protease inhibitor cocktail (Sigma P1860) at 500-fold dilution; do not apply antibiotics, cells are highly sensitive to antibiotics
at 37 °C with 5% CO2
ca. 40-50 hours
cell harvest of ca. 15 x 106 cells/175 cm2
frozen with 90% medium (including FGF-2), 10% DMSO
DSMZ Scientific Data:
negative in microbiological culture, PCR assays
To inquire about expression of EpCAM and intermediate filaments, contact firstname.lastname@example.org.
STR analysis according to the global standard ANSI/ATCC ASN-0002.1-2021 (2021) resulted in an authentic STR profile of the reference STR database.