Ccl 92

Cryopreserved hPSC-LSCs were thawed and plated onto Ø13mm cell-culture treated plastic coverslips (Thermanox™, Thermo Fisher Scientific) coated with Col IV/LN521, and cultured to a confluent stage in CnT-30 (with pen/strep). Coverslips with confluent cultures were thereafter transferred to a coculture with mitotically inactivated 3T3-Swiss albino feeder cells (CCL-92, ATCC, Manassas, VA), and CnT-30 medium was supplemented with 5% fetal bovine serum (FBS) and 1 mM calcium dichloride (CaCl₂; both from Sigma-Aldrich) to promote further differentiation. Cells were cultured in the enriched differentiation conditions for 7–21 days, changing the medium every or every other day and replacing the 3T3 feeder layers every 7 days. Samples were fixed at 7, 14 and 21-day time points and analyzed for their expression of CK3 and CK12, following the basic IF protocol described in the following chapter. Human PSC-LSCs from one line were used to demonstrate the capacity of hPSC-LSCs to differentiate further towards corneal epithelial cells. Detailed description of the experiment is provided in Supplemental methods within Additional file 1.

Cryopreserved marmoset hepatocytes were obtained from Bioreclamation IVT Inc. (Westbury, NY). The co-culture model consists of a mixture of marmoset hepatocytes and non-parenchymal mouse embryonic fibroblast 3T3-J2 cells (CCL-92, ATCC, Manassas, VA) were built as previously described^{36 (link),55 (link)}. Briefly, cryopreserved hepatocytes were thawed and centrifuged at 150 × g for 10 min and then the cells were re-suspended in Hµrel plating medium™ (Visikol Inc., Hampton, NJ). Hepatocyte number and cell viability were assessed using trypan blue exclusion. 3T3-J2 cells were cultured in normal DMEM medium (10% FBS, 200 U/mL penicillin/streptomycin) at 37 °C with 5% CO₂. Hepatocytes were seeded at a density of 30,000 cells in each well of a 96-well plate, respectively. 3T3-J2 cells were added the next day at 15,000 per well of 96-well plate, respectively. HµREL™−96 SACC-PMH are distributed by the Visikol, Inc. (Hampton, NJ). Cells were maintained in 100 μl Hµrel maintenance medium™ (Visikol, Inc., Hampton, NJ). Medium was replaced every 2 days. The cells were co-cultured at 37 °C in a 5% CO₂ for 10 days prior to HBV infections.

NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC^® CRL-1658™) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC^® CCL-92™) and the inbred BALB/c 3T3 (ATCC^® CCL-163™) [64 (link)]. The NIH/3T3 cells are highly sensitive to sarcoma virus focus formation and leukemia virus propagation. In confluent cultures, NIH/3T3 proliferation is contact-inhibited.

P31 cells were a kind gift from Prof. K. Grankvist (University of Umea, Sweden). 3T3 cells were obtained from American Type Culture Collection (ATCC; CCL-92). Primary hippocampal culture was a kind gift of Katalin Schlett and Krisztian Tarnok (Eötvös University, Hungary), and was established according to the animal welfare permit PEI/001/1108-4/2013 and PEI/001/1109-4/2013, in agreement with European Union and Hungarian legislation.
Cells were grown at 37°C in a humidified, 5% CO₂, 95% air atmosphere. For the cell lines, Dulbecco’s Modified Eagle Medium (DMEM, Lonza) containing L-glutamine was supplemented with 10% fetal bovine serum (Invitrogen) and penicillin-streptomycin-amphotericin B (Lonza). Primary cultures of embryonic hippocampal cells were prepared from CD1 mice on embryonic day 17/18 according to previously published protocol [15 (link)] using Neurobasal (NB) medium supplemented with B27 (Invitrogen) containing 5% fetal calf serum (FCS, Sigma), 0.5 μM GlutaMAX (Invitrogen), 40 μg/ml gentamicin (Hungaropharma, Budapest, Hungary), and 2.5 μg/ml amphotericin B (Sigma).