Cellartis enhanced hiPS-HEP FAQs

Cellartis enhanced hiPS-HEP cells are differentiated from human induced pluripotent stem (hiPS) cell lines ChiPSC12, ChiPSC18, and ChiPSC22. These lines were created by reprogramming human dermal fibroblasts with defective polycistronic retrovirus technology, which includes a retroviral vector containing Oct4, Sox2, Klf4, and c-Myc (pRV-OCT4-SOX2-KLF4-MYC; the vector map is proprietary). Reverse transcriptase activity testing shows that the hiPS cell lines do not contain replication-competent retroviruses. In addition, the viral reprogramming genes are silenced.

No. In some hepatocytes genetic engineering is used to improve hepatic function. Instead, we developed a culture protocol that substantially improves functionality. Each hiPS cell line—the source of the Cellartis hepatocytes—was reprogrammed using an integrative, retroviral technique. We tested for reverse transcriptase activity in the hiPS cell lines to confirm the absence of replication-competent retroviruses. We also confirmed that the factors used for reprogramming were silenced in the hiPS cell lines.

Yes, Cellartis enhanced hiPS-HEP cells are available from three different cell lines: ChiPSC12, ChiPSC18, and ChiPSC22. Hepatocytes derived from different hiPS cell lines model the genetic variation between individuals, therefore, the cells display phenotypic variation.

In our tests, high and constant CYP enzyme activities, plus albumin and urea secretion, were detected at Days 4–19 post-thawing. CYP activity (top panel) was measured by LC/MS at 4, 12, and 19 days post-thawing. Albumin and urea secretion (bottom panel) was measured by ELISA at 2, 4, 12, and 20 days post-thawing. Both were compared to human primary hepatocytes (hphep) after 20 (top) or 24 (bottom) hours. 

With Cellartis enhanced hiPS-HEP v2 kits, we are fulfilling a need for longer-lasting, functional, and mature hiPS cell-derived hepatocytes as a better alternative to primary and tumor-based cell models. Cellartis enhanced hiPS-HEP v2 kits contain the same high-performance Cellartis enhanced hiPS-HEP cells as the original kits. Both versions also contain hepatocyte thawing medium, coating, plating medium, and washing medium. However, the newer v2 kits contain long-term maintenance medium for extended culture of enhanced hiPS-HEP cells, ideal for disease modeling and drug discovery experiments.

Yes. In our tests, Cellartis enhanced hiPS-HEP cells derived from ChiPSC12, ChiPSC18, and ChiPSC22 expressed several transporters: NTCP, OCT1, OATP1B1, and OATP1B3 (uptake, Panel A); plus MRP2, MDR1, and BSEP (efflux, Panel B). Some transporters were expressed at similar levels as cryopreserved human primary hepatocytes (hphep); other transporters were expressed at lower levels than hphep. mRNA expression of OATP1B3 was not observed in enhanced hiPS-HEP cells derived from ChiPSC18. In Panel C, mRNA expression from hphep is shown as a dashed line. 

Yes. In our tests, Cellartis enhanced hiPS-HEP cells showed mRNA expression of UGT1A1 and UGT2B7 (Panel A) at similar or higher levels than cryopreserved human primary hepatocytes (Panel B). Enhanced hiPS-HEP cells displayed phase II enzyme activities, e.g., UGT and sulfotransferase activities, at similar or higher levels as in cryopreserved human primary hepatocytes cultured for 24 hours, as measured by LC/MS (Panel C). 

The cells have basal CYP enzyme activities that are measured during quality control and presented in the Certificate of Analysis. Minimal CYP induction is observed in Cellartis enhanced hiPS-HEP cells in 2D culture.

Whereas a CYP activity assay cannot discriminate between CYP3A4 (adult), CYP3A5 (adult), and CYP3A7 (fetal), qPCR can. In our tests, Cellartis enhanced hiPS-HEP cells expressed high mRNA levels of CYP3A4 and CYP3A5 and very low levels of CYP3A7—comparable to cryopreserved human primary hepatocytes cultured for 24 hours (Panel A). Interestingly, interindividual variation in CYP expression can be observed in Cellartis enhanced hiPS-HEP cells derived from different hiPS cell lines. In Panel B, mRNA expression of CYP enzymes in human primary hepatocytes is shown as a dashed line.

This subpopulation is likely a mixture of other endodermal cells, but the exact composition is unknown. We start with high-purity endodermal cells in the first step of differentiation, and we confirm through staining that there are no undifferentiated stem cells in the final product.

To determine the maturity of the cells, we investigated genes known to be expressed in fetal and adult hepatocytes and tested by multiple assays how they compare to primary hepatocytes. We observed high expression and activity of multiple adult hepatocyte markers such as albumin, CYP1A2, CYP2C9, CYP2C19, CYP3A4, HNFα, and ASGPR1. We confirmed this by immunofluorescence, activity, and qPCR analyses. We also observed a low expression level of the fetal gene CYP3A7—similar to the level in primary hepatocytes. View a technical note with these data »

We have not been able to show the formation of bile canaliculi in these cells.

Cellartis enhanced hiPS-HEP cells are frozen in a cryovial and packaged as a part of a Cellartis enhanced hiPS-HEP v2 kit. This kit also contains the Cellartis Enhanced hiPS-HEP Thawing and Plating Kit and Cellartis Enhanced hiPS-HEP Long-Term Maintenance Medium. All items are shipped together on dry ice.

96-well and 24-well plates are recommended.

We recommend using the provided Cellartis Enhanced hiPS-HEP Thawing and Plating Kit and Cellartis Enhanced hiPS-HEP Long-Term Maintenance Medium. These components are included within the Cellartis enhanced hiPS-HEP v2 kits, and they are crucial for optimal performance and results with the included Cellartis enhanced hiPS-HEP cells. Other coatings or media can be used, however, we cannot guarantee optimal performance. If you have questions or specific requirements regarding media or coating, please contact us.

Yes, Cellartis Enhanced hiPS-HEP Long-Term Maintenance Medium contains all three of these components. We have not evaluated the performance of Cellartis Enhanced hiPS-HEP cells in the absence of dexamethasone, hydrocortisone, or DMSO.

Washing with Hepatocyte Washing Medium on Day 1 after plating is necessary to remove nonadherent cells. When washing, carefully remove 100% of the media from only a few wells at a time. Do not let the wells dry.

Repeated drying, which easily happens when changing 100% of the media, detaches the cells at the edges of the well. Changing only 90% of the media ensures that liquid is always left on the cells, preventing desiccation and detachment. For washing steps in assays or immunocytochemical staining, it is better to leave a small amount of buffer or washing solution on the cells and increase the number of washing steps.

Yes, but add 50% more media on Friday (i.e., 1.5 ml/well of a 24-well plate and 230 µl/well of a 96-well plate). It is best to change the media late on Friday and early on Monday.

No, Cellartis enhanced hiPS-HEP cells are terminally differentiated hepatocytes that are unable to proliferate.

Liquid chromatography/mass spectrometry (LC/MS) was used to measure the formation of these specific metabolites: acetaminophen (CYP1A), 4-OH-Diclofenac (CYP2C9), OH-Bufuralol (CYP2D6), and 1-OH-Midazolam (CYP3A). The CYP activities of Cellartis Enhanced hiPS-HEP cells were analyzed at Days 4, 12, and 19 after thawing as part of routine quality control. LC/MS analysis was performed at Pharmacelsus GmbH, Germany.

To measure CYP activity, Cellartis Enhanced hiPS-HEP cells were washed twice with prewarmed William medium E (+0.1% PEST). Then, the activity assay was started by adding 110 µl per cm² culture area of prewarmed William medium E containing 0.1% PEST, 25 mM HEPES, 2 mM L-Glutamine, and the following probe-substrate cocktail:

After two hours at 37°C, 100 µl of supernatant was collected and kept at –80°C until LC/MS analysis. The metabolite concentrations measured by LC/MS were normalized to the amount of protein per well (determined using the Pierce BCA Protein Assay Kit) and assay duration (120 min).

These liquid extrusions are normal. In time-lapse recordings, we have observed that hepatocytes release these drops into the media and remain healthy afterward. This is not a sign of cell death, and cultures with these drops perform normally in activity assays. We have stained the cultures with Oil Red O, and these drops were not stained, indicating that they are not lipid droplets. Although the exact nature of the drops is unknown, cell performance remains intact despite their presence.

Occasionally, these domes appear, but they do not affect overall cell performance. In these structures, the hepatocytes have detached from the coating, possibly because they were growing very densely. To prevent cell loss, please be careful when changing the media; always leave 10% of the media in each well.

Yes, we have successfully generated 3D spheroids using ultra-low attachment plates. Spheroids can be maintained for 3–4 weeks in culture.