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Products >  Stem_Cell_Research >  Resources >  FAQs >  Enhanced_hiPS-HEP

FAQs

Hepatocytes Derived from Human iPS Cells—
Cellartis® Enhanced hiPS-HEP

General FAQs

What is the source of Cellartis Enhanced hiPS-HEP cells?

Cellartis Enhanced hiPS-HEP cells are differentiated from the human induced pluripotent stem (hiPS) cell line ChiPSC18. This line was created by reprogramming human dermal fibroblasts with defective polycistronic retrovirus technology. This 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 line does not contain replication-competent retroviruses. In addition, the viral reprogramming genes are silenced in ChiPSC18.

Are Cellartis Enhanced hiPS-HEP cells genetically engineered?

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

Are Cellartis Enhanced hiPS-HEP cells available from several hiPS cell lines?

Cellartis Enhanced hiPS-HEP cells derived from several different hiPS cell lines will soon be available. Hepatocytes derived from different hiPS cell lines model the genetic variation between individuals; therefore, the cells display phenotypic variation.

How soon after thawing Cellartis Enhanced hiPS-HEP cells can I begin testing?

High and constant CYP enzyme activity is detected 6–11 days in culture after thawing. For applications not requiring high CYP activity, the cells can be used as early as day 5 after thawing.

Can other maintenance media or coatings be used for culturing Cellartis Enhanced hiPS-HEP cells?

We recommend using the provided Cellartis HEP Coat and Cellartis HEP Supplement. The coating and supplement are crucial for optimal performance and results when using 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 tech support.

Does the maintenance supplement for these cells contain dexamethasone, hydrocortisone, and DMSO?

Yes, the Cellartis HEP Supplement contains all three components. Since DMSO is added separately when preparing the maintenance medium, the amount of DMSO can be reduced or omitted. However, we have not evaluated the performance of Cellartis Enhanced hiPS-HEP cells in the absence of dexamethasone, hydrocortisone, or DMSO.

Do Cellartis Enhanced hiPS-HEP cells express transporter proteins?

Yes, Cellartis Enhanced hiPS-HEP cells express several transporters: BSEP, MRP2, NTCP, OATP1B1, OCT1, and BCRP. Some transporters are expressed at similar levels as cryopreserved human primary hepatocytes; other transporters are expressed at lower levels than cryopreserved human primary hepatocytes.

Do Cellartis Enhanced hiPS-HEP cells express phase II enzymes?

Yes, Cellartis Enhanced hiPS-HEP cells express several phase II enzymes: UGT1A1, 2B7, and GSTA1-1. Some are expressed at lower levels whereas others are expressed at similar levels as cryopreserved human primary hepatocytes directly after thawing.

Can CYP expression be induced in Cellartis Enhanced hiPS-HEP cells?

The cells have basal CYP activity that is measured during quality control and presented in the datasheet provided. Minimal CYP induction is observed in Cellartis Enhanced hiPS-HEP cells in 2D culture.

Does fetal CYP3A7 contribute to overall CYP activity in these cells?

No. qPCR and Western blotting allow discrimination between CYP3A4 (adult), CYP3A5 (adult), and CYP3A7 (fetal). Cellartis Enhanced hiPS-HEP cells express high mRNA and protein levels of CYP3A4 and CYP3A5 and very low levels of CYP3A7.

Are there cellular contaminants in Cellartis Enhanced hiPS-HEP cells?

Yes, <10% of the population 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.

Can Cellartis Enhanced hiPS-HEP cells be used for hepatitis virus research?

We have not looked at the entry receptors for hepatitis C virus in Cellartis Enhanced hiPS-HEP cells. However, in Cellartis hiPS-HEP cells (the previous generation of hepatocytes derived from human induced pluripotent stem cells), CD81 was expressed in over 95% of the cells (as analyzed by FACS in at least three different batches of fresh hiPS-HEP cells). Cellartis Enhanced hiPS-HEP cells express NTCP transporter, an important entry receptor for hepatitis B virus.

Handling FAQs

How are Cellartis Enhanced hiPS-HEP cells shipped?

Cellartis Enhanced hiPS-HEP cells, Cellartis HEP Coat, and Cellartis HEP Supplement are transported frozen in vials on dry ice.

How do I thaw multiple vials of Cellartis Enhanced hiPS-HEP cells at a time?

Scale up the volume of thawing media according to the number of vials (e.g., 80 ml for four vials) and pre-warm in an appropriate tube or bottle. Pour the thawed cell suspensions from all vials into the total volume of thawing media, and mix very carefully by inversion 10 times (do not pipette). To count the cells (optional), take a sample from the total volume of thawing media. Next, distribute the cell suspension into 50 ml centrifuge tubes, adding no more than 40 ml per tube. Incubate for 15–20 min. Centrifuge at 100 x g for 2 min at room temperature using the slowest possible deceleration setting. For each tube, remove the media with a pipette without disturbing the cell pellet. Loosen the cell pellet by flicking, and then resuspend the pellets very carefully by slowly adding pre-warmed plating media (15 ml per thawed vial). Cell suspensions in different tubes can be pooled before seeding by combining into one bottle or tube.

Which culture vessels are suitable for Cellartis Enhanced hiPS-HEP cells?

96-well and 24-well plates are recommended. Other formats can be used, such as 6-well plates, 384-well plates, and T25 flasks.

How do I prepare the Y27632 stock solution?

Prepare a 5 mM Y27632 stock solution by dissolving 10 mg Y27632 in 5.92 ml sterile distilled water. Aliquot in appropriate volumes and store at ≤15°C for up to 12 months. Thawed aliquots can be stored at 2–8°C and should be used within one week.

Why should the cells be washed on day 1 after plating?

Washing with base maintenance medium (not included; contains WME basal medium + antibiotics) on day 1 after plating is necessary to remove non-adherent cells. When washing, carefully remove 100% of the media from only a few wells at a time; do not let the wells dry.

Why should partial media changes be performed from day 3 onward?

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.

Can Cellartis Enhanced hiPS-HEP cells go without changing media over the weekend?

Yes, but add 50% more media on Friday (e.g., 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.

Is it possible to proliferate Cellartis Enhanced hiPS-HEP cells?

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

Which assay is used to measure CYP activity in Cellartis Enhanced hiPS-HEP cells?

LC/MS is used to measure the formation of these specific metabolites: Acetaminophen (CYP1A), 4-OH-Diclofenac (CYP2C9), 4-OH-Mephenytoin (CYP2C19), OH-Bufuralol (CYP2D6) and 1-OH-Midazolam (CYP3A). The CYP activities of Cellartis Enhanced hiPS-HEP cells are analyzed at day 6 and 11 after thawing as part of routine quality control. LC/MS analysis is performed at Pharmacelsus GmbH, Germany.

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

CYP Substrate Concentration
1A Phenacetin 10 µM
2B6 Bupropion 10 µM
2C19 Mephenytoin 50 µM
2C9 Diclofenac 10 µM
2D6 Bufuralol 10 µM
3A Midazolam 5 µM

After 2 hr at 37°C, 100 µl of supernatant is collected and kept at -80°C until LC/MS analysis. The metabolite concentrations measured by LC/MS are normalized to the amount of protein per well (determined using the Pierce BCA Protein Assay Kit) and the assay duration (120 min).

I observe droplets in Cellartis Enhanced hiPS-HEP cell culture. Is this abnormal?

ChIP-Seq products

These liquid extrusions are not abnormal. In time-lapse recordings, we have observed that hepatocytes release these drops into the media and remain healthy afterwards. 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.

I observe dome-like structures in Cellartis Enhanced hiPS-HEP cell culture. Is this abnormal?

ChIP-Seq products

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. Please be careful when changing media by always leaving 10% of the media in each well.

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