5 Important Cell Culture Rules

Description

Hepatocytes are essential for critical processes like metabolism, detoxification, protein synthesis, and innate immunity. A robust cellular model is required to study these hepatocyte processes, as well as liver development, toxicity, disease and more. However, current models have significant limitations.

Primary hepatocytes are inconvenient to isolate, do not tolerate cryopreservation or proliferate in culture, and senesce rapidly. Alternatively, cell lines like HepG2 can divide in vitro, but are cancer cells and do not display normal hepatocyte function. After about 1 week in standard cell culture format, cell lines like HepG2 becomes overgrown with necrotic colonies, and are no longer suitable for experiments. Furthermore, the majority of current hepatocyte models are 2-dimensional, a format that is unlikely to mimic the 3-dimensional niche that hepatocytes occupy in the body.

To address these liver research needs, Cell Applications now offers a breakthrough, first-of-its kind hepatocyte system. Using our DISCUS™ Technology* to generate true three-dimensional tissue constructs, Cell Applications iPSC-derived Human Hepatocytes (i-HH) recapitulate hepatic development, generating 3D organoids with polarized structures capable of multiweek culture. The i-HH are well-characterized, and are demonstrated to express xenobiotic metabolizing enzymes, and demonstrate other key liver specific functions, including albumin secretion and ammonia detoxification.

 i-HH expresses liver markers HNF4A and ASPGR1, which are characteristic of functioning hepatocytes.1 Above Left: 3D i-HH organoid stained with HNF4A (red) and ASPGR1 (green) taken with a standard fluorescent microscope. The in-focus plane demonstrates a clear nuclear HNF4A signal on the apical exterior and basolateral ASPGR1 interior. Above Right: The 3D i-HH derived from two separate cell lines (1709 and 1439) have up to 87% of the cells express double positive HNF4A/ASPGR1 markers for up to four weeks.

i-HH demonstrates extended album secretion, urea production, and CYP3A4 activity, outperforming HepG2 in the DISCUS™ 3D system. Above graphs: i-HH is comparable in Albumin secretion (4-20 ug/mL)2, Urea production (48-255 nmol)3, and CYP3A4 activity (0.3-3.60E6 RFU)4 to primary human hepatocytes. HepG2 cells embedded in the DISCUS system as a 3D construct (HepG2-3D) performed markedly better compared to adherent HepG2 cell cultures. Adherent HepG2-2D cell cultures could only be cultured for one week before becoming overgrown.

The Cell Applications i-HH DISCUS total kit includes an all-in-one package that includes cryopreserved i-HH (iPSC-derived Human Hepatocytes), media, and DISCUS reagents that allow for the culture of the cells in a 3-D culture.

*DISCUS™ is a cell-specific system of 3D discs that address the challenges of anoxia and diffusion limits in 3D tissue engineering. Measuring 3mm across and only 600 microns in depth at the thickest point, these versatile units need only common, inexpensive laboratory equipment to generate your 3D engineered tissue of choice.

1.    Peters, et al. Development (2016) 1475-1481
2.    Lubberstedt, et al. J Pharm Tox Methods (2011) 59-63
3.    Donato, et al. Cell Transplantation (2008) 1211-1219
4.    Gramignoli, et al. Cell Tranplantation (2014) 1545-1556

 

i-HH in 3D culture are self-organizing epithelial structures that display apical-basolateral polartity.

Figure 1: i-HH in 3D culture:

A) 20x (amalgam). B) 4x.

C) HNF4A (apical) and DAPI staining.

D) HNF4A (apical) and ASGPR1 (basolateral) staining.

Details

SourceDerived from Human iPSC of a single donor
QCNo bacteria, yeast, fungi, mycoplasma, virus
CryovialApproxiately 500,000 equivalents of i-HH (2nd passage) in freezing medium
MediumCells maintained in serum-free, feeder-free culture conditions
StorageLiquid Nitrogen
BioassayAlbumin secretion, Urea production, CY3A4 activity
ApplicationsLaboratory research use only (RUO). Not for human, clinical, diagnostic or veterinary use.

 

 

Products

Product Size CAT.# Price Quantity
Cryopreserved Human Hepatocytes Total Kit, iPSC-derived: Cells, Medium & Reagents (See Details tab for specifics) Size: 1 Kit CAT.#: i780K-05 Price: $1,394.00
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Cryopreserved Human Hepatocytes, iPSC-derived: Frozen Human iPSC-Derived Human Hepatocytes (5x10^5) Size: 1 Cryovial CAT.#: i780-05 Price: $998.00
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Related Products

Product Size CAT.# Price Quantity
i-HH Recovery Medium Kit: Basal medium & supplements sold together packaged separately. Combine all for complete medium. Size: Yields 30 ml CAT.#: i71103K-30 Price: $78.00
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i-HH Maturation Medium Kit: Basal medium & supplements sold together packaged separately. Combine all for complete medium.   Size: Yields 100 ml CAT.#: i71105K-100 Price: $118.00
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i-HH Metabolic Medium Kit: Basal medium & supplements sold together packaged separately. Combine all for complete medium.   Size: Yields 30 ml CAT.#: i71107K-30 Price: $78.00
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i-HH DISCUS 3D Gel Kit: Cell Applications 3D system for iPSC-derived human hepatocytes. Size: 1 Kit CAT.#: DISC1053K Price: $68.00
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DISCUS Dissolution Reagent: Reagent for DISCUS dissolution. Size: 10 ml CAT.#: DISC1050 Price: $32.00
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DISCUS Non-Stick Solution: Reagent for cell culture. Size: 5 ml CAT.#: 1025-05 Price: $32.00
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Resources/Documents

5 Important Cell Culture Rules

Format: PDF

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