A novel maintenance medium for the long-term culture of human primary hepatocytes

Extend the usability of primary hepatocyte cultures for drug metabolism and disease modeling studies— without complicated culture methods.

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Jan 08, 2018
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Challenges in culturing primary hepatocytes

The liver is a vital organ, consisting of mostly hepatocytes, which performs over 500 functions including protein synthesis and detoxification of various metabolites. The functions carried out by a given hepatocyte are largely driven by the location of the cell within the liver. Periportal hepatocytes, for example, are largely responsible for gluconeogenesis, while drug metabolism occurs mostly within perivenous hepatocytes [1]. In order to be used as a predictive model for studying liver disease or the metabolic activity of drugs, in vitro primary liver cell culture models should not only perform both periportal and perivenous functions, but the cultured hepatocytes need to maintain these functionalities for short-term metabolism and long-term toxicity analyses [2].

For in vitro primary liver cell models, the gold standard for the evaluation of drug metabolism, drug-drug interactions, safety assessment of drug candidates, and disease modeling are human primary hepatocytes (hphep cells). However, a significant limitation of human primary hepatocytes is their rapid loss of function when cultured in vitro [3]. This fundamental limitation restricts the applications for which the cells can be used. To address this problem, 3D sandwich cultures with matrix overlays [4], bioreactor-based cultures [5], and 3D spheroid cultures [6] have been developed. Although these approaches can maintain some hepatocyte functions for several weeks in vitro, they do not entirely overcome the limitations of human primary hepatocytes because these culture systems restrict the types of assays that can be performed. Further, they require advanced and expensive lab equipment, are not easy to use, or are not generally applicable to primary cells from different donors.

To enable long-term cultures of primary hepatocytes in user-friendly 2D-culture formats, we developed Cellartis® Power™ Primary HEP Medium (Power HEP medium), a new medium that maintains healthy, functional human primary hepatocytes for up to four weeks in conventional 2D cultures—without the need for overlays or sandwich cultures—overcoming a key limitation of human primary hepatocytes. Furthermore, the recommended culture schedule is weekend free, requiring media changes only on Mondays, Wednesdays, and Fridays.

Maintained viability and morphology

The first basic requirement for successful long-term culture is maintained cell viability. Measurements of ATP content of the primary hepatocytes cultured in Power HEP medium showed that the cells were viable for four weeks. Furthermore, hphep cells from several donors, cultured in Power HEP medium, maintained typical hepatocyte morphology for 28 days in culture—a strong indicator of the terminal differentiation status.

Preserved periportal function—albumin secretion

In the liver, the tissue region located upstream of the terminal portal vein and hepatic arteriole is known as the periportal zone. According to the model of metabolic zonation, one of the predominant functions of periportal hepatocytes is albumin secretion [7]. Albumin is the main protein of human blood plasma, and albumin secretion is a commonly used characteristic to evaluate the quality of in vitro hepatocyte models, particularly for periportal hepatocytes. 

Overall, we show that not only was albumin already secreted after one day in culture, but secretion was also stably maintained throughout the 28-day culture period (Panel A). Interestingly, we also observed that while total albumin secretion stayed stable for all 28 days, total protein content increased from Day 1 to Day 7 (Panel B); therefore, once albumin was normalized to protein content (Panel C), we saw an initial drop in the normalized albumin content before it stabilized to a constant level that was maintained out to 28 days. We hypothesize that the initial rise of protein content correlates with a recovery period after thawing and plating.


Preserved perivenous function—CYP activity

Another major function of hepatocytes is the expression and activity of drug metabolizing CYP enzymes, which are important for the detoxification of drug compounds. This function occurs in hepatocytes from the perivenous zone and is often used to evaluate the functionality of in vitro hepatocyte models.

To investigate how the performance of hphep cells cultured in Power HEP medium compares to other commercially available hepatocyte maintenance media, we cultured primary hepatocytes from two different donors either in Power HEP medium or in hepatocyte media from three other vendors. The hepatocytes cultured in Power HEP medium showed sustained CYP activities for 28 days (Panel A), which is in sharp contrast to the rapid loss of activity in hphep cells cultured in other commercially available media (Panels B, C, and D). The loss of CYP function indicates that those media were unable to maintain cell viability past a week (data not shown). Interestingly, when we observed the behavior of different donors in Power HEP medium, CYP activity levels were found to be donor-dependent, in agreement with the interindividual variation found in the general population.


Preserved CYP induction

Drugs can increase CYP enzyme levels by inducing their mRNA expression, which can cause a change in the effects of coadministered drugs, leading to serious problems for patients taking multiple medications. Consequently, the assessment of potential drug-drug interactions is important in drug development, and hphep cells are the gold standard for assessing potential CYP inductions by a drug candidate.

To evaluate whether this important hepatocyte feature is preserved in hphep cells cultured with Power HEP medium, we exposed hphep cells to typical inducers (Table I) for 48 hr after they had reached 26 days in culture. We then analyzed CYP1A2 (Panel A)CYP2B6 (Panel B)CYP3A4 (Panel C), and CYP2C9 (Panel D) mRNA expression on Day 28 and compared them to levels in untreated cells. Even after the extended time in culture prior to induction, all four CYPs tested were induced at high levels comparable to the fold induction shown for primary hepatocytes induced earlier, at 2–4 days post-thawing [8]. The only exception to the pattern of high levels of CYP inducibility across donors is that CYP2C9 induction by Rif was observed in only 2 out of 3 donors (Panel D); however, this is consistent with reports that CYP2C9 is not inducible in some donors [8].

 Table I. Overview of CYP enzymes induced by three typical inducers.

CYP

Induced by

1A2

Omeprazole (Ome)

2B6

Phenobarbital (PB), Rifampicin (Rif)

3A4

Phenobarbital (PB), Rifampicin (Rif)

2C9

Phenobarbital (PB), Rifampicin (Rif)


More information

Cellartis Power Primary HEP Medium changes the game for drug metabolism and disease modeling studies by enabling maintenance of viable and functional hepatocytes for four weeks in conventional 2D cultures. Key hepatocyte functions such as albumin secretion, CYP activities, and CYP inducibility are maintained for the four-week culture period. Notably, both periportal and perivenous features, typical for the different hepatocyte phenotypes present in the different zones of the liver lobe, are preserved. The extended lifespan of primary hepatocytes cultured with Power HEP medium in user-friendly 2D cultures and with a weekend-free feeding schedule significantly enhances the utility of primary hepatocytes in multiple applications, such as drug discovery and safety toxicology studies.

To view the complete data set for the findings presented here, please read our detailed technical note.

References

1.Colnot, S. & Perret, C. Molecular Pathology of Liver Diseases. Springer, Boston, MA. 2011b. 5, 7–16 (2011). doi:10.1007/978-1-4419-7107-4_2   

2.Zeilinger, K., Freyer, N., Damm, G., Seehofer, D. & Knöspel, F. Cell sources for in vitro human liver cell culture models. Exp. Biol. Med. (Maywood). 241, 1684–98 (2016).           

3.Richert, L., Liquori, M.J., Abadie, C.et al. Gene expression in human hepatocytes in suspension after isolation is similar to the liver of origin, is not affected by hepatocyte cold storage and cryopreservation, but is strongly changed after hepatocyte plating. Drug Metab. Dispos. 34, 870–9 (2006).

4.Liu, X., Chism, J. P., LeCluyse, E. L., Brouwer, K. R. & Brouwer, K. L. Correlation of biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats. Drug Metab. Dispos. 27, 637–44 (1999).

5.Hoffmann, S. A., Müller-Vierira, U., Biemel, K. et al. Analysis of drug metabolism activities in a miniaturized liver cell bioreactor for use in pharmacological studies. Biotechnol. Bioeng. 109, 3172–81 (2012).

6.Proctor, W. R., Foster, A.J., Vogt, J. et al. Utility of spherical human liver microtissues for prediction of clinical drug-induced liver injury. Arch. Toxicol. 91, 2849–2863 (2017).

7.Jungermann, K. & Kietzmann, T. Oxygen: modulator of metabolic zonation and disease of the liver. Hepatology 31, 255–60 (2000).

8.Yajima, K., Uno, Y., Murayama, N. et al. Evaluation of 23 lots of commercially available cryopreserved hepatocytes for induction assays of human cytochromes P450. Drug Metab. Dispos. 42, 867–71 (2014)

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Takara Bio USA, Inc

Takara Bio USA, Inc. provides kits, reagents, and services that help researchers explore questions about gene discovery, regulation, and function. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

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