Khetani, S. | bioRxiv Science

bioRxiv2019

Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in 3-Dimensional Models

Human liver models that are 3-dimensional (3D) in architecture are proving to be indispensable for diverse applications, including compound metabolism and toxicity screening during preclinical drug development, to model human liver diseases for the discovery of novel therapeutics, and for cell-based therapies in the clinic; however, further development of such models is needed to maintain high levels of primary human hepatocyte (PHH) functions for weeks to months in vitro. Therefore, here we determined how microscale 3D collagen-I presentation and fibroblast interaction could affect the long-term functions of PHHs. High-throughput droplet microfluidics was utilized to rapidly generate reproducibly-sized (~300 m diameter) microtissues containing PHHs encapsulated in collagen-I +/- supportive fibroblasts, namely 3T3-J2 murine embryonic fibroblasts or primary human hepatic stellate cells (HSCs); self-assembled spheroids and bulk collagen gels (macrogels) containing PHHs served as gold-standard controls. Hepatic functions (e.g. albumin and cytochrome-P450 or CYP activities) and gene expression were subsequently measured for up to 6 weeks. We found that collagen-based 3D microtissues rescued PHH functions within static multi-well plates at 2- to 30-fold higher levels than self-assembled spheroids or macrogels. Further coating of PHH microtissues with 3T3-J2s led to higher hepatic functions than when the two cell types were either coencapsulated together or when HSCs were used for the coating instead. Additionally, the 3T3-J2-coated PHH microtissues displayed 6+ weeks of relatively stable hepatic gene expression and function at levels similar to freshly thawed PHHs. Lastly, microtissues responded in a clinically-relevant manner to drug-mediated CYP induction or hepatotoxicity. In conclusion, fibroblast-coated collagen microtissues containing PHHs display hepatic functions for 6+ weeks without any fluid perfusion at higher levels than spheroids and macrogels, and such microtissues can be used to assess drug-mediated CYP induction and hepatotoxicity. Ultimately, microtissues may find broader utility for modeling liver diseases and as building blocks for cell-based therapies.

bioengineering↗

bioRxiv2019

Intermittent Starvation Extends the Functional Lifetime of Primary Human Hepatocyte Cultures

Primary human hepatocyte (PHH) cultures have become indispensable to mitigate the risk of adverse drug reactions in human patients. In contrast to de-differentiating monocultures, co-culture with non-parenchymal cells maintains PHH functions for 2-4 weeks. However, since the functional lifespan of PHHs in vivo is 200-400 days, it is desirable to further prolong PHH functions in vitro towards modeling chronic drug exposure and disease progression. Fasting has benefits on the longevity of organisms and the health of tissues such as the liver. We hypothesized that a culturing protocol that mimics dynamic fasting/starvation could activate starvation pathways and prolong PHH functional lifetime. To mimic starvation, serum and hormones were intermittently removed from the culture medium of micropatterned co-cultures (MPCC) containing PHHs organized onto collagen domains and surrounded by 3T3-J2 murine fibroblasts. A weekly 2-day starvation optimally prolonged PHH functional lifetime for 6+ weeks in MPCCs versus a decline after 3 weeks in non-starved controls. The 2-day starvation also enhanced the functions of PHH monocultures for 2 weeks, suggesting direct effects on PHHs. In MPCCs, starvation activated adenosine monophosphate activated protein kinase and restricted fibroblast overgrowth onto PHH islands, thereby maintaining hepatic polarity. The effects of starvation on MPCCs were partially recapitulated by activating adenosine monophosphate activated protein kinase using metformin or growth-arresting fibroblasts via mitomycin-C. Lastly, starved MPCCs demonstrated lower false positives for drug toxicity tests and higher drug-induced cytochrome-P450 activities versus non-starved controls even after 5 weeks. In conclusion, intermittent serum/hormone starvation extends PHH functional lifetime towards enabling clinically-relevant drug screening.

cell biology↗