Bioprinted liver provides early insight into the role of Kupffer cells in TGF-β1 and methotrexate-induced fibrogenesis

Abstract

Hepatic fibrosis develops from a series of complex interactions among resident and recruited cells making it a challenge to replicate using standard in vitro approaches. While studies have demonstrated the importance of macrophages in fibrogenesis, the role of Kupffer cells (KCs) in modulating the initial response remains elusive. Previous work demonstrated utility of 3D bioprinted liver to recapitulate basic fibrogenic features following treatment with fibrosis-associated agents. In the present study, culture conditions were modified to recapitulate a gradual accumulation of collagen within the tissues over an extended exposure timeframe. Under these conditions, KCs were added to the model to examine their impact on the injury/fibrogenic response following cytokine and drug stimuli. A 28-day exposure to 10 ng/mL TGF-β1 and 0.209 μM methotrexate (MTX) resulted in sustained LDH release which was attenuated when KCs were incorporated in the model. Assessment of miR-122 confirmed early hepatocyte injury in response to TGF-β1 that appeared delayed in the presence of KCs, whereas MTX-induced increases in miR-122 were observed when KCs were incorporated in the model. Although the collagen responses were mild under the conditions tested to mimic early fibrotic injury, a global reduction in cytokines was observed in the KC-modified tissue model following treatment. Furthermore, gene expression profiling suggests KCs have a significant impact on baseline tissue function over time and an important modulatory role dependent on the context of injury. Although the number of differentially expressed genes across treatments was comparable, pathway enrichment suggests distinct, KC- and time-dependent changes in the transcriptome for each agent. As such, the incorporation of KCs and impact on baseline tissue homeostasis may be important in recapitulating temporal dynamics of the fibrogenic response to different agents.

3D Primary Hepatocyte Culture Systems for Analyses of Liver Diseases, Drug Metabolism, and Toxicity: Emerging Culture Paradigms and Applications

“…Furthermore, Organovo developed a bioprinting platform, in which, PHH, stellate cells, and KCs are bioprinted using nondisclosed bioink, where the cells showed five to ten times increased viability and albumin secretion compared to 2D culture after two weeks . Furthermore, the authors recently demonstrated that the model can recapitulate aspects of methotrexate‐induced fibrogenesis …”

A Microphysiological System for Studying Nonalcoholic Steatohepatitis

“…(17) Therefore, to date, most in vitro NAFLD studies involving PHHs have focused on short-term exposure (48-72 hours) to free fatty acids (FFAs), allowing the study of transient responses to triglyceride challenge. (14)(15)(16) Some studies using advanced in vitro platforms, including micropatterned co-cultures of primary hepatocytes and murine fibroblasts, (18) a hemodynamic co-culture system (19) or bioprinted cultures of primary liver cells, (20) have started to demonstrate how exposure to glucose and FFAs can affect human hepatic cell types. Moreover, with the addition of transforming growth factor β (TGF-β), early stages of fibrosis can be detected.…”

“…Moreover, with the addition of transforming growth factor β (TGF-β), early stages of fibrosis can be detected. (20) We previously developed a model of hepatic steatosis using a 3D perfused microphysiological system (MPS), which enabled PHHs to be cultured for 2 weeks in the presence of FFAs, allowing the chronic effects of triglyceride accumulation to be analyzed. (21) The perfused MPS maintains highly metabolically active PHHs for extended periods (up to 40 days) (22)(23)(24) and has been demonstrated to support PHHs and human Kupffer cell (HK) co-cultures to study the effects of liver inflammation on drug metabolism, drug-drug interactions, and liver toxicity.…”

Liver Bioreactor Design Issues of Fluid Flow and Zonation, Fibrosis, and Mechanics: A Computational Perspective

“…These were cultured for several days and allowed to coalesce into liver tissue for studies of drug-induced toxicity. This same technique was also applied to the study of various compound-induced fibroses [93] and further explored by focusing on the role of Kupffer cells [94]. Ma et al [95] developed a 3D hydrogel-based hepatic bioprinting system using human iPSC stem cells to explore the liver tissue maturation process.…”