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Recellularization of rat liver: An in vitro model for assessing human drug metabolism and liver biology.

ABSTRACT: Liver-like organoids that recapitulate the complex functions of the whole liver by combining cells, scaffolds, and mechanical or chemical cues are becoming important models for studying liver biology and drug metabolism. The advantages of growing cells in three-dimensional constructs include enhanced cell-cell and cell-extracellular matrix interactions and preserved cellular phenotype including, prevention of de-differentiation. In the current study, biomimetic liver constructs were made via perfusion decellularization of rat liver, with the goal of maintaining the native composition and structure of the extracellular matrix. We optimized our decellularization process to produce liver scaffolds in which immunogenic residual DNA was removed but glycosaminoglycans were maintained. When the constructs were recellularized with rat or human liver cells, the cells remained viable, capable of proliferation, and functional for 28 days. Specifically, the cells continued to express cytochrome P450 genes and maintained their ability to metabolize a model drug, midazolam. Microarray analysis showed an upregulation of genes involved in liver regeneration and fibrosis. In conclusion, these liver constructs have the potential to be used as test beds for studying liver biology and drug metabolism.

SUBMITTER: Robertson MJ

PROVIDER: S-EPMC5788381 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Recellularization of rat liver: An in vitro model for assessing human drug metabolism and liver biology.

Robertson Matthew J MJ Soibam Benjamin B O'Leary Jacqueline G JG Sampaio Luiz C LC Taylor Doris A DA

PloS one 20180129 1

Liver-like organoids that recapitulate the complex functions of the whole liver by combining cells, scaffolds, and mechanical or chemical cues are becoming important models for studying liver biology and drug metabolism. The advantages of growing cells in three-dimensional constructs include enhanced cell-cell and cell-extracellular matrix interactions and preserved cellular phenotype including, prevention of de-differentiation. In the current study, biomimetic liver constructs were made via per ...[more]

PMID: 29377912

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Project description:Due to its central role in glucose homeostasis, the liver is an important target for drug development efforts for type 2 diabetes mellitus (T2DM). Significant differences across species in liver metabolism necessitate supplementation of animal data with assays designed to assess human-relevant responses. However, isolated primary human hepatocytes (PHHs) display a rapid decline in phenotypic functions in conventional monolayer formats. Cocultivation of PHHs with specific stromal cells, especially in micropatterned configurations, can stabilize some liver functions for ~4 weeks in vitro. However, it remains unclear whether coculture approaches can stabilize glucose metabolism that can be modulated with hormones in PHHs. Thus, in this study, we compared commonly employed conventional culture formats and previously developed micropatterned cocultures (MPCCs) of cryopreserved PHHs and stromal fibroblasts for mRNA expression of key glucose metabolism genes (i.e., phosphoenolpyruvate carboxykinase-1 [PCK1]) and sensitivity of gluconeogenesis to prototypical hormones, insulin and glucagon. We found that only MPCCs displayed high expression of all transcripts tested for at least 2 weeks and robust gluconeogenesis with responsiveness to hormones for at least 3 weeks in vitro. Furthermore, MPCCs displayed glycogen storage and lysis, which could be modulated with hormones under the appropriate feeding and fasting states, respectively. Finally, we utilized MPCCs in proof-of-concept experiments where we tested gluconeogenesis inhibitors and evaluated the effects of stimulation with high levels of glucose as in T2DM. Gluconeogenesis in MPCCs was decreased after stimulation with drugs (i.e., metformin) and the PHHs accumulated significant amount of lipids following incubation with excess glucose (i.e., 340% in 50 mM glucose relative to physiologic 5 mM glucose controls). In conclusion, MPCCs provide a platform to study glucose metabolism and hormonal responsiveness in cryopreserved PHHs from multiple donors for several weeks in vitro. This model is also useful to study the effects of drugs and overnutrition for applications in T2DM.

Project description:BackgroundMany patients suffering from peripheral arterial disease (PAD) are dependent on bypass surgery. However, in some patients no suitable replacements (i.e. autologous or prosthetic bypass grafts) are available. Advances have been made to develop autologous tissue engineered vascular grafts (TEVG) using endothelial colony forming cells (ECFC) obtained by peripheral blood draw in large animal trials. Clinical translation of this technique, however, still requires additional data for usability of isolated ECFC from high cardiovascular risk patients. Bovine carotid arteries (BCA) were decellularized using a combined SDS (sodium dodecyl sulfate) -free mechanical-osmotic-enzymatic-detergent approach to show the feasibility of xenogenous vessel decellularization. Decellularized BCA chips were seeded with human ECFC, isolated from a high cardiovascular risk patient group, suffering from diabetes, hypertension and/or chronic renal failure. ECFC were cultured alone or in coculture with rat or human mesenchymal stromal cells (rMSC/hMSC). Decellularized BCA chips were evaluated for biochemical, histological and mechanical properties. Successful isolation of ECFC and recellularization capabilities were analyzed by histology.ResultsDecellularized BCA showed retained extracellular matrix (ECM) composition and mechanical properties upon cell removal. Isolation of ECFC from the intended target group was successfully performed (80% isolation efficiency). Isolated cells showed a typical ECFC-phenotype. Upon recellularization, co-seeding of patient-isolated ECFC with rMSC/hMSC and further incubation was successful for 14 (n = 9) and 23 (n = 5) days. Reendothelialization (rMSC) and partial reendothelialization (hMSC) was achieved. Seeded cells were CD31 and vWF positive, however, human cells were detectable for up to 14 days in xenogenic cell-culture only. Seeding of ECFC without rMSC was not successful.ConclusionUsing our refined decellularization process we generated easily obtainable TEVG with retained ECM- and mechanical quality, serving as a platform to develop small-diameter (< 6 mm) TEVG. ECFC isolation from the cardiovascular risk target group is possible and sufficient. Survival of diabetic ECFC appears to be highly dependent on perivascular support by rMSC/hMSC under static conditions. ECFC survival was limited to 14 days post seeding.

Dataset Information

Recellularization of rat liver: An in vitro model for assessing human drug metabolism and liver biology.

Publications

Recellularization of rat liver: An in vitro model for assessing human drug metabolism and liver biology.

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