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Efficient One-Step Production of Microencapsulated Hepatocyte Spheroids with Enhanced Functions.

ABSTRACT: Hepatocyte spheroids microencapsulated in hydrogels can contribute to liver research in various capacities. The conventional approach of microencapsulating spheroids produces a variable number of spheroids per microgel and requires an extra step of spheroid loading into the gel. Here, a microfluidics technology bypassing the step of spheroid loading and controlling the spheroid characteristics is reported. Double-emulsion droplets are used to generate microencapsulated homotypic or heterotypic hepatocyte spheroids (all as single spheroids <200 ?m in diameter) with enhanced functions in 4 h. The composition of the microgel is tunable as demonstrated by improved hepatocyte functions during 24 d culture (albumin secretion, urea secretion, and cytochrome P450 activity) when alginate-collagen composite hydrogel is used instead of alginate. Hepatocyte spheroids in alginate-collagen also perform better than hepatocytes cultured in collagen-sandwich configuration. Moreover, hepatocyte functions are significantly enhanced when hepatocytes and endothelial progenitor cells (used as a novel supporting cell source) are co-cultured to form composite spheroids at an optimal ratio of 5:1, which could be further boosted when encapsulated in alginate-collagen. This microencapsulated-spheroid formation technology with high yield, versatility, and uniformity is envisioned to be an enabling technology for liver tissue engineering as well as biomanufacturing.

SUBMITTER: Chan HF

PROVIDER: S-EPMC4982767 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Efficient One-Step Production of Microencapsulated Hepatocyte Spheroids with Enhanced Functions.

Chan Hon Fai HF Zhang Ying Y Leong Kam W KW

Small (Weinheim an der Bergstrasse, Germany) 20160401 20

Hepatocyte spheroids microencapsulated in hydrogels can contribute to liver research in various capacities. The conventional approach of microencapsulating spheroids produces a variable number of spheroids per microgel and requires an extra step of spheroid loading into the gel. Here, a microfluidics technology bypassing the step of spheroid loading and controlling the spheroid characteristics is reported. Double-emulsion droplets are used to generate microencapsulated homotypic or heterotypic h ...[more]

PMID: 27038291

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Project description:Candida parapsilosis CCTCC M203011 catalyzes the stereoinversion of (R)-1-phenyl-1,2-ethanediol (PED) through oxidation and reduction. Its NAD(+)-linked (R)-carbonyl reductase (RCR) catalyzes the oxidization of (R)-PED to 2-hydroxyacetophenone (HAP), and its NADPH-dependent (S)-carbonyl reductase (SCR) catalyzes the reduction of HAP to (S)-PED. The reactions require NAD(+) and NADPH as cofactors. However, even if NAD(+) and NADPH are added, the biotransformation of (S)-PED from the (R)-enantiomer by an Escherichia coli strain co-expressing RCR and SCR is slow and gives low yields, probably as a result of insufficient or imbalanced redox cofactors. To prepare (S)-PED from the (R)-enantiomer in one-step efficiently, plus redox cofactor regeneration, we introduced pyridine nucleotide transhydrogenases (PNTs) from E. coli to the metabolic pathway of (S)-PED.The PNTs were successfully introduced into the E. coli strain RSAB. Most of the PNT activities occurred in the cell membrane of E. coli. The introduction of PNTs increased intracellular NAD(+) and NADH concentrations and decreased the NADPH pool without affecting the total nucleotide concentration and cell growth properties. The presence of PNTs increased the NADH/NAD(+) ratio slightly and reduced the NADPH/NADP(+) ratio about two-fold; the ratio of NADPH/NADP(+) to NADH/NAD(+) was reduced from 36 to 17. So, the PNTs rebalanced the cofactor pathways: the rate of RCR was increased, while the rate of SCR was decreased. When the ratio of NAD(+)/NADPH was 3.0 or higher, the RSAB strain produced (S)-PED with the highest optical purity, 97.4%, and a yield of 95.2% at 6 h. The introduction of PNTs stimulated increases of 51.5% and 80.6%, respectively, in optical purity and yield, and simultaneously reduced the reaction time seven-fold.In this work, PNTs were introduced into E. coli to rebalance the cofactor pools within the engineered (S)-PED pathways. The efficient one-step production of (S)-PED plus NAD(+)-NADPH in-situ regeneration was realized. This work provided new insights into cofactor rebalancing pathways, using metabolic engineering methods, for efficient chiral alcohol production.

Dataset Information

Efficient One-Step Production of Microencapsulated Hepatocyte Spheroids with Enhanced Functions.

Publications

Efficient One-Step Production of Microencapsulated Hepatocyte Spheroids with Enhanced Functions.

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