High‐throughput production of liver parenchymal microtissues and enrichment of organ‐specific functions in gelatin methacrylamide microenvironment

• Published in: Biotechnology and bioengineering Vol. 119; no. 3; pp. 1018 - 1032
• Main Authors: Roopesh, Ramesh Pai, Muthusamy, Senthilkumar, Velayudhan, Shiny, Sabareeswaran, Arumugham, Anil Kumar, Pallickaveedu RajanAsari
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01-03-2022
• Subjects: Albumins; bioprinting; Cell culture; Cell size; Cell viability; Chambers; Cytochrome P450; Cytochromes P450; Droplets; Drug metabolism; Drug screening; Gelatin; gelatin methacrylamide; hanging‐drop culture; Hepatocytes; Hydrogels; Insulin; Liver; Mass production; Methacrylamide; Microenvironments; microtissues; Multidrug resistance; Polarity; Polyethylene; Polyethylene terephthalate; sandwich culture; Structural integrity; Structure-function relationships; Substrates; Three dimensional printing; Tissue engineering; Urea; Zonula occludens-1 protein; sandwich culture; hanging-drop culture; gelatin methacrylamide; bioprinting; microtissues
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.28010

Liver parenchymal microtissues (LPMTs) are three‐dimensional (3D) aggregates of hepatocytes that recapitulate in vivo‐like cellular assembly. They are considered as a valuable model to study drug metabolism, disease biology, and serve as ideal building blocks for liver tissue engineering. However, their integration into the mainstream drug screening process has been hindered due to the lack of simple, rapid techniques to produce a large number of uniform microtissues and preserve their structural–functional integrity over the long term. Here, we present a high‐throughput methodology to produce LPMTs in a novel, economic, and reusable Hanging‐drop Culture Chamber (HdCC). A drop‐on‐demand bioprinting approach was optimized to generate droplets of HepG2 cell suspension on a polyethylene terephthalate substrate. The substrates carrying droplets were placed inside a novel HdCC and incubated to obtain 1600 LPMTs having a size of 200–300 μm. Tissue size, cell viability, cellular arrangement and polarity, and insulin‐mediated glucose uptake by LPMTs were analyzed. The microtissues were viable and exhibited an active response to insulin stimulation. Cells within the microtissue reorganized to form hepatic plate‐like structures and expressed apical (Multidrug Resistance Protein 2 [MRP2]) and epithelial (Zonula Occludens 1 [ZO1]) markers. Further to maintain the structural integrity and enhance the functional capabilities, LPMTs were sandwiched within gelatin methacrylamide (GelMA) hydrogel and the liver‐specific functions were monitored for 2 weeks. The results showed that the 3D structure of LPMTs in GelMA sandwich was maintained while the albumin secretion, urea synthesis, and cytochrome P450 activity were enhanced compared with LPMTs in suspension. In conclusion, this study presents a novel culture chamber for mass production of microtissues and a method for enhancing organ‐specific functions of LPMTs in vitro.
A novel, cost‐effective hanging‐drop culture chamber (HdCC) device for high‐throughput microtissue production was developed. Droplets of HepG2 cell suspension generated by microvalve bioprinting technology, formed viable, polarized, and histomimicking liver parenchymal microtissues (LPMT) inside the device. The liver‐specific functions of LPMTs were improved by placing them in a sandwich culture with gelatin methacrylamide (GelMA) hydrogel. The proposed HdCC device, as well as the GelMA sandwich culture model, will be immensely useful in liver tissue engineering and drug testing.