Controlled Apoptosis of Stromal Cells to Engineer Human Microlivers

Abstract

Engineered tissue models comprise a variety of multiplexed ensembles in which combinations of epithelial, stromal, and immune cells give rise to physiologic functions. Engineering spatiotemporal control of cell-cell and cell-matrix interactions within these 3D multicellular tissues would represent a significant advance for tissue engineering. In this work, a new method, entitled CAMEO (Controlled Apoptosis in Multicellular tissues for Engineered Organogenesis) enables the noninvasive triggering of controlled apoptosis to eliminate genetically engineered cells from a pre-established culture. Using this approach, the contribution of stromal cells to the phenotypic stability of primary human hepatocytes is examined. 3D hepatic microtissues, in which fibroblasts can enhance phenotypic stability and accelerate aggregation into spheroids, are found to rely only transiently on fibroblast interaction to support multiple axes of liver function, such as protein secretion and drug detoxification. Due to its modularity, CAMEO has the promise to be readily extendable to other applications that are tied to the complexity of 3D tissue biology, from understanding in vitro organoid models to building artificial tissue grafts.

Gene signatures and host-parasite interactions revealed by dual single-cell profiling of Plasmodium vivax liver infection

“…Primary human hepatocytes were seeded on collagen-micropatterned 96-well plates and surrounded with murine embryonic fibroblasts 3T3-J2s as detailed previously (March et al, 2015). For the scRNA-seq analysis, MPCCs were established using 3T3-J2s expressing an inducible apoptosis switch (Chen et al, 2020). MPCCs were infected with fresh sporozoites obtained through dissection of P. vivax -infected mosquitoes.…”

Ebola virus infection induces a type I interferon response and the shutdown of key liver functions in human iPSC-derived hepatocytes

“…Fibroblast-depleted micropatterned PHH cultures were obtained by incorporation of 3T3-J2s expressing an inducible apoptosis switch (inducible caspase-9, Casp9). 3T3-J2s were lentivirally transduced with the pMSCV-F-del Casp9.IRES.GFP plasmid (15567, Addgene) followed by FACS puri cation (top 15%, FACSAria II, BD Biosciences) (47). All EBOV infection studies were performed with puri ed virions.…”

Engineering Modular 3D Liver Culture Microenvironments In Vitro to Parse the Interplay between Biophysical and Biochemical Microenvironment Cues on Hepatic Phenotypes

“…We adapted an alginate microwell system based on previous strategies that allowed the generation of hundreds to thousands of uniform spheroids but modifying the mold manufacturing method and design to accommodate oxygen-sensitive hepatic spheroids (Figure 1b). [36,37,47] To accommodate a target spheroid size of %100-150 μm diameter (%200-500 cells), comparable to other studies, [48][49][50] we designed Figure 1. Engineering 3D hepatic liver models using a perfused bioreactor and 3D-printing based alginate microwells.…”

“…Other literature reports employ cell numbers in the 200-1500 per spheroid range. [30,50] Hepatocytes can contain thousands of mitochondria per cell, and inadequate oxygenation drastically influences viability and functionality. [52][53][54] Models of optimal spheroid size have estimated that oxygen diffusion limitations typically arise when spheroids reach 200-300 μm.…”

“…We compared our alginate microwell system to three commercially available spheroid generation platforms that have been previously used for primary hepatic spheroids: Aggrewell 400, Aggrewell 800, and round-bottom 96-well low attachment plates. [30,50,63,64] Following protocols previously published for these other platforms, we seeded 1500 cells per spheroid for the 96-well plate format and 120 000 cells total for the alginate microwell arrays and Aggrewell plates (all in 24-well format), yielding cell/medium ratios of %7.5-15 cells μL À1 for typical 96-well plate spheroids [30] compared to 300-400 cells μL À1 for the 24-well plate microwells and 375 cells μL À1 for the LiverChip. [23] Given the order of magnitude differences, it was more accessible to use the microwell array system as a comparison to LiverChip or other mesoscale 3D bioreactor metrics.…”