Single-cell transcriptomics elucidates in vitro reprogramming of human intestinal epithelium cultured in a physiodynamic gut-on-a-chip

Abstract

The microphysiological human gut-on-a-chip has demonstrated in vivo-relevant cellular fidelity of intestinal epithelium compared to its cultures in a static condition. Microfluidic control of morphogen gradients and mechanical cues robustly induced morphological histogenesis with villi-like three-dimensional (3D) microarchitecture, lineage-associated cytodifferentiation, and physiological functions of a human intestinal Caco-2 epithelium. However, transcriptomic dynamics that orchestrates morphological and functional reprogramming of the epithelium in a microphysiological culture remains elusive. Single-cell transcriptomic analysis revealed that a gut-on-a-chip culture that offers physiological motions and flow drives three distinctive subclusters that offer distinct gene expression and unique spatial representation in 3D epithelial layers. The pseudotemporal trajectory of individual cells visualized the evolutionary transition from ancestral genotypes in static cultures into more heterogeneous phenotypes in physiodynamic cultures on cell cycles, differentiation, and intestinal functions including digestion, absorption, drug transport, and metabolism of xenobiotics. Furthermore, the inversed transcriptomic signature of oncogenes and tumor-suppressor genes of Caco-2 cells verified that a gut-on-a-chip culture drives a postmitotic reprogramming of cancer-associated phenotypes. Thus, we discovered that a physiodynamic on-chip culture is necessary and sufficient for a cancer cell line to be reprogrammed to elicit in vivo-relevant heterogeneous cell populations with restored normal physiological signatures.

3D in vitro morphogenesis of human intestinal epithelium in a gut-on-a-chip or a hybrid chip with a cell culture insert

“…The formed 3D epithelial layers using both a gut-on-a-chip and a hybrid chip have regenerative potential, confirmed in various studies 1,2,4,6,12,15,19 . For instance, when the basolateral flow is ceased, established 3D epithelial microstructure is destroyed, which can regenerate once the basolateral medium flow is resumed 4 .…”

“…In addition, multiple organ-on-a-chip products have been commercialized [16][17][18] . Hence, the validation of our in vitro morphogenesis method can be accelerated and potentially adopted in many research laboratories, industries or government and regulatory agencies to understand cellular reprogramming of in vitro intestinal morphogenesis at the transcriptome level 19 , to test absorption and transport of pharmaceutical or biotherapeutic candidates using the 3D gut surrogates or to assess reproducibility of the intestinal morphogenic process using customized or commercialized organ-on-a-chip models.…”

“…3b). Expression of differentiation markers may be assessed by performing quantitative PCR 5 or single-cell RNA sequencing 19 . In this case, epithelial cells of a 3D layer grown in either a gut-on-a-chip or a hybrid chip are harvested by trypsinization, then used for molecular or genetic analyses.…”

Capturing and Quantifying Particle Transcytosis with Microphysiological Intestine‐on‐Chip Models

“…[33] The dramatic influence of the microfluidic environment on Caco-2 cell phenotype was also shown in recent transcriptomic studies. [26,27] Heterogeneous phenotypes regarding cell cycles, differentiation, and intestinal functions including digestion, absorption, drug transport, and metabolism of xenobiotics were observed. It is also noteworthy that increased mitochondria biogenesis was observed in Caco-2 cells cultured in the IOC compared to the static permeable support culture.…”

“…[22][23][24] These models offer the ability to apply biochemical and mechanical stimuli to cells within the microfluidic environment, which translates into distinctly different phenotypes and functions. [25][26][27] For example, Caco-2 cells cultured within intestine-on-chip (IOC) models differentiate into all cellular subtypes of the intestinal epithelium. [28,29] IOC models are increasingly employed to examine the effects of drugs, as well as for personalized medicine and disease modeling.…”