A microfluidic gut-on-chip advances the in vitro development of Cryptosporidium hominis and Cryptosporidium parvum

Samantha Gunasekera

This thesis describes enhanced development of Cryptosporidium parvum and Cryptosporidium hominis using a microfluidic gut-on-chip. Once the growth conditions for both species were established, parasite development was analysed using quantitative PCR, immunofluorescence assays, scanning electron microscopy and bulk transcriptomics. Species-specific differences in the in vitro development of C. homins and C. parvum were further investigated using morphological data acquired from scanning electron micrographs. Chapter 1 provides a comprehensive literature review of bioengineered intestinal models and stem cell-based culture systems for the study of Cryptosporidium biology and introduces the concept of organ-on-chip technology as an accessible and affordable culture system for Cryptosporidium. Chapter 2 and Chapter 3 describe the use of a microfluidic gut-on-chip to support C. parvum and C. hominis growth for up to ten days respectively. Total parasite load within the microfluidic gut-on-chip was determined using quantitative PCR, and immunofluorescence assays demonstrated the presence of foci of infection throughout the duration of each experiment. Scanning electron micrographs revealed the presence of asexual and sexual Cryptosporidium life cycle stages and cultures utilising purified sporozoites were subject to downstream immunofluorescence assays to determine the presence of oocysts produced in vitro. These oocysts were then used to establish fresh cultures to assess their infectivity. The host-parasite interaction occurring within the microfluidic gut-on-chip, was further interrogated in Chapter 3 through an additional bulk transcriptomics analysis of the HCT-8 transcriptome of cells cultured under fluid shear stress conditions, both with, and without C. hominis (IdA15G1) infection. To determine a rationale for extended Cryptosporidium development using the microfluidic gut-on-chip, Chapter 4 includes an analysis of the transcriptomes of the uninfected HCT-8 cell line cultured with and without fluid shear stress, and a morphological comparison of the apical cell surface using scanning electron microscopy. Chapter 5 utilises semi-automated batch image acquisition of scanning electron micrographs to compare the proportion of each Cryptosporidium lifecycle stage occurring at 24 h post-infection, and 48 h post-infection across three different Cryptosporidium subtypes in the HCT-8 transwell system. Chapter 6 provides a synthesis of how each finding across all experimental chapters contributes to the broader understanding of Cryptosporidium biology. Ultimately, the findings of this thesis provide evidence that a microfluidic gut-on-chip can support the extended development of both species of Cryptosporidium most important to human health and puts forward a compelling argument that the simple addition of fluid shear stress to Cryptosporidium-infected HCT-8 cell cultures can dramatically enhance their longevity. This thesis also provides evidence of species-specific differences in the in vitro growth characteristics and life cycle progression of C. hominis compared to C. parvum. The present work enhances the current knowledge and understanding of Cryptosporidium biology and provides an affordable bioengineered intestinal model with many diverse applications in Cryptosporidium research.

A microfluidic gut-on-chip advances the in vitro development of Cryptosporidium hominis and Cryptosporidium parvum

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