Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

Abstract

Cryptosporidium spp. are apicomplexan parasites of global importance that cause human diarrheal disease. In vitro culture models that may be used to study this parasite and that have physiological relevance to in vivo infection remain suboptimal. Thus, the pathogenesis of cryptosporidiosis remains poorly characterized, and interventions for the disease are limited. In this study, we evaluated the potential of a novel bioengineered three-dimensional (3D) human intestinal tissue model (which we developed previously) to support long-term infection by Cryptosporidium parvum Infection was assessed by immunofluorescence assays and confocal and scanning electron microscopy and quantified by quantitative reverse transcription-PCR. We found that C. parvum infected and developed in this tissue model for at least 17 days, the extent of the study time used in the present study. Contents from infected scaffolds could be transferred to fresh scaffolds to establish new infections for at least three rounds. Asexual and sexual stages and the formation of new oocysts were observed during the course of infection. Additionally, we observed ablation, blunting, or distortion of microvilli in infected epithelial cells. Ultimately, a 3D model system capable of supporting continuous Cryptosporidium infection will be a useful tool for the study of host-parasite interactions, identification of putative drug targets, screening of potential interventions, and propagation of genetically modified parasites.

Keywords: 3D model; Cryptosporidium; in vitro culture; intestinal epithelial cells.

FIG 1

Schematic of the fabrication process, cell seeding strategy, and C. parvum infection process for the 3D human intestinal model. (A and B) Silk cocoons (A) were regenerated into a 4 to 5% (wt/vol) viscous silk solution (B). (C) The silk solution was poured into PDMS molds, and a Teflon-coated stainless steel wire was inserted through the cross section of the cylinder to develop a hollow channel. (D and E) Caco-2 and HT29-MTX cells (D) were seeded into the hollow channel (E), while the porous bulk space was used to house H-InMyoFibs. (F) The Caco-2 and HT29-MTX cells in the scaffolds were infected with C. parvum oocysts or purified sporozoites, and intracellular development was allowed to proceed through asexual and sexual cycles to complete the life cycle with the formation of oocysts. (Republished from reference with permission of the publisher as well as from Scientific Reports [26].)

FIG 2

IFA and confocal microscopy of Caco-2 and HT29-MTX cells in scaffolds at various time points during infection with C. parvum oocysts. The infected scaffolds were fixed, permeabilized, and stained with MAb 4E9. Uninfected scaffolds (A) and infected scaffolds obtained at 1 day (B), 2 days (C), 3 days (D), 8 days (E), 11 days (F), and 17 days (G) postinfection are shown.

FIG 3

IFA and confocal microscopy of Caco-2 and HT29-MTX cells in scaffolds infected with purified C. parvum sporozoites at various time points. Following infection, scaffolds were fixed, permeabilized, and stained with MAb 4E9. (A) Infected scaffolds at 5 days postinfection; (B) infected scaffolds at 10 days postinfection; (C) infected scaffolds at 15 days postinfection; (D) uninfected control scaffolds.

FIG 4

IFA and confocal microscopy of oocyst production following infection with purified C. parvum sporozoites at various time points. (A to C) Oocysts within scaffolds; (E to G); oocysts within luminal material; (I to K) oocysts within culture medium; (D, H, L), uninfected controls. At each time point, scaffolds or material from the lumen or culture medium was fixed, permeabilized, and stained with an oocyst-specific MAb (Crypt-a-Glo).

FIG 5

Quantification of C. parvum infection with oocysts (A) and purified sporozoites (B) in scaffolds by qRT-PCR. At the indicated times, the luminal contents and infected cells were harvested and infection was quantitated by qRT-PCR. Results are expressed as number of copies of C. parvum 18S cDNA obtained from a standard curve. Bars represent the means from six replicates with standard errors of the means. In panel A, P was <0.006 for day 1 compared to days 14 and 17 and P was <0.05 for day 2 compared to days 14 and 17. In panel B, P was <0.0005 for day 1 compared to day 10.

FIG 6

Scanning electron micrographs of C. parvum oocyst-infected Caco-2 and HT29-MTX cells in scaffolds. (A) Uninfected cells; (B) type I meronts (thick arrows) and empty parasitophorous vacuoles (thin arrows) on day 3; (C and D) type I meronts containing eight merozoites on day 3 (the images are enlargements of the image in panel B); (E) a type I meront excysting on day 2.

FIG 7

Scanning electron micrographs of C. parvum sporozoite-infected scaffolds. (A) A trophozoite on day 5; (B) early penetration of type I merozoites on day 1; (C to E) type I meronts excysting on days 5 (C and D) and 2 (E); (F and G) free merozoites on day 5; (H and I) macrogamonts on day 3.

FIG 8

C. parvum can be passaged from an infected scaffold to an uninfected one to establish a new infection. (A to C) Following infection, scaffolds were fixed, permeabilized, and stained with MAb 4E9. Images are from 3 days after the first passage (A), second passage (B), or third passage (C). (D) Scanning electron micrograph of a C. parvum-infected scaffold at 3 days after the first passage.