The Oesophageal Squamous Cell Carcinoma Cell Line COLO-680N Fails to Support Sustained Cryptosporidium parvum Proliferation

Abstract

Cryptosporidium parvum is an important diarrhoea-associated protozoan, which is difficult to propagate in vitro. In 2017, a report described a continuous culture of C. parvum Moredun strain, in the oesophageal squamous cell carcinoma cell line COLO-680N, as an easy-to-use system for C. parvum propagation and continuous production of oocysts. Here, we report that-using the Köllitsch strain of C. parvum-even though COLO-680N cells, indeed, allowed parasite invasion and early asexual parasite replication, C. parvum proliferation decreased after the second day post infection. Considering recurring studies, reporting on successful production of newly generated Cryptosporidium oocysts in the past, and the subsequent replication failure by other research groups, the current data stand as a reminder of the importance of reproducibility of in vitro systems in cryptosporidiosis research. This is of special importance since it will only be possible to develop promising strategies to fight cryptosporidiosis and its ominous consequences for both human and animal health by a continuous and reliable methodological progress.

Keywords: COLO-680N; Cryptosporidium parvum; cell culture; immunofluorescence; qPCR.

Figure 1

Exemplary illustration of Cryptosporidium parvum in vitro cell cultures at 24 hpi (A) Vicia villosa (VV) lectin-based detection of C. parvum (green), illustration of cell membranes via anti-β-catenin-mediated staining (red) and of cell nuclei via DAPI staining (blue). Scale bar 20 µm. (B) Infection rates of C. parvum-infected HCT-8 and COLO-680N cells after application of three different infection protocols. Infection rates are expressed as mean ± SD of six replicates per condition. For statistical analysis, a one-way analysis of variance (ANOVA) with Tukey’s test was performed using GraphPad^® Prism 8 software (San Diego, CA, USA), with a significance level of 5%. The significance levels are as follows: *** = p ≤ 0.001, ** = p ≤ 0.01 and presented within the table in (B).

Figure 2

Replication of C. parvum in HCT-8- and COLO-680N cells using infection protocol III. Parasite intracellular replication was quantified by both qPCR- (A) and Vicia villosa (VV) lectin (VVL)-based immunofluorescence analyses (B). (C) Exemplary illustration of both in vitro cell culture systems: fluorescence-based detection of C. parvum via VVL (green) and cell nuclei via DAPI (blue). To assess parasite development, a two-tailed t-test was performed, comparing the infection rate per day, measured in HCT-8- and COLO-680N cells. The significance values were as follows: *** = p ≤ 0.001, ** = p ≤ 0.01. Scale bar 20 µm.