Interlaboratory comparison of an intestinal triple culture to confirm transferability and reproducibility

Abstract

The development and improvement of advanced intestinal in vitro models has received increasing attention in recent years. While the availability of relevant in vitro models is pivotal to advance the replacement and reduction of animal use in research, their robustness is a crucial determinant for intra- and interlaboratory reproducibility. We have developed a standard protocol to build a triple culture model combining two types of human intestinal epithelial cells (Caco-2, HT29-MTX-E12) and macrophages (THP-1), which was tested for transferability and reproducibility between three laboratories. The epithelial tissue barrier development and triple culture stability were investigated as well as the models’ responses to the non-steroidal anti-inflammatory drug diclofenac in terms of barrier integrity, cytotoxicity, and cytokine release. The results of two partner laboratories were compared to previously established benchmark results and quality criteria. For the epithelial co-cultures, the results were overall highly comparable between the laboratories. The addition of THP-1 cells resulted in increased variability and reduced reproducibility. While good correlation was achieved in several endpoints, others showed substantial response differences between the laboratories. Some variations may be addressed with training or demonstrations, whereas others might be related to fundamental differences in the cell lines introduced during routine cell culture and maintenance. Our results underline the importance of interlaboratory transfer studies using standardised experimental procedures, including defined quality criteria and benchmarks, as well as of training when newly establishing complex in vitro models in laboratories.

Virus-like silica nanoparticles enhance macromolecule permeationin vivo

“…Caco-2/MTX-HT29 co-culture monolayer: The Caco-2 cells and MTX-HT29-E12 cells were seeded on the apical chamber of Transwell with 9 : 1 ratio at the density of 1 × 10 5 cells per well with DMEM supplied with 10% FBS, 1% MEM, 1% glutamine, and 1% P/S and cultured for 8 days. 38,39 The culture medium was replaced every 2 days.…”

Investigating nanoplastics toxicity using advanced stem cell-based intestinal and lung in vitro models

“…Uniquely, these spheroids represent all major epithelial cell lineages within the organoid (Sato et al, 2011), and allow to skew them towards specific cell types and rare lineages like tuft cells (Gerbe et al, 2016;Treveil et al, 2020;Inaba et al, 2021). Even the most sophisticated non-stem cell models still fail to replicate the lineage complexity to this extent (Lozoya-Agullo et al, 2017;Lehner et al, 2020;Kämpfer et al, 2022). For the purpose of hazard testing of nanoplastics, both overall effects on the tissue as well as effects in specific individual cell types may be relevant.…”

“…The cultures can replicate the complexity of various regions of the intestinal tract-from duodenum to colon-as well as healthy or diseased tissue depending on the biopsy origin or growth/differentiation protocol (Sato et al, 2011;Tsai et al, 2017;Bandi et al, 2020). In contrast, standard cell models mostly rely on cancer-derived cell lines in mono-or co-culture that often require extensive differentiation times and further manipulation to obtain rather mature cell types (Hilgers et al, 1990;Lesuffleur et al, 1990) and disease-like phenotypes (Susewind et al, 2016;Kämpfer et al, 2017). Inherently diseased, these cell lines may be of limited physiological relevance for specific research questions due to biochemical and genetic differences compared to healthy intestinal tissue, e.g.…”

Development of an Inflammation-Triggered In Vitro “Leaky Gut” Model Using Caco-2/HT29-MTX-E12 Combined with Macrophage-like THP-1 Cells or Primary Human-Derived Macrophages

“…Intestinal macrophages in the subepithelial lamina propria (LP) are the most abundant mononuclear phagocytes in the body and play a critical role in maintaining intestinal homeostasis. Thus, some researchers have started to combine intestinal cell lines with human macrophage-like cells (e.g., differentiated monocytic THP-1 cells), or monocyte-derived macrophages from peripheral blood mononuclear cells (PBMCs) [ 38 , 39 , 40 ]. However, each of these IBD models has its disadvantages, because of either (1) the lack of mucus-producing cells [ 41 , 42 , 43 , 44 ], (2) a spatial distance between the intestinal cells and macrophages [ 45 , 46 , 47 ], or (3) being specifically adapted for buoyant particles [ 48 ].…”