Culture media and format alter cellular composition and barrier integrity of porcine colonoid-derived monolayers

ABSTRACT

Intestinal organoid technology has revolutionized our approach to in vitro cell culture due in part to their three-dimensional structures being more like the native tissue from which they were derived with respect to cellular composition and architecture. For this reason, organoids are becoming the new gold standard for undertaking intestinal epithelial cell research. Unfortunately, their otherwise advantageous three-dimensional geometry prevents easy access to the apical epithelium, which is a major limitation when studying interactions between dietary or microbial components and host tissues. To overcome this problem, we developed porcine colonoid-derived monolayers cultured on both permeable Transwell inserts and tissue culture treated polystyrene plates. We found that seeding density and culture format altered the expression of genes encoding markers of specific cell types (stem cells, colonocytes, goblets, and enteroendocrine cells), and barrier maturation (tight junctions). Additionally, we found that changes to the formulation of the culture medium altered the cellular composition of colonoids and of monolayers derived from them, resulting in cultures with an increasingly differentiated phenotype that was similar to that of their tissue of origin.

SUMMARY

In vitro models of the intestine are used to study the complex in vivo intestinal processes in a simplified context. As such, these models need to be representative of their tissue of origin. Here, we demonstrate that porcine colonoids and colonoid-derived monolayers that have comparable stem cells and differentiated cell types to those of the native tissue can be developed but are influenced by cell seeding density, culture format, and medium formulation.

KEYWORDS:

• Barrier integrity
• colonoid-derived monolayers
• differentiation
• medium formulation
• Porcine colonoids
• stem cells

Abbreviations

ADF, Advanced DMEM/F12; Anova, analysis of variance; ATCC, American Type Culture Collection; BM, Basal medium; BSA, bovine serum albumin; CA2, carbonic anhydrase 2; CM conditioned media; CGA, chromogranin A; CLDN, claudin; DMSO, dimethyl sulfoxide; EDTA, ethylene diamine tetra acetic acid; FBS, fetal bovine serum; IDT, Integrated DNA Technologies; Ins, insert; ISC, intestinal stem cell; LGR5, Leucine-rich repeat-containing G protein-coupled receptor 5; MAPK, p38 Mitogen-activated protein kinase; MUC2, mucin 2; OCLN, occludin; PBS, Phosphate-buffered saline; Quantitative Real-Time PCR (qPCR); RIN, RNA integrity number; ROCK, Rho-associated Coiled-coil Kinase; RRID, research resource identifier, RPL4, Ribosomal protein L4; RT, room temperature; SGLT1, sodium-glucose transporter 1; SOX9, Sex-determining region Y (SRY) – box 9; TBP, Tata box protein; TC, tissue culture; TEER, trans epithelial electrical resistance, TGFβ, transforming growth factor beta; ZO1, zonula occludens 1.

Author contributions

Alicia M. Barnett conceived and designed the experiments; Alicia M. Barnett performed the experiments and analyzed the data; Alicia M. Barnett evaluated the literature; Alicia M. Barnett, Jane A Mullaney, Nicole C. Roy, and Warren C. McNabb have contributed to the interpretation of results and the writing of the manuscript. Nicole C. Roy and Warren C. McNabb sourced the funding for the research.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Institutional review board statement

The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of Massey University, Palmerston North, New Zealand (protocol code #19/83).

Data availability statement

The datasets and raw data used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Additional information

Funding

The research was funded by the Riddet Institute Centre of Research Excellence. Alicia Barnett, Warren McNabb, and Nicole Roy were funded by the New Zealand Ministry of Business, Innovation and Employment Endeavour research program New Zealand Milks Mean More (MAUX1803).