Abstract
Mucin glycoproteins are secreted in large quantities by mucosal epithelia and cell surface mucins are a prominent feature of the glycocalyx of all mucosal epithelia. Currently, studies investigating the gastrointestinal mucosal barrier use either animal experiments or non-in vivo like cell cultures. Many pathogens cause different pathology in mice compared to humans and the in vitro cell cultures used are suboptimal because they are very different from an in vivo mucosal surface, are often not polarized, lack important components of the glycocalyx, and often lack the mucus layer. Although gastrointestinal cell lines exist that produce mucins or polarize, human cell line models that reproducibly create the combination of a polarized epithelial cell layer, functional tight junctions and an adherent mucus layer have been missing until now. We trialed a range of treatments to induce polarization, 3D-organization, tight junctions, mucin production, mucus secretion, and formation of an adherent mucus layer that can be carried out using standard equipment. These treatments were tested on cell lines of intestinal (Caco-2, LS513, HT29, T84, LS174T, HT29 MTX-P8 and HT29 MTX-E12) and gastric (MKN7, MKN45, AGS, NCI-N87 and its hTERT Clone5 and Clone6) origins using Ussing chamber methodology and (immuno)histology. Semi-wet interface culture in combination with mechanical stimulation and DAPT caused HT29 MTX-P8, HT29 MTX-E12 and LS513 cells to polarize, form functional tight junctions, a three-dimensional architecture resembling colonic crypts, and produce an adherent mucus layer. Caco-2 and T84 cells also polarized, formed functional tight junctions and produced a thin adherent mucus layer after this treatment, but with less consistency. In conclusion, culture methods affect cell lines differently, and testing a matrix of methods vs. cell lines may be important to develop better in vitro models. The methods developed herein create in vitro mucosal surfaces suitable for studies of host-pathogen interactions at the mucosal surface.
Highlights
The mucosal surfaces of the gastrointestinal tract are the first site where invading pathogens encounter the host
We have previously demonstrated that the intestinal cell lines Caco-2, LS513 and PCAA/C11 and the gastric cell lines MKN1, MKN7, MKN28 and HFE-145 are capable of forming firmly adherent continuous cell layers, either spontaneously or after cultivation on special matrices, whereas other cell lines such as MKN45 and Kato III do not have this ability [22]
The cell lines varied in mucin expression with MKN7 being most similar to gastric mucosa and Caco-2 and LS513 to the intestinal mucosa with regards to mucin expression, none of them produced more than 1% of the amount of the mucin glycoproteins that build up the mucus layer in the in vivo gastrointestinal mucosa [22]
Summary
The mucosal surfaces of the gastrointestinal tract are the first site where invading pathogens encounter the host. Mucin glycoproteins secreted by mucus producing cells in the epithelium or submucosal glands produce a layer of viscous mucus which acts as a lubricant, physical barrier and a trap for pathogens, as well as creating a matrix for other antimicrobial molecules [1,2]. The thickness of mucus layer is variable along the gastrointestinal tract and is thickest in the colon and thinnest in the jejunum [1]. MUC2 is the major component of the intestinal mucus layer. In the healthy human stomach the MUC5AC and MUC6 mucins are secreted and together they produce a laminated mucus layer in which the majority of layers are MUC5AC [5]
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