Ultrathin Alumina Membranes as Scaffold for Epithelial Cell Culture from the Intestine of Rainbow Trout

Abstract

Permeable membranes are indispensable for in vitro epithelial barrier models. However, currently available polymer-based membranes are low in porosity and relatively thick, resulting in a limited permeability and unrealistic culture conditions. In this study, we developed an ultrathin, nanoporous alumina membrane as novel cell culture interface for vertebrate cells, with focus on the rainbow trout (Onchorynchus mykiss) intestinal cell line RTgutGC. The new type of membrane is framed in a silicon chip for physical support and has a thickness of only 1 μm, with a porosity of 15% and homogeneous nanopores (Ø = 73 ± 21 nm). Permeability rates for small molecules, namely lucifer yellow, dextran 40, and bovine serum albumin, exceeded those of standard polyethylene terephthalate (PET) membranes by up to 27 fold. With the final goal to establish a representative model of the fish intestine for environmental toxicology, we engineered a simple culture setup, capable of testing the cellular response toward chemical exposure. Herein, cells were cultured in a monolayer on the alumina membranes and formed a polarized epithelium with apical expression of the tight junction protein ZO-1 within 14 days. Impedance spectroscopy, a noninvasive and real time electrical measurement, was used to determine cellular resistance during epithelial layer formation and chemical exposure to evaluate barrier functionality. Resistance values during epithelial development revealed different stages of epithelial maturity and were comparable with the in vivo situation. During chemical exposure, cellular resistance changed immediately when barrier tightness or cell viability was affected. Thus, our study demonstrates nanoporous alumina membranes as promising novel interface for alternative in vitro approaches, capable of allowing cell culture in a physiologically realistic manner and enabling high quality microscopy and sensitive measurement of cellular resistance.

Fish-gut-on-chip: development of a microfluidic bioreactor to study the role of the fish intestinein vitro

“…Fixed staining of ZO-1, f-actin and DAPI followed the same procedure as described previously. 13 Briefly, cells were fixed in 3.7% paraformaldehyde (Invitrogen, Switzerland) in PBS for 10 min, followed by a quick washing step and permeabilization with 0.2% Triton X-100 in PBS for 15 min. After a further washing step with PBS, containing 0.1% Triton X-100, cells were incubated in Image-iT (Invitrogen, Switzerland) for 30 min and washed with PBS.…”

“…Overall, the resistance values of RTgutGC monolayer cultured under static conditions in the microfluidic bioreactor are in accordance with previously reported values obtained in static cell culture inserts 8,9 and on ultrathin alumina membranes. 13 For freshwater adapted Atlantic salmon (Salmo salar), TEER values between 30 and 150 Ω cm 2 have been reported from isolated gut-sac preparations, which are composed of the epithelial cell layer and the underlying fibroblasts; 32 thus RTgutGC cell cultures under static and flow conditions generally closely reflect the in vivo TEER in salmonids. The reconstruction of the epithelial-mesenchymal interface by combining RTgutGC and RTgutF cells on opposite membrane sides under flow conditions resulted in stable TEER values of ∼55-60 Ω cm 2 from day 3-9.…”

“…These results are consistent with previous studies demonstrating that RTgutGC remain in monolayer for at least 14 days when seeded at a similar density on permeable membranes. 8,13 In the fish-gut-on-chip device, RTgutGC cells possessed a height of ∼6 μm at day 1. During the culture period of 9 days, cells increased only marginally in height to 7-9 μm, which was independent of flow and co-culturing (Fig.…”

“…10 Yet, commercial inserts have limitations, such as low permeability of the polymeric cell culture membrane and limited visibility of cells. 11,12 These limitations have led to the development of ultrathin nanoporous alumina membranes, 13 which allowed the establishment of an intestinal co-culture model of epithelial RTgutGC and newly developed fibroblastic RTgutF cell lines to partially reconstruct the complexity of the intestinal wall for improved barrier functionality. 14 However, culture conditions are stagnant which is not reflective of the in vivo situation.…”

Establishment and long-term maintenance of primary intestinal epithelial cells cultured from the rainbow trout,Oncorhynchus mykiss

“…Investigations are continuing to improve fish cell models and the current proposed models through investigations of double seeding techniques ( Langan et al, 2017 ; Schnell et al, 2016 ), application of artificial basement membranes (e.g. Drieschner et al, 2017 ; Vllasaliu et al, 2014 ), co-culture (e.g. Nollevaux et al, 2006 ) or through chemically induced differentiation.…”