Hollow fiber culture accelerates differentiation of Caco-2 cells

Abstract

Caco-2 cells usually require 21 days of culture for developing sufficient differentiation in traditional two-dimensional Transwell culture, deviating far away from the quick differentiation of enterocytes in vivo. The recently proposed three-dimensional cultures of Caco-2 cells, though imitating the villi/crypt-like microstructure of intestinal epithelium, showed no effect on accelerating the differentiation of Caco-2 cells. In this study, a novel culture of Caco-2 cells on hollow fiber bioreactor was applied to morphologically mimic the human small intestine lumen for accelerating the expression of intestine functions. The porous hollow fibers of polyethersulfone (PES), a suitable membrane material for Caco-2 cell culture, successfully promoted cells to form confluent monolayer on the inner surface. The differentiated functions of Caco-2 cells, represented by alkaline phosphatase, γ-glutamyltransferase, and P-glycoprotein activity, were greatly higher in a 10-day hollow fiber culture than in a 21-day Transwell culture. Moreover, the Caco-2 cells on PES hollow fibers expressed higher F-actin and zonula occludens-1 protein than those on Transwell culture, indicative of an increased mechanical stress in Caco-2 cells on PES hollow fibers. The accelerated differentiation of Caco-2 cells on PES hollow fibers was unassociated with membrane chemical composition and surface roughness, but could be stimulated by hollow fiber configuration, since PES flat membranes with either rough or smooth surface failed to enhance the differentiation of Caco-2. Therefore, the accelerated expression of Caco-2 cell function on hollow fiber culture might show great values in simulation of the tissue microenvironment in vivo and guide the construction of intestinal tissue engineering apparatus.

Are synthetic scaffolds suitable for the development of clinical tissue‐engineered tubular organs?

“…Unlike cellulose acetate, polysulfone, and polyacrylonitrile, polyethersulfone (PES) hollow fibers (inner diameter of 900 µm; thickness of 330 µm; inner surface covered with microwells with 5–10 µm diameter) allowed cells to symmetrically form confluent monolayer on the inner lumen surface and to accelerate the cellular differentiation within a short time period (6 days of culture instead of 13 days of standard culture). Moreover, hollow 3D fiber cultures enhanced the activities of intestinal transports, like P‐glycoprotein, suggesting that, when cultured on curvy surface similar to the topography of the small intestine, Caco‐2 cells might become rapidly differentiated and polarized, sustaining the enterocyte‐like structures morphologically and functionally …”

“…Moreover, hollow 3D fiber cultures enhanced the activities of intestinal transports, like P-glycoprotein, suggesting that, when cultured on curvy surface similar to the topography of the small intestine, Caco-2 cells might become rapidly differentiated and polarized, sustaining the enterocyte-like structures morphologically and functionally. 67 In vivo scaffold assessment An ideal intestinal replacement should not only contain the different cellular components in each layer (such as circular and longitudinal smooth muscle components with intramuscular interstitial cells) but also integrate with the existing muscular and neuronal layers to receive cues from the central nervous system to facilitate peristalsis, gut motility, digestion, and excretion. Collagen [type I (70-80%) and type III (20-30%) atelocollagen] sponge grafts, freeze-dried and cross-linked, seeded with autologous MSCs, just before implantation, were used to reconstruct a 5-cm segment of dog small intestine.…”

A dynamic in-vitro model of the intestinal epithelium for the investigation of P-glycoprotein activity

“…On the other hand, Deng et al (37) reported that Caco-2 cells cultured in a hollow ber bioreactor revealed higher expression of P-gp, which was attributed to the topographic features of the substrate. Reporting a similar result, Schweinlin et al (38) observed that the expression of P-gp and e ux of Rh-123 increased in a decellularized biological scaffold-based multicellular model of the intestinal barrier cultured in a perfusion bioreactor, comparing with the values obtained for Caco-2 monoculture from another study.…”

Hollow Fiber Bioreactors for <em>In Vivo</em>-like Mammalian Tissue Culture

“…Human neural stem cell derived neurons and glia cultured in a commercially available polystyrene scaffold exhibited both high (action potential) and low-frequency (local field potential) spontaneous activity whereas no neuronal activity was detected in the 2D cultured cells 21 . Caco-2 cells demonstrated enhanced differentiation in a HFB compared to 2D culture measured by increased alkaline phosphatase, γ-glutamyltransferase and P-glycoprotein activity and higher expression of F-actin and zona occludens-1 protein 16 . Despite the advantages, the routine culturing of cells in systems other than a 2D tissue culture flask surface is still not practiced in many laboratories, although the number of publications citing 3D cell culture is growing (8-fold increase in the last 10 years.…”

“…This creates a versatile culture system with superior mass transport in which high cell densities can be reached 11 . The HFB system is well suited for the maintenance of anchorage dependent cell types and has been used to culture a variety of cells including rat pancreatic islets of Langerhans , Madin Darby canine kidney cells (MDCK) 15 and Caco-2 cells 16 to name a few.…”