Short Term Caco-2/TC7 Cell Culture: Comparison between of Conventional 21-d and a Commercially Available 3-d System

Abstract

The Caco-2 cell model is a valuable tool for studying intestinal biotransformation of xenobiotics and to evaluate the potential of human intestinal absorption of new compounds. These properties were evaluated with Caco-2/TC7 cells in accelerated conditions to reduce maturation lag time from 21-d to 3-d in order to increase time and labor efficiency. Transmission electron and fluorescent microscopy were used for morphological characterization. Alkaline phosphatase and lactate dehydrogenase activities were assessed within time. Cytochrome P450 expression was studied by RT-PCR. Apparent permeabilities of a set of passively absorbed molecules across Caco-2/TC7 cell monolayers were determined to evaluate potential of both systems for prediction of human intestinal absorption. Microscopic images revealed that cells under both conditions differentiated as enterocyte-like cells but did so heterogeneously in the 3-d model. TEER values have shown that the 3-d model is a leakier cell system with higher mannitol Papp (cm/s). Biochemical characterization (hydrolase activities, CYP450 expression) suggested that the 3-d model was at a lower maturation level than the 21-d model. Carrier-mediated uptake of L-Phe was lower in the 3-d model suggesting that this model has limited application for mechanistic studies. Reasonable correlation was obtained between the two models (r2=0.88, p>0.01) for 11 passively absorbed compounds with high potential of rank ordering of compounds. Although results suggested that the 3-d cells are under-differentiated, they could be usable to estimate the oral absorption of passively absorbed compounds.

Optimizing culture conditions of a porcine epithelial cell line IPEC-J2 through a histological and physiological characterization

“…Secondly, the decrease of TEER values after day 20 (Fig. 2b) indicates a limited culture time, which is similar to Caco-2, and most probably caused by deterioration of tight junctions relative to the culture's age and initiated apoptosis (Da Violante et al 1994). …”

Sublingual Delivery of Insulin: Effects of Enhancers on the Mucosal Lipid Fluidity and Protein Conformation, Transport, and in Vivo Hypoglycemic Activity

“…Calculation and Statistical Analysis The parameters of secondary structure of mucosal proteins, viz., a-helix (%), bsheet (%), b-turn (%), random coil (%) were obtained by simulating analysis from the Jasco J-700 software. Apical to basolateral apparent permeability (P app ) values of insulin across HIOEC monolayer were calculated with the equation according to Violante (2004), P app ϭdQ/dt/A/C 0 . Where dQ/dt is the rate of permeation, A is the diffusion area (cm 2 ), C 0 is the initial concentration of insulin in the donor compartment.…”

“…Where dQ/dt is the rate of permeation, A is the diffusion area (cm 2 ), C 0 is the initial concentration of insulin in the donor compartment. 30) In order to compare the glucose-lowering effects of various treatments, the difference value of the area above the plasma glucose curve (DAAC) was calculated by the trapezoidal method. 31) All DAAC values were estimated versus the glucose level after insulin was sublingually administered in absence of any enhancer.…”

Multiparametric temporal analysis of the Caco-2/TC7 demonstrated functional and differentiated monolayers as early as 14 days of culture

“…Various studies were carried out; high seeding density, specific extracellular matrix composition and/or chemical addition in culture medium were shown to drastically decrease the Caco-2 differentiation period by 3-5 days (Alsenz and Haenel, 2003;Chong et al, 1997;Da Violante et al, 2004). However, comparison between conventional (21 days) and fast (3-7 days) culture methods revealed that the differentiation of Caco-2 and Caco-2/TC7 cells were not optimal (sub differentiated) with the faster protocol.…”

“…However, comparison between conventional (21 days) and fast (3-7 days) culture methods revealed that the differentiation of Caco-2 and Caco-2/TC7 cells were not optimal (sub differentiated) with the faster protocol. Furthermore, it appeared that the short protocol was unable to induce mature P-gp efflux (Liang et al, 2000;Da Violante et al, 2004). Despite the advantages associated with the fast culture protocols, the technique resulted in the generation of sub-optimal Caco-2 functional epithelium as compared with conventionally grown 21 day Caco-2 monolayers.…”