New and better protocols for a short-term Caco-2 cell culture system

Yamashita, Shinji; Konishi, Keisuke; Yamazaki, Yukako; Taki, Yoko; Sakane, Toshiyasu; Sezaki, Hitoshi; Furuyama, Yukihiro

doi:10.1002/jps.10050

Cited by 119 publications

(98 citation statements)

References 19 publications

(16 reference statements)

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“…Even the barrier function of these monolayers is low compared to the traditional 21-day culture; it has been claimed that permeability coefficients across this monolayer correlate reasonably well with the extent of absorption in man. 20,21 However, our results indicate that the BioCoat™ HTS Caco-2 Assay System does not seem to be adequate for the prediction of human absorption. The regression at pH 7.4 is not satisfactory, whereas at pH 6.5, it has no biological significance, as the curve cannot predict any intermediate values of human absorption.…”

Section: Discussionmentioning

confidence: 84%

Comparison of in Vitro Models for the Prediction of Compound Absorption across the Human Intestinal Mucosa

Miret

Abrahamse²,

Groene³

2004

SLAS Discovery

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Several in vitro assays have been developed to evaluate the gastrointestinal absorption of compounds. Our aim was to compare 3 of these methods: 1) the bio-mimetic artificial membrane permeability assay (BAMPA) method, which offers a highthroughput, noncellular approach to the measurement of passive transport; 2) the traditional Caco-2 cell assay, the use of which as a high-throughput tool is limited by the long cell differentiation time (21 days); and 3) The BioCoat™ high-throughput screening Caco-2 Assay System, which reduces Caco-2 cell differentiation to 3 days. The transport of known compounds (such as cephalexin, propranolol, or chlorothiazide) was studied at pH 7.4 and 6.5 in BAMPA and both Caco-2 cell models. Permeability data obtained was correlated to known values of human absorption. Best correlations (r = 0.9) were obtained at pH 6.5 for BAMPA and at pH 7.4 for the Caco-2 cells grown for 21 days. The Caco-2 BioCoat™ HTS Caco-2 Assay System does not seem to be adequate for the prediction of absorption. The overall results indicate that BAMPA and the 21-day Caco-2 system can be complementary for an accurate prediction of human intestinal absorption. (Journal of Biomolecular Screening 2004:598-606) Key words: BAMPA, bioavailability, Caco-2, permeability A LTHOUGH BIOLOGICAL EFFICACY is the key issue during the discovery process of active compounds, other factors such as bioavailability are equally important when the action of the ingredient is not limited to the gastrointestinal tract. An efficacious compound must be sufficiently bioavailable to perform its expected activity. The gastrointestinal absorption of an orally administered compound is one of the key factors for its bioavailability. Because absorption potential has become a more important criterion in the discovery process, there is a need for reliable screening methods to assess for compound permeability. 1Intestinal absorption screening assays should be highly predictive, fast, reliable, cost-effective, and require a small amount of compound. 2,3Current approaches include physicochemical models, in silico computational models, in vitro models (cell and tissue based), in situ models of intestinal perfusion, and in vivo animal models. 1The intestinal absorption of a compound depends on 2 major parameters: 1) permeability across the epithelial mucosa and 2) gastrointestinal transit. Because gastrointestinal transit can be evaluated only in vivo, all in vitro systems can determine only mucosal permeability.The main barrier for the absorption of a compound is formed by the intestinal epithelium, and several routes can be followed to cross this barrier. The passive transcellular pathway involves the movement of solute molecules across the apical membrane, through the cytoplasm, and across the basolateral membrane. This is the predominant route for hydrophobic compounds. Another passive route is via the tight junctions between the enterocytes: the paracellular pathway. Small hydrophilic compounds are mostly transported via the paracellular route...

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Section: Discussionmentioning

confidence: 84%

Comparison of in Vitro Models for the Prediction of Compound Absorption across the Human Intestinal Mucosa

Miret

Abrahamse²,

Groene³

2004

SLAS Discovery

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“…In addition, the Biocoat™ HTS system, which includes medium supplemented with MITO+™ serum extender, is recommended for accelerated differentiation of Caco-2 cells in 3 days (BD Biocoat™ HTS Caco-2 assay system). However, in this assay, differentiation medium also includes butyrate, which was shown to promote cellular differentiation but also causes toxicity when used longer than 5 days (Yamashita et al, 2002). Serum-free medium containing MITO+™ serum extender has, to the best of our knowledge, never before been tested alone for differentiation of intestinal cell lines.…”

Section: B Amentioning

confidence: 99%

“…The introduction of a commercially available Caco-2 assay system, the BD Biocoat™ HTS, employing filters coated with fibrillary collagen I and using a differentiation medium containing a mixture of hormones and growth factors (MITO+™ serum extender), reported to allow full differentiation of Caco-2 cells in 3 days, compared to the 21-day standard protocol, has encouraged research to verify the company's claims. Overall, the results of these studies concluded that the 3-day accelerated differentiation protocol did not allow full expression of active membrane transporters and differentiation-dependent enzyme activities, although passive permeability properties were comparable to those obtained with the standard 21-day protocol employing medium supplemented with 10% FBS (Chong et al, 1997;Miret et al, 2004;Yamashita et al, 2002). It is unfortunate, however, that these studies only compared the 3-day serum-free protocol with the standard 21-day 10% FBS-medium, while failing to investigate the serum-free protocol over an extended period of time.…”

mentioning

confidence: 93%

Serum-reduced and serum-free media for differentiation of Caco-2 cells

Ferruzza

2013

ALTEX

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“…Drug Absorption and Disposition after Nasal and Oral Administration medium supplemented with 10% fetal bovine serum, 1% Lglutamine, 1% non-essential amino acid, and 5% antibioticantimycotic solution in a culture flask. 22) Caco-2 monolayers were prepared according to the short-term culture method 23) using the culture kit, BIOCOAT ® HTS Caco-2 Assay System (Beckton Dickinson Bioscience, Bedford, MA, U.S.A.).…”

Section: Methodsmentioning

confidence: 99%