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Evidence for Diminished Functional Expression of Intestinal Transporters in Caco-2 Cell Monolayers at High Passages
Abstract
Purpose To investigate the effects of passaging on the intrinsic membrane transport parameters of compounds absorbed by means of passive and carrier-mediated processes in the Caco-2 cell line.Methods Caco-2 cells at low (28-36) and high (93-108) passage numbers were used to evaluate the transport characteristics of model compounds for paracellular diffusion (mannitol), transcellular diffusion (progesterone) and carrier-mediated transport (cephalexin, cephradine, phenylalanine, proline, and taurocholic acid) using side-by-side diffusion chambers. Intrinsic intestinal transport parameters were determined by correcting the effective permeability for potential biases introduced by the microporous filter and aqueous boundary layer. Intrinsic maximal flux (Jmax), Michaelis constant (K(m)) and carrier permeability (Pc) were determined as a function of passage number.Results Compared to the low passaged cells, the high passaged Caco-2 cells were characterized by less morphological heterogeneity, higher transepithelial electrical resistance, higher transcellular diffusion, lower paracellular diffusion, lower carrier-mediated transport and lower alkaline phosphatase activity. The use of effective transport parameters overestimated the K(m) and underestimated Pc but had no effect on Jmax.Conclusions The current results provide experimental evidence that the passaging process significantly affects the biological characteristics and transport properties of Caco-2 cell monolayers. The effects are consistent with a reduction in the functional expression of a brush border enzyme and several transport proteins as passage number is increased. The underlying basis for this appears to be a selection of fast-growing subpopulations from the original heterogeneous Caco-2 cell line during passaging.
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“However, cell lines can undergo many changes as a result of short or prolonged cultivation including increased genetic and phenotypic instability, which may alter cell morphology and gene expression, chromosomal duplication and rearrangements, potential mutations in genomic and mitochondrial DNA, and epigenetic modifications. 3 , 4”
From Cell line authentication and validation is a key requirement for Journal of Cell Communication and Signaling publications (2025) · cites this paper as [3]
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“As a result, there has been a growing emphasis on validating the authenticity and integrity of both experimental and established cell lines prior to publication.”
From Cell line authentication and validation is a key requirement for Journal of Cell Communication and Signaling publications (2025) · cites this paper as [3]
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Background
Metadata
- Authors
- Yu, H., Cook, T. J., & Sinko, P. J.
- Affiliations
- Rutgers, The State University of New Jersey (US)
- Corresponding author
- Sinko, Patrick J.
- ORCID
- Sinko, Patrick J.
- Venue
- Pharmaceutical Research
- Published
- 1997-06
- Volume
- 14
- Issue
- 6
- Pages
- 757-762
- ISSN
- 0724-8741 (ISSN-L)
- 1573-904X
- OpenAlex
- W294850324
- Cited by
- 133
- References
- 26
- Citations by year
- 2026: 3 · 2025: 4 · 2024: 5 · 2023: 3 · 2022: 9 · 2021: 8 · 2020: 2 · 2019: 3 · 2018: 4 · 2017: 2 · 2016: 3 · 2015: 5 · 2014: 7 · 2013: 6 · 2012: 6
- FWCI
- 1.21
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- Locations
- Pharmaceutical Research (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmaceutical Science › Drug Solubulity and Delivery Systems
- Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
- Keywords
- Caco-2 cells, cell passage number, intestinal transporters, paracellular diffusion, carrier-mediated transport, effective permeability, Michaelis constant, alkaline phosphatase, brush border enzymes, transepithelial electrical resistance, mannitol, progesterone, cephradine, taurocholate
- MeSH terms
- Alkaline Phosphatase, Biological Transport, Carrier Proteins, Cell Count, Cell Membrane Permeability, Diffusion, Epithelium, Humans, Electric Impedance, Caco-2 Cells
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