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Caco-2

The Caco-2 cell line is an immortalized human epithelial cell line derived from a colorectal adenocarcinoma tumor in the colon of a 72-year-old Caucasian male patient, originally established in 1977 at the Sloan-Kettering Institute for Cancer Research using explant culture techniques.^[1]^[2]^[3] When cultured to confluence, Caco-2 cells spontaneously differentiate into polarized monolayers resembling small intestinal absorptive enterocytes, complete with microvilli, brush borders, tight junctions, and dome formation indicative of ion transport.^[3]^[4] This differentiation process typically requires 15–21 days post-confluence on permeable supports, such as Transwell inserts with 0.4 μm pore size filters, resulting in high transepithelial electrical resistance (TEER) values often exceeding 300 Ω·cm², which confirms the formation of a functional barrier.^[3]^[4] Caco-2 cells express a range of intestinal enzymes (e.g., sucrase-isomaltase, lactase, alkaline phosphatase) and transporters (e.g., P-glycoprotein, peptide transporters), though they lack significant cytochrome P450 activity and represent primarily enterocytic cells without the heterogeneity of other intestinal cell types like goblet or Paneth cells.^[3]^[5] The cell line is heterogeneous, with subpopulations exhibiting varying differentiation potential, and its properties can be influenced by passage number, culture conditions, and subclone selection.^[6]^[4] Caco-2 monolayers are extensively employed in pharmaceutical research as a predictive model for oral drug absorption, nutrient transport, and intestinal permeability, correlating well (r > 0.8) with human in vivo bioavailability data for passively transported compounds.^[7]^[8]^[5] They facilitate bidirectional transport studies to distinguish apical-to-basolateral (absorptive) from basolateral-to-apical (secretory) flux, aiding in the identification of efflux transporter substrates like those recognized by MDR1.^[8] Beyond pharmacology, Caco-2 cells support investigations into food bioactives, toxicology, inflammation, and viral infections (e.g., susceptibility to HIV-1 and noroviruses), as well as 3D spheroid cultures for more complex tissue modeling.^[1]^[3]^[9] Despite these strengths, limitations include the absence of mucus production, an unstirred water layer, and non-enterocyte cell types, which can overestimate permeability for hydrophilic compounds or underestimate metabolism-dependent absorption; co-cultures with mucus-secreting cells like HT29-MTX are often used to address these gaps.^[3]^[5]^[10] Regulatory bodies such as the FDA and EMA endorse Caco-2 assays as a standard for Biopharmaceutics Classification System (BCS) assignments, underscoring their role in drug development workflows.^[11]^[12]

Origin and Development

Establishment of the Cell Line

The Caco-2 cell line was isolated in 1977 from a heterogeneous colorectal adenocarcinoma tumor excised from a 72-year-old Caucasian male patient at the Sloan–Kettering Institute for Cancer Research in New York.^[2] The isolation was performed by Jorgen Fogh and colleagues using the explant culture technique, in which small pieces of the tumor tissue were directly cultured in vitro to allow outgrowth of epithelial cells. This method facilitated the initial establishment of the line without the need for enzymatic dissociation, preserving the heterogeneous nature of the tumor source, which included a mix of poorly differentiated adenocarcinoma cells.^[2] Following isolation, the Caco-2 cells were characterized as an adherent epithelial cell line capable of forming monolayers in standard culture conditions. The line achieved immortality spontaneously through continuous serial passaging, relying on the inherent tumorigenic properties of the source material rather than artificial transformation agents such as viruses or chemical mutagens.^[3] No exogenous interventions were required to maintain indefinite proliferation, distinguishing it from finite primary cell cultures. Early post-isolation viability was robust, with the cells demonstrating consistent attachment and expansion in nutrient-rich media.^[2] Initial growth kinetics post-establishment showed a population doubling time of approximately 20-30 hours, reflecting the proliferative vigor of low-passage cells derived from the aggressive adenocarcinoma.^[13] This rapid early replication supported the line's suitability for long-term maintenance and initial experimental use as a model of human colonic epithelium. The cell line was subsequently provided to Alain Zweibaum's laboratory at INSERM in France, where it was further characterized and optimized for research. Subsequent studies by Zweibaum's group further explored its potential for differentiation research.^[14]

Historical Milestones

The Caco-2 cell line, derived from a human colorectal adenocarcinoma in 1977, underwent initial characterization in the early 1980s that revealed its potential for modeling intestinal epithelial behavior. A landmark study in 1983 by Pinto et al., led by Alain Zweibaum at INSERM in France, demonstrated that prolonged culture of Caco-2 cells induces spontaneous enterocyte-like differentiation, including the formation of brush border enzymes and dome structures indicative of polarization, marking the first recognition of its utility in studying intestinal cell maturation. Throughout the 1980s, Zweibaum's group at INSERM advanced understanding of Caco-2 differentiation, establishing it as a model for enterocytic functions through serial publications on polarization and enzyme expression.^[15] Concurrently, researchers Ismael Hidalgo at the University of Kansas and Tom Raub at the Upjohn Company explored its applications in permeability, adapting the cells to permeable supports to mimic intestinal barriers for drug transport studies.^[15] The pivotal 1989 publication by Hidalgo, Raub, and Borchardt validated Caco-2 monolayers as a predictive model for human intestinal absorption, showing that permeability coefficients for passively transported compounds correlated strongly with in vivo data from rat perfusion models, thereby shifting focus toward pharmaceutical applications.^[16] By the 1990s, Caco-2 had gained widespread adoption in pharmaceutical screening for drug absorption predictions, with high-throughput assays integrated into early drug discovery pipelines across industry labs to evaluate compound bioavailability efficiently.^[17] Standardization efforts accelerated in the early 2000s, as evidenced by the U.S. FDA's 2000 guidance on biowaivers, which endorsed Caco-2 permeability assays alongside in situ models for supporting bioavailability classifications under the Biopharmaceutics Classification System (BCS).^[18] In the 2020s, advancements in genetic engineering have enhanced Caco-2's precision, with CRISPR/Cas9-mediated knockouts of transporters like ABCG2 enabling targeted studies of drug efflux and uptake mechanisms, as demonstrated in models developed for improved absorption predictions.^[19] These modifications, including validations in Caco-2 subclones like TC7 for lipoprotein-related investigations, continue to refine the model's relevance for personalized pharmacotherapy.^[20]

Biological Characteristics

Morphology and Differentiation

Caco-2 cells in their undifferentiated state exhibit a characteristic cobblestone morphology, forming polygonal, tightly packed colonies with epithelial-like adherence to the culture substrate.^[21] This appearance reflects their origin as polarized epithelial cells derived from a human colorectal adenocarcinoma, though they initially lack the specialized features of mature enterocytes.^[3] Upon reaching confluence, Caco-2 cells undergo spontaneous differentiation, typically over 14–21 days in culture, transitioning into polarized monolayers with distinct apical and basolateral domains.^[3] The apical surface develops densely packed microvilli forming a brush border, while the basolateral membrane establishes vectorial polarity, mimicking the organization of intestinal epithelium.^[22] This process follows a mosaic pattern, where subpopulations of cells progressively acquire differentiated traits.^[23] As differentiation advances, tight junctions assemble between adjacent cells, evidenced by the expression and localization of zonula occludens-1 (ZO-1), which stabilizes the paracellular barrier and prevents leakage across the monolayer.^[24] In fully confluent cultures, these tight junctions contribute to dome formation, where fluid accumulation beneath the monolayer creates hemispherical elevations indicative of functional polarity and transepithelial transport.^[25] Ultrastructural analysis via electron microscopy confirms the presence of a well-developed brush border on the apical domain, complete with microvilli rich in actin filaments, and desmosomes facilitating strong cell-cell adhesion.^[22] These features closely resemble those of small intestine enterocytes, including the glycocalyx-covered microvilli and junctional complexes, despite the cells' colonic derivation.^[3] The quality and consistency of differentiation are passage-dependent, with optimal monolayer formation and polarization observed between passages 30 and 60, beyond which heterogeneity increases, leading to variable dome formation and reduced barrier integrity.^[26]

Molecular and Functional Properties

Differentiated Caco-2 cells express a range of intestinal brush border enzymes that recapitulate key aspects of enterocyte function, including sucrase-isomaltase for carbohydrate digestion, alkaline phosphatase for phosphate hydrolysis, and dipeptidyl peptidase IV for peptide processing.^[27]^[28] These enzymes are upregulated during spontaneous differentiation, typically peaking 14-21 days post-confluence, supporting the cells' role as a model for absorptive processes.^[29] The cells exhibit polarized expression of transporter proteins essential for nutrient and drug handling, with apical transporters such as PEPT1 facilitating peptide uptake and P-glycoprotein (MDR1) mediating efflux of xenobiotics back into the lumen.^[30]^[31] Basolateral transporters, including MRP3, enable secretion toward the bloodstream, contributing to vectorial transport across the monolayer.^[32] This asymmetric distribution aligns with the morphological polarization of the cells into enterocyte-like structures.^[29] Barrier integrity in differentiated Caco-2 monolayers is quantified by transepithelial electrical resistance (TEER) values typically ranging from 200 to 500 Ω·cm², reflecting robust tight junctions and low paracellular permeability.^[33] Metabolically, the cells support Phase I oxidation via cytochrome P450 enzymes (e.g., CYP1A1, CYP3A4 at low levels) and Phase II conjugation through glucuronidation and sulfation pathways, though expression is generally lower than in native human intestine.^[34]^[29] Genetically, Caco-2 cells display karyotype abnormalities, resulting in a modal chromosome number of approximately 96; despite this aneuploidy, the line maintains phenotypic consistency across passages and laboratories, ensuring reproducible functional properties.^[1]^[35]

Cultivation Methods

Culture Conditions and Protocols

Caco-2 cells are routinely cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10-20% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), 1 mM sodium pyruvate, and antibiotics such as 100 U/mL penicillin and 100 μg/mL streptomycin.^[11] Cultures are maintained at 37°C in a humidified atmosphere of 5% CO₂.^[1] This formulation supports robust proliferation while mimicking physiological nutrient conditions.^[2] For maintenance, cells are seeded at a density of 1-5 × 10⁴ viable cells/cm² to achieve optimal growth without overcrowding.^[1] Subculturing occurs every 3-5 days when cultures reach 70-80% confluence, using 0.25% trypsin-EDTA for detachment followed by neutralization with complete medium.^[2] A split ratio of 1:4 to 1:6 is typically employed, with medium renewed 1-2 times per week to prevent nutrient depletion and accumulation of metabolic waste.^[1] To minimize phenotypic drift and dedifferentiation, Caco-2 cells should be used within passages 20-50, as higher passages can alter gene expression and barrier properties.^[11] Regular mycoplasma testing is essential, often performed via PCR or indicator cell culture methods, to ensure contamination-free stocks.^[11] For enhanced differentiation toward enterocytic phenotypes, variations include reducing serum to 2% FBS after initial attachment or supplementing with 1-5 mM sodium butyrate during the differentiation period.^[36]^[37] These adjustments promote polarization without compromising viability, though standard protocols suffice for routine propagation.^[37] Viability and health are monitored using trypan blue exclusion, targeting >90% viable cells prior to subculturing, alongside visual inspection for uniform morphology and absence of contamination.^[1]

Monolayer Formation Techniques

Caco-2 monolayers are typically formed on porous membrane inserts to enable studies of transcellular and paracellular transport across a polarized epithelial barrier. These inserts, such as Transwell or Snapwell systems, feature polycarbonate or polyester membranes with pore sizes ranging from 0.4 to 3 μm, which allow nutrient diffusion while preventing cell migration and supporting bidirectional access to apical and basolateral compartments for transport assays.^[38]^[39] Cells are seeded at densities of 5 × 10⁴ to 1 × 10⁵ per insert onto these supports, followed by a culture period of 14-21 days to achieve full differentiation into a confluent monolayer with tight junctions and brush border enzymes.^[40]^[41] During this time, transepithelial electrical resistance (TEER) stabilizes as an indicator of barrier integrity.^[42] To enhance physiological relevance, co-cultures integrate mucus-producing HT29-MTX cells at ratios of 10-20% with Caco-2 cells, forming a heterogeneous epithelium that secretes a mucus layer mimicking the intestinal environment.^[43] Monolayer integrity is validated using permeability assays with paracellular markers: apparent permeability (P_app) for Lucifer yellow should be below 1 × 10^{-6} cm/s to confirm low leakage, while mannitol serves as a standard probe for tight junction functionality.^[44]^[45] Commercial kits like CacoReady provide pre-differentiated Caco-2 monolayers on inserts, shipped at day 13 of culture for immediate use up to day 21, thereby minimizing inter-laboratory variability in differentiation and barrier formation.^[46]^[47]

Research Applications

Drug Permeability and Absorption Studies

Caco-2 monolayers serve as a primary in vitro model for evaluating drug permeability across the intestinal epithelium, enabling predictions of oral bioavailability early in pharmaceutical development. These cells form polarized barriers mimicking the human small intestine, allowing assessment of passive diffusion and active transport mechanisms. Permeability studies typically involve measuring the flux of test compounds across the monolayer under controlled conditions, with results providing insights into absorption potential.^[8] Bidirectional transport assays are routinely employed to distinguish between absorptive and secretory pathways, particularly to identify substrates of efflux transporters like P-glycoprotein (P-gp). In these experiments, drug flux is quantified from the apical-to-basolateral (A-B) direction, simulating intestinal absorption, and from the basolateral-to-apical (B-A) direction, indicating potential efflux. The efflux ratio, calculated as the B-A permeability divided by A-B permeability, helps classify compounds; ratios greater than 2 typically suggest P-gp-mediated efflux, guiding further investigation into drug-transporter interactions.^[48]^[49] The apparent permeability coefficient (P_app) is the key metric derived from these assays, providing a standardized measure of transport efficiency. It is computed using the formula:

P_{app} = \frac{dQ/dt}{A \times C_0}

where dQ/dt represents the steady-state flux rate (amount transported per unit time), A is the surface area of the monolayer, and C_0 is the initial donor concentration. This calculation assumes sink conditions on the receiver side and is performed after verifying monolayer integrity via transepithelial electrical resistance (TEER).^[50]^[51] Caco-2-derived P_app values correlate strongly with in vivo human intestinal absorption, with high permeability (P_app > 10^{-5} cm/s) predicting greater than 90% absorption for many compounds. This relationship has been validated against the Biopharmaceutics Drug Disposition Classification System (BDDCS), where high permeability aligns with extensive metabolism and distribution, aiding in the classification of drugs into BDDCS Classes 1 and 2. Such correlations support the model's reliability for forecasting bioavailability without relying solely on animal data.^[7]^[52] In pharmaceutical screening, Caco-2 assays are integral to Biopharmaceutics Classification System (BCS) evaluations, categorizing compounds based on solubility and permeability to inform formulation strategies and biowaiver applications. For instance, high P_app values confirm BCS Class 1 or 3 status for immediate-release products, streamlining regulatory approvals. Similarly, for peptide-based therapeutics, such as insulin analogs, the model assesses paracellular and transcellular routes, highlighting low permeability challenges for oral delivery of biologics.^[53]^[54] Post-2018 advancements have enhanced Caco-2's predictive accuracy, including hybrid models combining Caco-2 with MDCK cells for refined transporter studies and integration of artificial intelligence for machine learning-based permeability predictions. These AI approaches, trained on large datasets, achieve up to 90% accuracy in classifying permeability classes, reducing experimental variability and accelerating drug discovery as of 2025. Microfluidic adaptations further mimic dynamic intestinal flow, improving correlations with in vivo absorption.^[48]^[55]^[56]

Toxicity and Other Biomedical Uses

Caco-2 cells are widely employed in cytotoxicity assays to evaluate the toxic potential of various compounds, leveraging their ability to form differentiated monolayers that mimic intestinal epithelial barriers. Common methods include the MTT assay, which measures mitochondrial dehydrogenase activity to assess cell viability, and the LDH release assay, which detects lactate dehydrogenase leakage as an indicator of plasma membrane damage. For instance, in studies of phycotoxins, the LDH assay demonstrated higher sensitivity in detecting cytotoxicity in Caco-2 cells compared to Alamar Blue, with IC50 values varying by toxin type and exposure duration. Similarly, nanotoxicology research has utilized these assays to investigate nanoparticle uptake and effects; colloidal silver nanoparticles exhibited dose- and time-dependent cytotoxicity in Caco-2 cells, with significant viability reduction at concentrations as low as 5 μg/mL after 48 hours, attributed to oxidative stress and membrane disruption.^[57]^[58] In infection models, Caco-2 monolayers serve as a platform to study pathogen invasion and host responses, particularly for enteric bacteria like Salmonella. Polarized Caco-2 cells infected apically with Salmonella typhimurium reveal bacterial adhesion and invasion mechanisms. These models have quantified invasion rates, modulated by host factors such as guanylate cyclase C signaling, which reduces invasion.^[59] For viral infections, Caco-2 cells support replication cycles of pathogens like hepatitis E virus genotype 1, achieving efficient transfection and propagation, enabling studies on viral entry and maturation without significant cytotoxicity at low multiplicities of infection.^[60] Caco-2 cells model nutrient transport processes, including lipid and peptide absorption, due to their expression of relevant transporters and receptors. In lipid studies, Caco-2 monolayers facilitate the uptake of fatty acids like oleic acid, with co-culture systems enhancing transport efficiency by simulating adipose interactions. Peptide transport assays have shown that lipidated peptides, such as those with varying chain lengths, undergo endocytosis-mediated translocation, with longer chains significantly increasing absorption rates across the monolayer.^[61] Regarding vitamin D, Caco-2 cells express the vitamin D receptor (VDR), and treatment with 1,25-dihydroxyvitamin D3 upregulates VDR-mediated uptake of associated nutrients; for example, it enhances folic acid transport via increased expression of folate receptors, while glycocholic acid and butyrate synergistically boost calcium absorption up to 9-fold through VDR activation.^[62]^[63] As a model for colorectal carcinoma, Caco-2 cells are used to investigate metastasis and chemotherapy resistance, reflecting their origin from a human colon adenocarcinoma. In metastasis studies, Caco-2 variants derived from serial liver transplantation exhibit enhanced invasive potential, with ROCK2 inhibition promoting collective cell migration and stromal invasion in 3D models. For chemotherapy resistance, Caco-2 cells overexpressing genes like RBCK1 or TIAM1 display reduced sensitivity to agents such as oxaliplatin due to stemness activation and anoikis resistance linked to KRAS mutations.^[64]^[65] Emerging applications in the 2020s integrate Caco-2 cells into organ-on-a-chip platforms to study gut microbiome interactions, providing dynamic models of host-microbe dynamics. These microfluidic systems co-culture Caco-2 monolayers with anaerobic bacteria, simulating peristalsis and revealing microbiome influences on barrier integrity, such as reduced invasion by Salmonella in the presence of protective postbiotics from Lactiplantibacillus plantarum. Such models have demonstrated that microbial metabolites modulate Caco-2 responses, enhancing research into dysbiosis-related toxicity and nutrient modulation in a physiologically relevant context.^[66]^[67]

Advantages, Limitations, and Comparisons

Strengths and Validation

The Caco-2 cell model offers significant high-throughput capabilities, enabling the cost-effective screening of thousands of drug compounds annually through automated protocols and scalable multi-well formats. This efficiency stems from optimized cultivation and permeability assays that reduce time and resource demands compared to traditional methods, facilitating early-stage drug discovery in pharmaceutical pipelines.^[68]^[69] Regulatory agencies, including the FDA and EMA, endorse the Caco-2 model for biowaiver applications under the Biopharmaceutics Classification System (BCS), allowing waiver of in vivo bioavailability studies when apparent permeability (P_app) values demonstrate strong correlation with human jejunal data for passively transported compounds. This acceptance is supported by extensive validation showing reliable prediction of intestinal absorption, as outlined in ICH M9 guidelines.^[70]^[71] Ethically, the Caco-2 model adheres to the 3Rs principle—replacement, reduction, and refinement—by serving as a human-relevant in vitro alternative to animal testing, while maintaining a consistent epithelial phenotype across laboratories worldwide. This reduces reliance on in vivo models and promotes reproducible results without ethical concerns associated with animal use.^[72] Quantitative validations confirm the model's robustness, with studies reporting 80-90% accuracy in predicting human absorption for passively diffused drugs, based on correlations between Caco-2 permeability and clinical data.^[73]

Drawbacks and Variability Issues

The Caco-2 cell line, derived from human colorectal carcinoma, exhibits incomplete mimicry of the small intestine epithelium, as it overexpresses certain colonic markers such as MUC2 and underrepresents key small intestine-specific transporters like organic anion-transporting polypeptides (OATPs), including OATP1A2 and OATP2B1, which are crucial for uptake of endogenous compounds and drugs.^[74] This colonic bias limits its accuracy in modeling jejunal or ileal absorption, where OATP expression is higher, leading to potential underestimation of transporter-mediated influx in permeability studies.^[75] Passage-dependent effects contribute significantly to variability, with dedifferentiation occurring after approximately passage 60, resulting in altered morphology, reduced enzyme activities like alkaline phosphatase, and variability in apparent permeability coefficients (P_app) for test compounds due to inconsistent monolayer integrity.^[76] Transepithelial electrical resistance (TEER) values, a marker of barrier function, peak around passages 30-50 but decline thereafter, exacerbating inconsistencies in transport measurements across experiments.^[77] Inter-laboratory differences further amplify variability, with TEER measurements fluctuating due to variations in media composition, fetal bovine serum lots, and seeding densities (typically 10^5 to 5x10^5 cells/cm²), yielding coefficients of variation (CV) up to 30% in permeability assays.^[11] These discrepancies arise from heterogeneous cell subpopulations and non-standardized protocols, making direct comparisons of P_app values challenging and reducing reproducibility across studies.^[78] Metabolic limitations are prominent, as Caco-2 cells display substantially lower CYP3A4 activity—responsible for metabolizing about 50% of drugs—compared to native human enterocytes, often requiring induction agents like 1α,25-dihydroxyvitamin D3 for even modest expression.^[79] This reduced enzymatic capacity hinders accurate prediction of prodrug activation and first-pass metabolism, potentially overestimating bioavailability for CYP3A4 substrates in absorption studies.^[80] Recent critiques, particularly in 2024-2025 literature, highlight overreliance on Caco-2 data in AI-driven drug discovery models, where historical data gaps and assay biases amplify prediction errors, such as in permeability classification, due to underrepresented chemical spaces and inherent experimental variability.^[81] This perpetuates flawed in silico extrapolations, underscoring the need for cautious integration of Caco-2-derived datasets in machine learning pipelines for ADMET forecasting.^[82]

Alternatives to Caco-2 Models

Madin-Darby canine kidney (MDCK) cells serve as a common alternative to Caco-2 models for assessing intestinal drug permeability, particularly in high-throughput screening scenarios. These cells differentiate more rapidly, forming tight monolayers in 3-5 days compared to the 21 days typically required for Caco-2, enabling faster experimental turnaround. MDCK cells, especially the MDCK-MDR1 variant overexpressing human P-glycoprotein, excel in evaluating efflux transporter activity, often providing permeability rankings that correlate well with Caco-2 for passively transported compounds. However, their canine origin limits human relevance, as they exhibit differences in transporter expression and may overestimate permeability for some substrates due to lower paracellular leakage.^[83]^[84]^[85] Co-cultures of Caco-2 with HT29 cells, particularly the mucus-secreting HT29-MTX subline, enhance model realism by incorporating a physiological mucus layer absent in standard Caco-2 monolayers. In typical 70:30 Caco-2:HT29 ratios, these systems better mimic the intestinal barrier's heterogeneity, with HT29 cells representing goblet cells that produce mucin and influence drug diffusion. Such co-cultures demonstrate improved prediction of nanoparticle and toxin interactions at the mucosal surface, though they increase experimental complexity due to variable mucus thickness and longer differentiation times. Permeability measurements in these models often align with Caco-2 for small molecules but provide more accurate barriers for mucoadhesive compounds.^[86]^[87]^[88] Induced pluripotent stem cell (iPSC)-derived enteroids offer a patient-specific, three-dimensional alternative that recapitulates the crypt-villus architecture and cellular diversity of human intestine, surpassing Caco-2's monoculture limitations. These organoid-derived monolayers express a broader range of transporters and metabolic enzymes, yielding permeability correlations with in vivo human data that are comparable or superior to Caco-2 for both passive and active transport. For instance, iPSC-enteroids better predict regional differences in absorption along the jejunum and duodenum. Despite their higher fidelity to native tissue, challenges include high production costs, variability from donor genetics, and technical demands for 3D-to-2D adaptation in permeability assays.^[89]^[90]^[91] Ussing chambers mounted with ex vivo animal intestinal tissues, such as porcine or rat jejunum, represent a gold standard for validating permeability models due to their retention of native architecture, including multiple cell types and innervation. These setups measure transepithelial electrical resistance and flux under physiological conditions, often providing more accurate predictions of drug absorption than Caco-2 for ion-sensitive or poorly soluble compounds. However, ethical concerns over animal use, short tissue viability (typically 1-3 hours), and inter-animal variability restrict their routine application, positioning them primarily as confirmatory tools rather than scalable alternatives.^[91]^[92]^[93] Computational models, including physiologically based pharmacokinetic (PBPK) simulations and quantitative structure-activity relationship (QSAR) approaches, emerge as cell-free alternatives for predicting drug absorption without relying solely on Caco-2 data. PBPK models integrate Caco-2 permeability inputs with in silico estimates of solubility and metabolism to simulate whole-body pharmacokinetics, achieving high accuracy for BCS class I-III drugs in regulatory submissions. Recent 2025 advancements in QSAR leverage machine learning on large datasets to forecast permeability directly from molecular descriptors, reducing experimental needs and enabling early-stage screening. These tools are particularly preferable for virtual prototyping but require validation against empirical data to account for transporter interactions.^[94]^[95]^[96]

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Cellosaurus cell line Caco-2 (CVCL_0025)
Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability.
[36]
Effect of Passage Number and Culture Time on the Expression ... - NIH
Conclusion: The use of Caco-2 cells at passage number range of 5-15 and culture time of 72 hours provides proper conditions for IDE-related studies.
[37]
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0245348
[38]
Colon cancer cell differentiation by sodium butyrate modulates ...
Jan 20, 2021 · The treatment of Caco-2 cells with NaBT increased production of ATP by oxidative phosphorylation, enhanced mitochondrial spare respiratory ...
[39]
[PDF] Permeable Supports Product Selection Guide - Corning
Transwell® permeable supports are available in a broad range of pore sizes from 0.4 to 8.0 µm. This high pore density membrane is suitable for a variety of ...Missing: bidirectional | Show results with:bidirectional<|separator|>
[40]
Caco‐2 Cells as a Model for Intestinal Absorption - Current Protocols
Feb 1, 2011 · Caco-2 cells were established from gastrointestinal tumors (Fogh et al., 1977); when grown as a monolayer on a permeable filter support for ∼3 ...
[41]
Caco-2 Monolayer Permeability and Stability of Chamaelirium ...
Apr 26, 2019 · Approximately 1 × 105 cells (passage 39) were seeded onto Transwell inserts. On days 1–8, cells were fed with plate media on alternate days.Results And Discussion · Figure 3 · Experimental Section
[42]
Caco-2 Cells for Measuring Intestinal Cholesterol Transport
Apr 11, 2020 · The LXR agonist GW3965 did not downregulate NPC1L1 at any of the time points (2–30 h) (Fig. 7c), which is in contrast to already published ...Missing: doubling | Show results with:doubling
[43]
Evaluation of Caco-2 and human intestinal epithelial cells as in vitro ...
Previous investigation of HIEC monolayers indicate superior drug absorption prediction [30] and better physiological representation of small intestinal ...Missing: doubling | Show results with:doubling
[44]
Defining conditions for the co-culture of Caco-2 and HT29-MTX cells ...
On the other hand, the seeding density and the Caco-2:HT29-MTX ratio exerted no significant influence on TEER values and the drug permeability coefficients.
[45]
Re-Use of Caco-2 Monolayers in Permeability Assays—Validation ...
Sep 26, 2021 · Caco-2 monolayers are a common in vitro model used to evaluate human intestinal absorption. The reference protocol requires 21 days ...
[46]
A Paracellular Modeling Approach | Molecular Pharmaceutics
The obtained acamprosate Papp, 1.56 ± 0.28 × 10–7 cm·s–1, is similar to the Papp of molecular markers for paracellular permeability, namely, mannitol (2.72 ± ...
[47]
[PDF] A Novel, Ready-to-Use System for In Vitro Intestinal Absorption ...
The CacoReady™ kit consists of differentiated Caco-2 cell barriers, shipped on day 13 of differentiation, and provides 14-day polarized cultures of Caco-2 ...
[48]
Application of Caco-2 Cell Line in Herb-Drug Interaction Studies - NIH
The MDCK has a shorter cultivation period (3 – 5 days) compared to 21 days of Caco −2 cell line. ... 2 × 105 cells/cm2 and cultured for 24 h. Investigated ...Caco-2 Cell Line · Comparison Of Caco-2 With... · Generalized Caco-2 Cells...Missing: 10⁴ 10⁵ stabilization
[49]
The pH-dependence of efflux ratios determined with bidirectional ...
MDCK/Caco-2 assays serve as essential in vitro tools for evaluating membrane permeability and active transport, especially mediated by P-glycoprotein (P-gp) ...2. Theory · 2.1. Multi-Barrier Model · 4.3. Model Fits
[50]
Caco-2 Method Validation - Bienta
When a compound has an efflux ratio of greater than 2, it suggests that the compound may be subject to active efflux. The data for compounds are mean of two ...
[51]
Caco-2 Permeability Assay - Evotec
... . The permeability coefficient (Papp) is calculated from the following equation: P app = dQ / dt C 0 x A. Where Papp is the apparent permeability ...
[52]
Caco-2 monolayers in experimental and theoretical predictions of ...
This review examines the use of Caco-2 monolayers in the prediction of intestinal drug absorption. First, the different routes of drug transport in Caco-2 ...
[53]
The Use of BDDCS in Classifying the Permeability of Marketed Drugs
They reported that the BCS classification using cimetidine as the reference permeability drug appeared to exhibit the best overall agreement with BDDCS, where ...
[54]
Caco-2 cells and Biopharmaceutics Classification System (BCS) for ...
Abstract. Objectives: The Caco-2 cell monolayer model is widely used as a standard screening tool for studying the mechanisms of cellular drug transport.
[55]
Drug-transporter mediated interactions between anthelminthic and ...
May 4, 2017 · Drug-transporter mediated interactions between anthelminthic and antiretroviral drugs across the Caco-2 cell monolayers. Gabriel Kigen ...Missing: example | Show results with:example
[56]
Permeation of Insulin, Calcitonin and Exenatide across Caco-2 ...
Caco-2 monolayers are one of the most widely used in vitro models for prediction of intestinal permeability of therapeutic molecules.<|separator|>
[57]
First report on machine learning based multiclass classification of ...
ABSTRACT. Evaluating the permeability of different molecular structures across the Caco-2 cell line is crucial for drug discovery and development.
[58]
The Caco-2 Model: Modifications and enhancements to improve ...
Aug 25, 2022 · The aim of this review is to discuss such modifications to enhance this model's utility, predictive performance, and reproducibility.Missing: integration | Show results with:integration
[59]
Higher-throughput screening for Caco-2 permeability utilizing a ...
By integrating these components together with automated method development and data processing, a system capable of screening 100 compounds per week for Caco-2 ...Missing: annually | Show results with:annually
[60]
https://pmc.ncbi.nlm.nih.gov/articles/PMC11261143/
[61]
[PDF] M9 Biopharmaceutics Classification System- Based Biowaivers - FDA
A validated Caco-2 method used for drug permeability determinations should employ conditions established during the validation and include a moderate and a high ...Missing: 2000s | Show results with:2000s
[62]
[PDF] ICH M9 guideline on biopharmaceutics classification system-based ...
Feb 10, 2020 · Furthermore, numerous laboratories have successfully validated Caco-2 cell line systems for BCS classification using these reference drug ...
[63]
Proliferation and Differentiation of Intestinal Caco-2 Cells Are ... - NIH
Nov 5, 2021 · The frequently used Caco-2 cell line is considered to reflect characteristics of differentiated intestinal epithelium.
[64]
Correlation between oral drug absorption in humans and apparent ...
A good correlation was obtained between data on aral absorption in humans and the results in the Caco-2 model. Drugs that are completely absorbed in humans had ...
[65]
[PDF] ICCVAM Biennial Progress Report 2020-2021
Aug 1, 2022 · The human Caco-2 cell line, derived from human colon epithelial cancer cells, is currently used in an in vitro model of human intestinal ...Missing: 2020s | Show results with:2020s
[66]
OATPs, OATs and OCTs: the organic anion and cation transporters ...
Predominant contribution of organic anion transporting polypeptide OATP-B (OATP2B1) to apical uptake of estrone-3-sulfate by human intestinal Caco-2 cells.
[67]
The Organic Anion-Transporting Peptide 2B1 Is Localized ... - PubMed
Apr 10, 2017 · It was the aim of this study to investigate its localization and function in the human jejunum and in the frequently used intestinal Caco-2 cell line.
[68]
The influence of culture time and passage number on the ... - PubMed
TEER values declined after about passage 60; sucrase remained elevated but variable. ... These results emphasize the inherent variability in Caco-2 cell models ...Missing: effects P_app
[69]
[PDF] The Influence of Culture Time and Passage Number on the ...
The purpose of the present study was to monitor the structural and functional characteristics of our. Caco-2 cells during the growth period and from different.
[70]
Interlaboratory Variability in the Madin–Darby Canine Kidney Cell ...
The monolayer TEER for Lab H was relatively high (within 850–1250 Ω·cm2) compared to that for Labs B and C (Table 1) and may reflect the general proteome or ...
[71]
Expression of enzymatically active CYP3A4 by Caco-2 cells grown ...
Treatment with 1alpha,25-dihydroxyvitamin D3 increases CYP3A4 expression in Caco-2 cells, requiring an extracellular matrix and serum for maximal expression.
[72]
In-Depth Characterization of EpiIntestinal Microtissue as a Model for ...
Due to the lack of cytochrome P450 3A4 (CYP3A4) activities, Caco-2 model is not suitable for the investigation of intestinal first-pass metabolism. The purpose ...
[73]
A Case Study from Bayer's Caco-2 Permeability Database
Nov 20, 2024 · In the discovery setting 'the rule of 5' predicts that poor absorption or permeation is more likely when there are more than 5 H-bond donors, 10 ...
[74]
ADMET evaluation in drug discovery: 21. Application and industrial ...
Jan 10, 2025 · The Caco-2 cell model has been widely used to assess the intestinal permeability of drug candidates in vitro, owing to its morphological and ...
[75]
Cell-Based Intestinal Permeability Models - Creative Bioarray
The Madin-Darby canine kidney (MDCK) is considered as an alternative to Caco- 2 cell for permeability studies. MDCK cells exhibit a shorter culture time (3 ...
[76]
Comparison of MDCK-MDR1 and Caco-2 cell based permeability ...
MDCK permeability based assays provide an attractive alternative to Caco-2 cells in terms of the increasing throughput, and reducing the time required to ...
[77]
Comparison of MDCK-MDR1 and Caco-2 cell based permeability ...
Results: Both Caco-2 and MDCK permeability assays produced similar Papp results for potential anti-malarial compounds with low and medium permeability.
[78]
Co-cultivation of Caco-2 and HT-29MTX - NCBI - NIH
The co-culture of Caco-2 and HT-29MTX is a model that is useful for the investigation of transport over intestinal epithelium and for bacterial adhesion and ...
[79]
Caco-2/HT29-MTX co-cultured cells as a model for studying ...
Oct 7, 2021 · The TEER values of ≈ 500 Ω * cm2 for co-cultured and Caco-2 cells and ≈ 300 Ω * cm2 for HT29-MTX cells are in accordance with previously ...
[80]
Morphofunctional properties of a differentiated Caco2/HT-29 co ...
At T14, HT-29 cells reduced to 18.4% and formed domes, indicative of transepithelial transport of nutrients. This Caco2/HT-29 co-culture could be considered a ...Introduction · Materials and methods · Results · Discussion
[81]
Comparison of iPSC-derived human intestinal epithelial cells with ...
Nov 2, 2024 · A promising alternative for Caco-2 cells are human stem cell-derived intestinal models, like human induced pluripotent stem cell (hiPSC)- ...
[82]
Alternative functional in vitro models of human intestinal epithelia - NIH
In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers ...
[83]
Human enteroid monolayers as a potential alternative for Ussing ...
Oct 1, 2024 · Human enteroid monolayers as a potential alternative for Ussing chamber and Caco-2 monolayers to study passive permeability and drug efflux.
[84]
Comparison of Ussing Chamber and Caco-2 Model in Evaluation of ...
Currently, Caco-2 and Ussing chamber are both used in the study of intestinal permeability of drugs at different stages of drug development. However, ...Missing: animal | Show results with:animal
[85]
Drug Transport across Porcine Intestine Using an Ussing Chamber ...
Mar 21, 2019 · Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: Permeabilities ... Ussing chamber system, equipped with intestinal tissue from rats and dogs ...
[86]
A Combination of Machine Learning and PBPK Modeling Approach ...
Jun 25, 2024 · This study developed a whole-body PBPK model and ML models of plasma protein fraction unbound ( ), Caco-2 cell permeability, and total plasma ...
[87]
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0257824
[88]
Evaluation of the Success of High-Throughput Physiologically ...
A novel high-throughput PBPK (HT-PBPK) approach was evaluated to assess the scalability of PBPK predictions for a larger number of compounds in drug discovery.