# Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity

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## Abstract

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_{app}values that are dependent on the sampling duration. The assay also leads to a small decrease in the cell monolayer TEER, which is fully recovered when cell monolayers are incubated with culture media for two full days. When this procedure is followed, the cell monolayers may be used for permeability assays on days 22, 25, and 28, triplicating the throughput of this important assay.

## 1. Introduction

_{app}) through Caco-2 monolayers and human jejunal absorption [7,8,9]. Caco-2 cells are not, however, intrinsically intestinal epithelial cells, and this in vitro model shows limitations regarding the metabolizing enzymes and the active transporters expressed [1,10]. When using cell monolayer permeability assays in drug screening, it may therefore be convenient to include more advanced models, such as those derived from intestinal epithelial stem cells [11]. Caco-2 permeability assays are nevertheless excellent in vitro models to predict permeability through passive routes and give important insight regarding active transport [7,8,9,10].

## 2. Materials and Methods

#### 2.1. Reagents and Materials

^{−1}/streptomycin 10,000 µg mL

^{−1}solution (Pen/Strep), 0.25% (w/v) Trypsin, ethylenediamine tetraacetic acid (EDTA, ≥98.5%) Triton X-100, sodium bicarbonate (≥99.7%), Hank’s balanced salt solution (HBSS), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), and Lucifer yellow CH di-potassium salt (≥99%) were purchased from Sigma-Aldrich Química S.A. (Sintra, Portugal). Fetal bovine serum (FBS) was obtained from Gibco-Life Technologies (Porto, Portugal). Corning

^{®}Transwell™ 12-well inserts with a polycarbonate membrane (1.12 cm

^{2}surface area, 0.4 µm pore size) and 12-well cell culture plates were obtained from VWR (Lisboa, Portugal). The primary and secondary antibodies were purchased from Alfagene (Lisboa, Portugal), Dako fluorescence mounting medium from Agilent (Lisboa, Portugal), and bovine serum albumin (BSA) from Applichem (Darmstadt, Germany). Ammonium formate (≥99%) and organic solvents of analytical grade were acquired from Fischer Scientific (Lisboa, Portugal).

#### 2.2. Caco-2 Cell Culture and Seeding

^{2}flasks in culture medium (DMEM supplemented with 1% (v/v) NEAA, 10% (v/v) heat-inactivated FBS), in a humidified atmosphere of 5% CO

_{2}at 37 °C. As recommended in the reference protocol, the permeability assays were performed using cells in passages 95–105. The cells were thawed on passage 89 and sub-cultured twice a week at or shortly before 90% confluency by a 1:8 split. To avoid seeding aggregated cells and the formation of multilayers in Transwell™ inserts, the cell clusters were disaggregated into single cells by passing them 3 times through a syringe needle of 23 gauges (BD Falcon) before cell counting. Caco-2 cells were seeded at 2.6 × 10

^{5}cells/cm

^{2}on 12-well polycarbonate filter inserts. The culture medium (with 1% (v/v) Pen/Strep) was replaced by fresh medium 6 h following seeding and then every 2–3 days thereafter until transport experiments.

#### 2.3. Permeability Assays and Transepithelial Electrical Resistance (TEER)

_{2}at 37 °C with an orbital shaker (IKA-Schüttler MTS

_{4}, JMGS, Lisboa, Portugal) at 50 rpm for 8 min. The washing step was repeated with new HBSS. The TEER was measured in HBSS at 37 °C using a Millicell

^{®}ERS-2 voltmeter equipped with a chopstick electrode pair (Merck, Lisboa, Portugal). A filter insert without cells was included in each set of experiments for correction of the TEER value for the background resistance. The resistance values (Ω cm

^{2}) of the cell monolayers were obtained by subtracting the TEER value from blank inserts and multiplying by the surface area of the insert. All cell monolayers presented TEER values above 200 Ω cm

^{2}. At least one insert of the set was fixed and stained for confocal microscopy analysis (see next section). The remaining ones were used to perform the apical to basolateral permeability assays with the paracellular marker LY. For this, the donor HBSS washing solution was decanted, and the inserts were placed into empty wells of a fresh 12-well plate. The permeability assay was initiated with the addition of 450 µL of the LY solution (20 µM in HBSS at 37 °C) simultaneously to the donor compartments of 3 to 4 insert, from which a 50 µL aliquot was immediately collected and stored for the calculation of the solute concentration in the donor compartment at t = 0. The inserts containing the LY solution were subsequently positioned in a pre-prepared 12-well plate containing HBSS (1.2 mL per well), and the lid-covered plate was placed on the incubator (without CO

_{2}, 50 rpm and 37 °C). At the defined sampling times, the plate was positioned into the laminar flow hood and the inserts were transferred into new wells containing fresh HBSS. The high fluorescence quantum yield of LY allows its detection by HPLC, even for the very small amount expected to permeate during short permeation intervals. In this work we used 10, 20, 30, and 60 min or 60 min only as sampling times. At the end of the assay, inserts were transferred to empty wells and 50 µL was taken from the donor compartment for the calculation of the solute mass balance. The remaining solution was decanted, the inserts were placed on new wells containing 1.5 mL of pre-warm HBSS, and 0.5 mL of pre-warmed HBSS was added to each insert for TEER measurements. Some selected monolayers were prepared for microscopy; the remaining were incubated with culture media for re-use, as described below.

_{2}at 37 °C. To maximize the use of the already established cell monolayers up to three times during the total period where the monolayers are stable (21–30 days) [16,17,33], the days selected for the permeability experiments were 22, 25, and 28 post-seeding.

_{app}) of LY was determined from the amount of solute transported across the monolayer per time:

^{2}), ${V}^{D}$ is the volume of the donor compartment (0.4 cm

^{3}), and ${Q}_{0}^{D}$ is the amount of solute in the donor compartment at the beginning of the time interval considered. When several sampling times were performed, the amount of solute in the donor compartment at the beginning of the time interval considered was calculated from that in the beginning of the experiment by subtracting the solute that reached the acceptor compartment in the preceding sampling times. This equation is equivalent to that indicated in the reference protocol for the instantaneous permeability coefficient [15]. The only difference is that, instead of directly considering the initial concentration of the tested compound in the donor compartment, we explicitly indicate the amount of compound and the volume of the compartment. This avoids subjectivity when using different units for the distinct variables. In Equation (1), the amount of solute may be in the most convenient units, provided that the same units are used for the solute in both compartments ($\Delta {Q}^{A}$ and ${Q}_{0}^{D}$). Additionally, it becomes more intuitive that the surface and the volume must use the same length units, which will be that of the resulting P

_{app}.

#### 2.4. LY Quantification

^{2}≥ 0.9998) was obtained in the concentration range from 0.001 to 1 µM and 0.001 to 0.2 µM, respectively (see Supplementary Material—Section S1 for details). The lower limit of quantification was 0.02 µM for 100 μL injections and 0.0005 µM for 900 μL. The corresponding limits of detection were 0.006 µM and 0.0002 µM.

#### 2.5. Confocal Laser Screening Microscopy

^{2}) was analyzed on at least 2 independent cell monolayers per condition. Each image was manually threshold to select nuclei and ZO-1 only fluorescence.

#### 2.6. Statistical Analysis

_{app}, and uncertainty is expressed as 95% confidence intervals, calculated from the standard deviation of LogP

_{app}[37,38].

_{95}and the p-value were calculated for each βi; p < 0.05 was considered as statistically different from zero. This statistical analysis was performed using GraphPad Prism (version 8.4.2, San Diego, CA, USA).

## 3. Results and Discussion

#### 3.1. Effect of Re-Use on the Cell Monolayer Integrity after Single Time Sampling Permeability Assays

^{2}) when compared to day 22 and day 25, which show a similar value of TEER (1036 ± 491 and 986 ± 432 Ω cm

^{2}, respectively), the overall distribution at all days being 1067 ± 473 Ω cm

^{2}. The distribution of TEER values after the execution of a permeability assay are represented in Figure 1B. It is observed that there is a small decrease in the mean value of TEER (766 ± 383 Ω cm

^{2}, for the best fit distribution of the cumulative data at all days), which is mostly due to a decrease in the TEER values obtained after the permeability experiment for cells on day 22 (from 1036 to 719 Ω cm

^{2}), but also for cells on day 28. Nevertheless, TEER values above the threshold of 200 Ω cm

^{2}, which is commonly accepted for a confluent cell monolayer [39,40], are obtained in all cases; see Supplementary Material—Table S1 for further details.

_{app}, calculated from a single 60 min sampling interval, are shown in Figure 3.

_{app}values. The quality of the best fit is significantly improved when a LogNormal distribution is considered (black line), which is in fact the statistical distribution expected for rate constants [37]. Due to the asymmetry in the distribution, the uncertainty associated with this parameter should be expressed as a confidence interval [37]. This interval may be calculated from the parameters of the LogNormal distribution that best describes the results, or directly from the average and standard deviation of the observed values of LogP

_{app}, leading, respectively, to [1.1, 4.0] × 10

^{−7}cm/s and [1.2, 3.9] × 10

^{−7}cm/s at 95% confidence (CI

_{95}), and an average value of P

_{app}equal to 2.1 × 10

^{−7}cm/s for both analyses.

_{app}values obtained with cell monolayers on days 25 and 28 is also shown in Figure 3. The number of assays performed with cell monolayers on day 25 that have been previously used on day 22 is large (N = 23) and leads to a well-defined distribution frequency (light green). However, the number of assays performed on day 25 for cell monolayers used on a single day (dark green, N = 6) is too small to allow the characterization of the frequency distribution. The characteristic value and confidence intervals must therefore be calculated from the average and standard deviation of the observed values of Log P

_{app}, leading to µ = 1.8 × 10

^{−7}cm/s and CI

_{95}equal to [0.6, 5.3] × 10

^{−7}cm/s. The same average value is also obtained for P

_{app}on re-used cell monolayers. In this case, the much larger number of assays leads to a smaller uncertainty, with the CI

_{95}being [1.1, 2.6] × 10

^{−7}cm/s. This shows that the two conditions do not lead to statistically different results and, thus, the cell monolayers that have been used on day 22 may be re-used on day 25 if following the proposed protocol. When this treatment is performed for cells on day 28 post-seeding, the parameters obtained are µ = 3.2 × 10

^{−7}cm/s and CI

_{95}equal to [1.7, 6.1] × 10

^{−7}cm/s for cell monolayers used a single time (N = 6) and µ = 1.6 × 10

^{−7}cm/s and CI

_{95}equal to [0.7, 3.6] × 10

^{−7}cm/s for cell monolayers previously used on day 22 and 25 (N = 18). As observed on day 25, the small number of assays performed with single use cell monolayers lead to a large uncertainty, and the two conditions are not statistically different. The collective result led to µ = 1.9 × 10

^{−7}cm/s and CI

_{95}equal to [0.8, 4.7] × 10

^{−7}cm/s. When the results obtained on all days are analyzed (right plot in Figure 3), it is observed that they are very well described by a single LogNormal distribution. This shows that, although a small decrease is observed in the P

_{app}values as cell monolayers are maintained on the inserts (specially at day 28), the distributions are not statistically independent. The parameters obtained from the collective values at all days and conditions are µ = 2.0 × 10

^{−7}cm/s and CI

_{95}equal to [1.0, 3.8] × 10

^{−7}cm/s. The small values obtained for LY permeability show that the cell monolayers are intact and tightly sealed at all conditions, P

_{app}< 5 × 10

^{−7}cm/s [15,39,43]. Thus, the results show that, when the cell monolayer is allowed to recover in culture media for 2 full days between assays, its integrity is not compromised, and they may be re-used on days 25 and 28.

#### 3.2. Effect of Multi-Time Sampling on the Paracellular Permeability through Caco-2 Monolayers

_{app}). Independent distributions, however, are observed for both sampling time intervals, with higher P

_{app}values obtained for the shorter sampling time interval (µ = 15 × 10

^{−7}cm/s and CI

_{95}equal to [7.6, 29] × 10

^{−7}cm/s).

_{A}) during the sample time interval (Figure 4, lower plots). In this case, it is observed that a Normal distribution centered at around −0.6 is obtained for LogQ

_{A}during both sampling intervals (10 or 60 min), corresponding to Q

_{A}around 0.2% (for details see the Supplementary Material—Table S4). A very similar amount of LY was also observed to permeate through Caco-2 monolayers on day 23 post-seeding during a 90 min sampling interval, 0.27% [30]. This shows that, for cell monolayers on day 22 post-seeding, manipulation of the cell monolayer during the permeability assay leads to a significant amount of LY transport and that very little permeation is observed during the sampling time interval, thus leading to P

_{app}values that are strongly dependent on the sampling time interval. When assessing the integrity of the cell monolayer using distinct incubation times, it is therefore preferable to refer to the observed permeability in terms of % of the control solute transported, which is usually indicated as below 0.5% for a tight Caco-2 monolayer [48].

_{app}are still dependent on the sampling time interval, but a significant overlap is observed between the frequency distribution obtained for both conditions. Conversely, the frequency distributions of the amount of LY that permeates shows significant differences between the two sampling time intervals, being higher for the longer sampling interval.

_{app}for different analytes, it is important that the same conditions are used in the permeability assays, namely the day post-seeding and, most importantly, the sampling time intervals.

_{app}; the results obtained for the amount of LY that reaches the acceptor compartment are shown in Figure 5. A small increase in Q

_{A}is observed for consecutive sampling with the same time interval: µ = 0.25, 0.29 and µ = 0.37% on day 22; µ = 0.19, 0.25 and µ = 0.25% on day 25; and µ = 0.09, 0.10, and µ = 0.13% on day 28. An increase is also observed on the width of confidence intervals (see Supplementary Material—Table S4). This shows that repeated manipulation of the cell monolayer leads to more significant perturbation and that the effect is not the same for all cell monolayers. When the cumulative amount of LY that reaches the acceptor compartment during the first 30 min (with sampling at each 10 min) is analyzed, an upward curvature is therefore observed (Supplementary Material—Figure S3). However, when the final sampling at 60 min (Δt = 30 min) is included in the cumulative transport, the non-proportionality between Q

_{A}and the sampling time interval dominates, and a downward curvature is observed for the whole 60 min sampling.

#### 3.3. Morphological Features and Integrity of the Cell Monolayer

_{app}values obtained in the present work suggest a pore radius of approximately 6 Å. Given the several orders of magnitude difference between the resolution of the confocal images, and the tight junctions´ pores through which LY permeates, it is not surprising that a continuous tight junction’s network was observed despite the increased LY permeability.

#### 3.4. Multivariate Analysis

_{app}obtained for cell monolayers at day 22 post-seeding). This introduces some uncertainty in the results, but significantly improves the robustness of the conclusions achieved. A multivariate analysis has been performed to evaluate the correlation between the permeability parameters and the independent variables. When TEER before the permeability assay is considered, the independent variables are the cell batch, cell passage number (between 95 and 105), day post-seeding, re-use of the cell monolayers, and sampling time points used on the previous permeability assays. For the TEER value after the permeability assay, the variable TEER before the assay was also included as an independent variable, and the TEER after the assay was also considered when the multivariate analysis was performed for LY transport (quantified by P

_{app}and Q

_{A}). The results obtained are presented in the Supplementary Material—Table S6, a brief discussion of the major findings is given here.

_{app}, a strong correlation is observed with the duration of the sampling interval, Δt, smaller sampling intervals leading to higher LY P

_{app}. As discussed in Section 3.2, a small negative correlation (not statistically significant) is observed for Q

_{A}. A significant correlation is also observed between P

_{app}and the day post-seeding (cell monolayers becoming more impermeable to LY from day 22 to 28).

^{2}), the area occupied by nuclei, and that occupied by tight junctions containing ZO-1. The results obtained are shown in the Supplementary Material—Tables S7 and S8. A high variability is observed for the cell density: 3.1 (±0.7) × 10

^{5}cells/cm

^{2}at day 22 before any permeability assay (N = 7) and 3.3 (±0.8) × 10

^{5}cells/cm

^{2}for all cell monolayers (N = 52). The correlation is not, however, statistically significant with cell batch, passage number, day post-seeding, or cell monolayer use/re-use. As expected, a positive correlation is obtained between the cell density and the area occupied by the nuclei. This correlation is, however, sub-linear, which indicates that cells (and their nucleus) are more elongated when at higher density and is consistent with a continuous cell monolayer in all situations. A positive correlation was also observed between the cell density and the cell monolayer TEER, suggesting that the thickness of the monolayer may influence the value of TEER obtained. This correlation was strong when only the cell monolayers at day 22 (N = 7) were considered (See Supplementary Material—Figure S5) but become not statistically significant when cell monolayers at all conditions were considered (for details see the Supplementary Material—Section S3). No correlation, however, is observed between the area occupied by nuclei and by tight junctions, or with the amount of LY that has permeated during the assay. This shows that TEER is affected by parameters not related to the cell monolayer tightness, this being better evaluated by the permeability of paracellular markers such as LY. The area occupied by tight junctions does not show statistically significant correlations (or any weak correlation) with any of the variables considered.

## 4. Conclusions

_{app}parameter, which was shown to be skewed towards higher values following a LogNormal distribution. The characteristic value and associated uncertainty cannot therefore be obtained from the average and standard deviation of the observed values of P

_{app}. Instead, the more probable P

_{app}should be calculated from the average of LogP

_{app}and the confidence intervals obtained from the standard deviation of LogP

_{app}and the number of assays [37,38].

## Supplementary Materials

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

## References

- Balimane, P.V.; Chong, S. Cell culture-based models for intestinal permeability: A critique. Drug Discov. Today
**2005**, 10, 335–343. [Google Scholar] [CrossRef] - Volpe, D.A. Advances in cell-based permeability assays to screen drugs for intestinal absorption. Expert Opin. Drug Discov.
**2020**, 15, 539–549. [Google Scholar] [CrossRef] [PubMed] - Hidalgo, I.J.; Raub, T.J.; Borchardt, R.T. Characterization of the Human-Colon Carcinoma Cell-Line (Caco-2) as a Model System for Intestinal Epithelial Permeability. Gastroenterology
**1989**, 96, 736–749. [Google Scholar] [CrossRef] - Hilgers, A.R.; Conradi, R.A.; Burton, P.S. Caco-2 Cell Monolayers as a Model for Drug Transport Across the Intestinal-Mucosa. Pharm. Res.
**1990**, 7, 902–910. [Google Scholar] [CrossRef] - van Breemen, R.B.; Li, Y. Caco-2 cell permeability assays to measure drug absorption. Expert Opin. Drug Metab. Toxicol.
**2005**, 1, 175–185. [Google Scholar] [CrossRef] - Artursson, P.; Palm, K.; Luthman, K. Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv. Drug Deliv. Rev.
**2012**, 64, 280–289. [Google Scholar] [CrossRef] - Lennernas, H.; Palm, K.; Fagerholm, U.; Artursson, P. Comparison between active and passive drug transport in human intestinal epithelial (Caco-2) cells in vitro and human jejunum in vivo. Int. J. Pharmaceut.
**1996**, 127, 103–107. [Google Scholar] [CrossRef] - Artursson, P.; Karlsson, J. Correlation Between Oral-Drug Absorption in Humans and Apparent Drug Permeability Coefficients in Human Intestinal Epithelial (Caco-2) Cells. Biochem. Biophys. Res. Commun.
**1991**, 175, 880–885. [Google Scholar] [CrossRef] - Di, L.; Artursson, P.; Avdeef, A.; Ecker, G.F.; Faller, B.; Fischer, H.; Houston, J.B.; Kansy, M.; Kerns, E.H.; Kramer, S.D.; et al. Evidence-based approach to assess passive diffusion and carrier-mediated drug transport. Drug Discov. Today
**2012**, 17, 905–912. [Google Scholar] [CrossRef] - Sun, H.; Chow, E.C.Y.; Liu, S.; Du, Y.; Pang, K.S. The Caco-2 cell monolayer: Usefulness and limitations. Expert Opin. Drug Metab. Toxicol.
**2008**, 4, 395–411. [Google Scholar] [CrossRef] - Kozuka, K.; He, Y.; Koo-Mccoy, S.; Kumaraswamy, P.; Nie, B.; Shaw, K.; Chan, P.; Leadbetter, M.; He, L.; Lewis, J.G.; et al. Development and Characterization of a Human and Mouse Intestinal Epithelial Cell Monolayer Platform. Stem Cell Rep.
**2017**, 9, 1976–1990. [Google Scholar] [CrossRef] [Green Version] - Oltra-Noguera, D.; Mangas-Sanjuan, V.; Centelles-Sanguesa, A.; Gonzalez-Garcia, I.; Sanchez-Castano, G.; Gonzalez-Alvarez, M.; Casabo, V.G.; Merino, V.; Gonzalez-Alvarez, I.; Bermejo, M. Variability of permeability estimation from different protocols of subculture and transport experiments in cell monolayers. J. Pharmacol. Toxicol. Methods
**2015**, 71, 21–32. [Google Scholar] [CrossRef] - Lee, J.B.; Zgair, A.; Taha, D.A.; Zang, X.W.; Kagan, L.; Kim, T.H.; Kim, M.G.; Yun, H.Y.; Fischer, P.M.; Gershkovich, P. Quantitative analysis of lab-to-lab variability in Caco-2 permeability assays. Eur. J. Pharm. Biopharm.
**2017**, 114, 38–42. [Google Scholar] [CrossRef] - Sambuy, Y.; Angelis, I.; Ranaldi, G.; Scarino, M.L.; Stammati, A.; Zucco, F. The Caco-2 cell line as a model of the intestinal barrier: Influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol. Toxicol.
**2005**, 21, 1–26. [Google Scholar] [CrossRef] [PubMed] - Hubatsch, I.; Ragnarsson, E.G.E.; Artursson, P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc.
**2007**, 2, 2111–2119. [Google Scholar] [CrossRef] [PubMed] - BriskeAnderson, M.J.; Finley, J.W.; Newman, S.M. The influence of culture time and passage number on the morphological and physiological development of Caco-2 cells. Proc. Soc. Exp. Biol. Med.
**1997**, 214, 248–257. [Google Scholar] [CrossRef] - Lu, S.; Gough, A.W.; Bobrowski, W.F.; Stewart, B.H. Transport properties are not altered across Caco-2 cells with heightened TEER despite underlying physiological and ultrastructural changes. J. Pharm. Sci.
**1996**, 85, 270–273. [Google Scholar] [CrossRef] - Liang, E.; Chessic, K.; Yazdanian, M. Evaluation of an accelerated Caco-2 cell permeability model. J. Pharm. Sci.
**2000**, 89, 336–345. [Google Scholar] [CrossRef] - Yamashita, S.; Konishi, K.; Yamazaki, Y.; Taki, Y.; Sakane, T.; Sezaki, H.; Furuyama, Y. New and better protocols for a short-term Caco-2 cell culture system. J. Pharm. Sci.
**2002**, 91, 669–679. [Google Scholar] [CrossRef] [PubMed] - Sevin, E.; Dehouck, L.; Fabulas-da Costa, A.; Cecchelli, R.; Dehouck, M.P.; Lundquist, S.; Culot, M. Accelerated Caco-2 cell permeability model for drug discovery. J. Pharmacol. Toxicol. Methods
**2013**, 68, 334–339. [Google Scholar] [CrossRef] [PubMed] - Cai, Y.K.; Xu, C.S.; Chen, P.Y.; Hu, J.Q.; Hu, R.; Huang, M.; Bi, H.C. Development, validation, and application of a novel 7-day Caco-2 cell culture system. J. Pharmacol. Toxicol. Methods
**2014**, 70, 175–181. [Google Scholar] [CrossRef] - Marino, A.M.; Yarde, M.; Patel, H.; Chong, S.H.; Balimane, P.V. Validation of the 96 well Caco-2 cell culture model for high throughput permeability assessment of discovery compounds. Int. J. Pharmaceut.
**2005**, 297, 235–241. [Google Scholar] [CrossRef] [PubMed] - Balimane, P.V.; Patel, K.; Marino, A.; Chong, S.H. Utility of 96 well Caco-2 cell system for increased throughput of P-gp screening in drug discovery. Eur. J. Pharm. Biopharm.
**2004**, 58, 99–105. [Google Scholar] [CrossRef] [PubMed] - Sevin, E.; Dehouck, L.; Versele, R.; Culot, M.; Gosselet, F. A Miniaturized Pump Out Method for Characterizing Molecule Interaction with ABC Transporters. Int. J. Mol. Sci.
**2019**, 20, 5529. [Google Scholar] [CrossRef] [Green Version] - Alsenz, J.; Haenel, E. Development of a 7-day, 96-well Caco-2 permeability assay with high-throughput direct UV compound analysis. Pharm. Res.
**2003**, 20, 1961–1969. [Google Scholar] [CrossRef] - Bu, H.Z.; Poglod, M.; Micetich, R.G.; Khan, J.K. High-throughput Caco-2 cell permeability screening by cassette dosing and sample pooling approaches using direct injection/on-line guard cartridge extraction/tandem mass spectrometry. Rapid Commun. Mass Spectrom.
**2000**, 14, 523–528. [Google Scholar] [CrossRef] - Laitinen, L.; Kangas, H.; Kaukonen, A.M.; Hakala, K.; Kotiaho, T.; Kostiainen, R.; Hirvonen, J. N-in-one permeability studies of heterogeneous sets of compounds across Caco-2 cell monolayers. Pharm. Res.
**2003**, 20, 187–197. [Google Scholar] [CrossRef] - Tannergren, C.; Langguth, P.; Hoffmann, K.J. Compound mixtures in Caco-2 cell permeability screens as a means to increase screening capacity. Pharmazie
**2001**, 56, 337–342. [Google Scholar] [PubMed] - Konsoula, R.; Barile, F.A. Correlation of in vitro cytotoxicity with paracellular permeability in Caco-2 cells. Toxicol. Vitr.
**2005**, 19, 675–684. [Google Scholar] [CrossRef] - Rodrigues, E.T.; Nascimento, S.F.; Pires, C.L.; Godinho, L.P.; Churro, C.; Moreno, M.J.; Pardal, M.A. Determination of intestinal absorption of the paralytic shellfish toxin GTX-5 using the Caco-2 human cell model. Environ. Sci. Pollut. Res.
**2021**. [Google Scholar] [CrossRef] - Yu, H.S.; Cook, T.J.; Sinko, P.J. Evidence for diminished functional expression of intestinal transporters in Caco-2 cell monolayers at high passages. Pharm. Res.
**1997**, 14, 757–762. [Google Scholar] [CrossRef] - Volpe, D.A. Application of Method Suitability for Drug Permeability Classification. Aaps J.
**2010**, 12, 670–678. [Google Scholar] [CrossRef] [Green Version] - Vachon, P.H.; Beaulieu, J.F. Transient Mosaic Patterns of Morphological and Functional-Differentiation in the Caco-2 Cell-Line. Gastroenterology
**1992**, 103, 414–423. [Google Scholar] [CrossRef] - Ujhelyi, Z.; Fenyvesi, F.; Varadi, J.; Feher, P.; Kiss, T.; Veszelka, S.; Deli, M.; Vecsernyes, M.; Bacskay, I. Evaluation of cytotoxicity of surfactants used in self-micro emulsifying drug delivery systems and their effects on paracellular transport in Caco-2 cell monolayer. Eur. J. Pharm. Sci.
**2012**, 47, 564–573. [Google Scholar] [CrossRef] [PubMed] - Corazza, F.G.; Ernesto, J.V.; Nambu, F.A.N.; de Carvalho, L.R.; Leite-Silva, V.R.; Varca, G.H.C.; Calixto, L.A.; Vieira, D.P.; Andreo-Filho, N.; Lopes, P.S. Papain-cyclodextrin complexes as an intestinal permeation enhancer: Permeability and in vitro safety evaluation. J. Drug Deliv. Sci. Technol.
**2020**, 55, 101413. [Google Scholar] [CrossRef] - Hellinger, E.; Veszelka, S.; Toth, A.E.; Walter, F.; Kittel, A.; Bakk, M.L.; Tihanyi, K.; Hada, V.; Nakagawa, S.; Thuy, D.H.D.; et al. Comparison of brain capillary endothelial cell-based and epithelial (MDCK-MDR1, Caco-2, and VB-Caco-2) cell-based surrogate blood-brain barrier penetration models. Eur. J. Pharm. Biopharm.
**2012**, 82, 340–351. [Google Scholar] [CrossRef] [PubMed] - Paketuryte, V.; Petrauskas, V.; Zubriene, A.; Abian, O.; Bastos, M.; Chen, W.Y.; Moreno, M.J.; Krainer, G.; Linkuviene, V.; Sedivy, A.; et al. Uncertainty in protein-ligand binding constants: Asymmetric confidence intervals versus standard errors. Eur. Biophys. J. Biophys. Lett.
**2021**, 50, 661–670. [Google Scholar] [CrossRef] [PubMed] - Kemmer, G.; Keller, S. Nonlinear least-squares data fitting in Excel spreadsheets. Nat. Protoc.
**2010**, 5, 267–281. [Google Scholar] [CrossRef] - Delie, F.; Rubas, W. A human colonic cell line sharing similarities with enterocytes as a model to examine oral absorption: Advantages and limitations of the Caco-2 model. Crit. Rev. Ther. Drug Carr. Syst.
**1997**, 14, 221–286. [Google Scholar] [CrossRef] - Hidalgo, I.J. Assessing the absorption of new pharmaceuticals. Curr. Top. Med. Chem.
**2001**, 1, 385–401. [Google Scholar] [CrossRef] - Madara, J.L. Regulation of the movement of solutes across tight junctions. Annu. Rev. Physiol.
**1998**, 60, 143–159. [Google Scholar] [CrossRef] [PubMed] - Tervonen, A.; Ihalainen, T.O.; Nymark, S.; Hyttinen, J. Structural dynamics of tight junctions modulate the properties of the epithelial barrier. PLoS ONE
**2019**, 14, e0214876. [Google Scholar] [CrossRef] [PubMed] - Adson, A.; Raub, T.J.; Burton, P.S.; Barsuhn, C.L.; Hilgers, A.R.; Audus, K.L.; Ho, N.F.H. Quantitative Approaches to Delineate Paracellular Diffusion in Cultured Epithelial-Cell Monolayers. J. Pharm. Sci.
**1994**, 83, 1529–1536. [Google Scholar] [CrossRef] [PubMed] - Press, B.; Di Grandi, D. Permeability for Intestinal Absorption: Caco-2 Assay and Related Issues. Curr. Drug Metab.
**2008**, 9, 893–900. [Google Scholar] [CrossRef] - Bohets, H.; Annaert, P.; Mannens, G.; Van Beijsterveldt, L.; Anciaux, K.; Verboven, P.; Meuldermans, W.; Lavrijsen, K. Strategies for absorption screening in drug discovery and development. Curr. Top. Med. Chem.
**2001**, 1, 367–383. [Google Scholar] [CrossRef] [Green Version] - Antonescu, I.E.; Rasmussen, K.F.; Neuhoff, S.; Frette, X.; Karlgren, M.; Bergstrom, C.A.S.; Nielsen, C.U.; Steffansen, B. The Permeation of Acamprosate Is Predominantly Caused by Paracellular Diffusion across Caco-2 Cell Monolayers: A Paracellular Modeling Approach. Mol. Pharm.
**2019**, 16, 4636–4650. [Google Scholar] [CrossRef] - Shimizu, M.; Tsunogai, M.; Arai, S. Transepithelial transport of oligopeptides in the human intestinal cell, Caco-2. Peptides
**1997**, 18, 681–687. [Google Scholar] [CrossRef] - Yee, S.Y. In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man—Fact or myth. Pharm. Res.
**1997**, 14, 763–766. [Google Scholar] [CrossRef] - Artursson, P. Epithelial transport of drugs in cell culture. I: A model for studying the passive diffusion of drugs over intestinal absorbtive (Caco-2) cells. J. Pharm. Sci.
**1990**, 79, 476–482. [Google Scholar] [CrossRef] - Broeders, J.J.W.; van Eijkeren, J.C.H.; Blaauboer, B.J.; Hermens, J.L.M. Transport of Chlorpromazine in the Caco-2 Cell Permeability Assay: A Kinetic Study. Chem. Res. Toxicol.
**2012**, 25, 1442–1451. [Google Scholar] [CrossRef] - Hidalgo, I.J.; Borchardt, R.T. Transport of Bile-Acids In a Human Intestinal Epithelial-Cell Line, Caco-2. Biochim. Biophys. Acta
**1990**, 1035, 97–103. [Google Scholar] [CrossRef] - Hosoya, K.; Kim, K.J.; Lee, V.H.L. Age-dependent expression of P-glycoprotein gp170 in Caco-2 cell monolayers. Pharm. Res.
**1996**, 13, 885–890. [Google Scholar] [CrossRef] - Saitoh, R.; Sugano, K.; Takata, N.; Tachibana, T.; Higashida, A.; Nabuchi, Y.; Aso, Y. Correction of permeability with pore radius of tight junctions in Caco-2 monolayers improves the prediction of the dose fraction of hydrophilic drugs absorbed by humans. Pharm. Res.
**2004**, 21, 749–755. [Google Scholar] [CrossRef] [PubMed] - Linnankoski, J.; Makela, J.; Palmgren, J.; Mauriala, T.; Vedin, C.; Ungell, A.L.; Lazorova, L.; Artursson, P.; Urtti, A.; Yliperttula, M. Paracellular Porosity and Pore Size of the Human Intestinal Epithelium in Tissue and Cell Culture Models. J. Pharm. Sci.
**2010**, 99, 2166–2175. [Google Scholar] [CrossRef] [PubMed]

**Figure 1.**Evaluation of the Caco-2 monolayer integrity when used for a single permeability assay at distinct days after seeding (22, 25, and 28). The TEER values obtained before are shown in Plot (

**A**), and after the permeability assay in Plot (

**B**). The lines are the best fit of a Normal distribution to the results obtained on each day (colored lines) or cumulatively at all days (black line). The grey line in Plot B is the overall distribution obtained before the permeability experiment.

**Figure 2.**Evaluation of the re-establishment of Caco-2 monolayer integrity when incubated in culture media for two full days after being used for a permeability assay. The TEER values for cell monolayers not used on permeability assays are shown in dark colors ( , , ), and those previously used are shown in light colors ( and , TEER on day 25 previously used on day 22; and , TEER on day 28 previously used on days 22 and 25, on single or multi-time sampling). The lines are the best fit of a Normal distribution to the results obtained on single use or re-used cell monolayers (colored lines) or cumulatively for each day (black line), with the parameters given in the Supplementary Material—Table S1. The cumulative results at all days are shown on the right plot ( , TEER before assay on days 22, 25, or 28; , TEER before assay on days 25 or 28 after being used on day 22 or on days 22 and 25).

**Figure 3.**Dependence of LY P

_{app}(single sampling at 60 min) on the day post-seeding and on the re-use of the monolayer for additional permeability assays after incubation with culture media for two full days. The left plots show the distribution of P

_{app}values obtained on the different days after cell seeding; dark colors represent results for single use and light colors for re-used cell monolayers; the lines are the best fit of a Normal distribution (grey dashed) or a LogNormal distribution (black continuous). The plot on the right shows the cumulative results at all days and conditions; the lines are the best fit of a LogNormal distribution for data from each day (colored) and collectively for all days (black), with the parameters given in the Supplementary Material—Table S2.

**Figure 4.**Effect of the sampling time interval on the paracellular permeability of LY through Caco-2 monolayers for cells at day 22, 25, or 28, post-seeding. The LY transport expressed as the Logarithm of the instantaneous permeability (LogP

_{app}) is shown in the upper plots, while that expressed as the Logarithm of the amount of LY that permeates (LogQ

_{A}) is shown in the lower plots, for a sampling interval of 10 min (light colors) or 60 min (dark colors). The lines are the best fit of a Normal distribution, with the parameters given in the Supplementary Material—Table S4, the black line corresponding to the cumulative data from both sampling time intervals.

**Figure 5.**Effect of multi-time sampling on the paracellular permeability of LY through Caco-2 monolayers for cells at day 22, 25, or 28, post-seeding. The LY transport is expressed as the Logarithm of the amount of LY that reaches the acceptor compartment (LogQ

_{A}) for 3 consecutive 10 min sampling intervals (at 10, 20, and 30 min): 1st 10 min sampling ( , , ), 2nd 10 min sampling ( , , ), and 3rd 10 min sampling ( , , ). The lines are the best fit of a Normal distribution, with the parameters given in Supplementary Material—Table S4, the black line corresponding to the sum of the results from the 3 sampling time points.

**Figure 6.**Immunofluorescence ZO-1 staining of Caco-2 monolayers. Representative images for cell monolayers at day 22, 25, and 28 are shown in the left, middle, and right panels, respectively. In the upper plots, the monolayer was not used in permeability assays, while in the additional plots the monolayers were previously used. The lower plots correspond to monolayers immediately after the LY permeability assay, and in the middle plots the images correspond to cell monolayers that were maintained in culture media for 2 days after the permeability assay. Scale bar 50 µm.

**Figure 7.**Staining of ZO-1 (red) and nuclei (blue) for Caco-2 monolayers at day 28 post-seeding. In the left plots, the monolayer was not used in permeability assays, in the middle plots the monolayer was previously used on days 22 and 25 and maintained in fresh media until day 28, and the right plots correspond to cell monolayer immediately after a permeability assay on day 28 (after being previously used on days 22 and 25). The lower plots correspond to the z-stacks at the cross section, indicated by the respective yellow triangle.

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**MDPI and ACS Style**

Pires, C.L.; Praça, C.; Martins, P.A.T.; Batista de Carvalho, A.L.M.; Ferreira, L.; Marques, M.P.M.; Moreno, M.J.
Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity. *Pharmaceutics* **2021**, *13*, 1563.
https://doi.org/10.3390/pharmaceutics13101563

**AMA Style**

Pires CL, Praça C, Martins PAT, Batista de Carvalho ALM, Ferreira L, Marques MPM, Moreno MJ.
Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity. *Pharmaceutics*. 2021; 13(10):1563.
https://doi.org/10.3390/pharmaceutics13101563

**Chicago/Turabian Style**

Pires, Cristiana L., Catarina Praça, Patrícia A. T. Martins, Ana L. M. Batista de Carvalho, Lino Ferreira, Maria Paula M. Marques, and Maria João Moreno.
2021. "Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity" *Pharmaceutics* 13, no. 10: 1563.
https://doi.org/10.3390/pharmaceutics13101563