Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity
Caco-2 monolayers are a common in vitro model used to evaluate human intestinal absorption. The reference protocol requires 21 days post-seeding to establish a stable and confluent cell monolayer, which is used in a single permeability assay during the period of monolayer stability (up to day 30). I...
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| Format: | Article |
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MDPI AG
2021-09-01
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| Series: | Pharmaceutics |
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| Online Access: | https://www.mdpi.com/1999-4923/13/10/1563 |
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| author | Cristiana L. Pires Catarina Praça Patrícia A. T. Martins Ana L. M. Batista de Carvalho Lino Ferreira Maria Paula M. Marques Maria João Moreno |
| author_facet | Cristiana L. Pires Catarina Praça Patrícia A. T. Martins Ana L. M. Batista de Carvalho Lino Ferreira Maria Paula M. Marques Maria João Moreno |
| author_sort | Cristiana L. Pires |
| collection | DOAJ |
| description | Caco-2 monolayers are a common in vitro model used to evaluate human intestinal absorption. The reference protocol requires 21 days post-seeding to establish a stable and confluent cell monolayer, which is used in a single permeability assay during the period of monolayer stability (up to day 30). In this work, we characterize variations in the tightness of the cell monolayer over the stable time interval and evaluate the conditions required for their re-use in permeability assays. The monolayer integrity was assessed through TEER measurements and permeability of the paracellular marker Lucifer Yellow (LY), complemented with nuclei and ZO-1 staining for morphological studies and the presence of tight junctions. Over 150 permeability assays were performed, which showed that manipulation of the cell monolayer in the permeability assay may contribute significantly to the flux of LY, leading to <i>P</i><sub>app</sub> 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. |
| first_indexed | 2024-03-10T06:16:56Z |
| format | Article |
| id | doaj.art-1c9b44e53e6b428289ca0f775d56e471 |
| institution | Directory Open Access Journal |
| issn | 1999-4923 |
| language | English |
| last_indexed | 2024-03-10T06:16:56Z |
| publishDate | 2021-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Pharmaceutics |
| spelling | doaj.art-1c9b44e53e6b428289ca0f775d56e4712023-11-22T19:37:37ZengMDPI AGPharmaceutics1999-49232021-09-011310156310.3390/pharmaceutics13101563Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer IntegrityCristiana L. Pires0Catarina Praça1Patrícia A. T. Martins2Ana L. M. Batista de Carvalho3Lino Ferreira4Maria Paula M. Marques5Maria João Moreno6Coimbra Chemistry Center, Department of Chemistry, University of Coimbra, 3004-535 Coimbra, PortugalCNC—Centro de Neurociências e Biologia Celular, CIBB—Centro de Inovação em Biomedicina e Biotecnologia, Universidade de Coimbra, 3004-504 Coimbra, PortugalCoimbra Chemistry Center, Department of Chemistry, University of Coimbra, 3004-535 Coimbra, PortugalMolecular Physical-Chemistry R&D Unit, Department of Chemistry, University of Coimbra, 3004-535 Coimbra, PortugalCNC—Centro de Neurociências e Biologia Celular, CIBB—Centro de Inovação em Biomedicina e Biotecnologia, Universidade de Coimbra, 3004-504 Coimbra, PortugalMolecular Physical-Chemistry R&D Unit, Department of Chemistry, University of Coimbra, 3004-535 Coimbra, PortugalCoimbra Chemistry Center, Department of Chemistry, University of Coimbra, 3004-535 Coimbra, PortugalCaco-2 monolayers are a common in vitro model used to evaluate human intestinal absorption. The reference protocol requires 21 days post-seeding to establish a stable and confluent cell monolayer, which is used in a single permeability assay during the period of monolayer stability (up to day 30). In this work, we characterize variations in the tightness of the cell monolayer over the stable time interval and evaluate the conditions required for their re-use in permeability assays. The monolayer integrity was assessed through TEER measurements and permeability of the paracellular marker Lucifer Yellow (LY), complemented with nuclei and ZO-1 staining for morphological studies and the presence of tight junctions. Over 150 permeability assays were performed, which showed that manipulation of the cell monolayer in the permeability assay may contribute significantly to the flux of LY, leading to <i>P</i><sub>app</sub> 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.https://www.mdpi.com/1999-4923/13/10/1563Caco-2 cellsintestinal permeabilitytight junctionslucifer yellowhigh-throughput |
| spellingShingle | Cristiana L. Pires Catarina Praça Patrícia A. T. Martins Ana L. M. Batista de Carvalho Lino Ferreira Maria Paula M. Marques Maria João Moreno Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity Pharmaceutics Caco-2 cells intestinal permeability tight junctions lucifer yellow high-throughput |
| title | Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity |
| title_full | Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity |
| title_fullStr | Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity |
| title_full_unstemmed | Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity |
| title_short | Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity |
| title_sort | re use of caco 2 monolayers in permeability assays validation regarding cell monolayer integrity |
| topic | Caco-2 cells intestinal permeability tight junctions lucifer yellow high-throughput |
| url | https://www.mdpi.com/1999-4923/13/10/1563 |
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