Epithelial Cells

Epithelial cells are found throughout the body, from skin to glandular formations within tissues. In vivo these cells are attached to a three dimensional basement membrane matrix.

The interactions between the epithelial cell and matrix proteins effect cell morphology and function. Two highly specified epithelial cell types have been discussed in the hepatocyte and endothelial cell sections, utilizing both 2-dimensional (2D) and three-dimensional (3D) culture systems. In vitro, 2D and 3D culture systems can be used to study different aspects of cell growth and differentiation. 2D culture systems are used for cell attachment and proliferation. 3D environments are utilized in studies requiring a more in vivo-like setting, such as mammary acini formation.

BD BioCoat™ cultureware provides a range of 2D surfaces for cell growth. Both keratinocytes1-2 and HEK-2933-4 cells are examples of epithelial cells that can be studied in 2D culture environments. Keratinocytes are a major component of the epidermis; BD BioCoat Collagen I supports growth of human neonatal keratinocytes (Figure 1). HEK-293 cells are a human epithelial kidney cell line which exhibit enhanced attachment to poly-lysine coated surfaces as compared to tissue culture-treated surfaces. This is particularly important if the cells need to remain attached during subsequent washes (Figure 2). The appropriate 2D surface is determined by the cell type.

Three-dimensional growth substrates can support certain cellular behaviors that are not observed when cells are cultured on a planar two-dimensional surface, as exemplified by mammary epithelial6-10 and caco-211-12 cells. In vivo, mammary epithelial cells form polarized acini. When tumorigenic human mammary carcinoma cells (T4-2) are cultured on a 3D substrate comprised of reconstituted basement membrane (Growth Factor Reduced BD Matrigel™ Matrix) they form large disorganized colonies, as shown with the T4-vector control in a study from Dr. Bissell’s laboratory7 (Figure 3). Epidermal growth factor receptor (EGFR) had previously been shown to be elevated in T4-2 cells, and downregulation of this signaling pathway in T4-2 cells cultured in 3D BD Matrigel Matrix is known to lead to phenotypic reversion to polarized acini. These cells exhibit polarized acinar architecture in the presence of the EGFR inhibitor AG1478 or when stably expressing dominant negative Rap1 (T4-DN-Rap1); reversion to a normal phenotype is shown by proper localization of α6-integrin (basal marker), β-catenin (basolateral marker) and GM130 (apical marker). These data show that three-dimensional BD Matrigel Matrix culture conditions are conducive to studying signaling pathways involved in regulating mammary acinar architecture.

Another example of the effect of 3D growth substrates on cellular phenotypes is the use of BD BioCoat Fibrillar Collagen Inserts in Caco-2 assays. Caco-2 cells are an epithelial cell line, derived from a colorectal adenocarcinoma, commonly used to measure compound permeability. The gold standard for modeling drug permeability across the intestinal epithelium in vitro is measuring permeability across differentiated Caco-2 cells, where the cells have been cultured for 21 days on cell culture inserts. BD BioCoat HTS Caco-2 Assay System and BD BioCoat Intestinal Epithelium Differentiation Environment utilize BD BioCoat Fibrilliar Collagen Inserts and a specialized media to enhance the rate of Caco-2 differentation from 21 to 3 days (Figures 4-5), thereby reducing the time and labor required for the analysis of compound permeability.

The 2D and 3D cell culture systems available from BD Biosciences provide multiple options to researchers studying epithelial cells in vitro.

Figure 1. Proliferation of Human Neonatal Keratinocytes on BD BioCoat™ Collagen I Cultureware

Figure 1. Proliferation of Human Neonatal Keratinocytes on BD BioCoat™ Collagen I Cultureware

Human neonatal keratinocytes cultured on BD BioCoat™ Collagen I cultureware.

Figure 1. Adherance of HEK-293 Cells to BD BioCoat Poly-D-Lysine Cultureware

Figure 2. Adherance of HEK-293 Cells to BD BioCoat Poly-D-Lysine Cultureware

HEK-293 cells have enhanced attachment to BD BioCoat Poly-D-Lysine Cultureware as compared to BD Falcon Tissue Culture-treated Cultureware. An equal number of cells were plated on BD BioCoat Poly-D-Lysine 384-well black/clear (right) and BD Falcon Tissue Culture-treated 384-well Black/Clear Plates (left) and grown under serum-free conditions. Before washing (top), there were a similar number of cells in the BD BioCoat Poly-D-Lysine coated wells and the BD Falcon Tissue Culture-treated wells. After washing, using a Skatron Washer (Molecular Devices) (middle), the cells remained attached to the BD BioCoat Poly-D-Lysine wells while few cells remained attached to the BD Falcon Tissue Culturetreated wells. Post-wash, the cells were visualized using Calcein AM (bottom).


 
Figure 3. Effect of RAP1 Activity on T4-2 Cell Polarity in 3D Growth Factor Reduced BD Matrigel Matrix Culture

Figure 3. Effect of RAP1 Activity on T4-2 Cell Polarity in 3D Growth Factor Reduced BD Matrigel Matrix Culture

Growth Factor Reduced BD Matrigel Matrix supports mammary acini formation in vitro. Malignant T4-2 cells were grown in three-dimensional culture on Growth Factor Reduced BD Matrigel Matrix. Cells were stably transfected with control (T4-vector) or dominant negative-Rap1 (T4-DN-Rap1). Inhibition of EGFR with AG1478 was used as a positive control for reversion of T4-2 to normal mammary acinar architecture. Indirect immunofluorescence was used to analyze cell polarity markers for basal (α6-interin), basolateral (β-catenin) and apical (GM130) membrane domains. Bar, 5 μm. Images kindly provided by Dr. Masahiko Itoh and Dr. Mina Bissell, originally published in Cancer Research 67(10):4759-47662. Reproduced with permission.

Figure 4. Permeability of Mannitol and Antibiotics Through CACO-2 Monolayers

Figure 4. Permeability of Mannitol and Antibiotics Through CACO-2 Monolayers

Barrier formation occurs three days postseeding in the BD BioCoat Intestinal Epithelium Differentiation Environment and two to four weeks with conventional methods. Monolayers formed using either the BD BioCoat Intestinal Epithelium Differentiation Environment or conventional methods are equally permeable for each of the three compounds tested.


 
Figure 5. P-Glycoprotein (P-GP) Function in CACO-2 Cells

Figure 5. P-Glycoprotein (P-GP) Function in CACO-2 Cells

Caco-2 cells were cultured using the three-day BD BioCoat HTS Caco-2 Assay System supplemented with BD™ MITO+ Serum Extender (A) or the traditional 21-day system (B). P-gp function was assessed by adding 10 nM 3H-labeled vinblastine in PBS to either the apical or basal side of the insert. Samples were withdrawn from the non-labeled side of the insert and counted by scintillation counting. To inhibit the P-gp with verapmil, 100 μM verapamil was added to the insert chambers.

References

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  2. Grose R, Hutter C, Bloch W, Thorey I, Watt FM, Fassler R, Brakebusch, Werner S. (2002) A crucial role of β1 integrins for keratinocyte migration in vitro and during cutaneous wound repair. Development. 129:2303.

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  6. Nelson CM, Inman JL, Bissell MJ. (2008) Three-dimensional lithographically defined organotypic tissue arrays for quantitative analysis of morphogenesis and neoplastic progression. Nat Protocol. 3(4):674.

  7. Itoh M, Nelson CM, Myers CA, Bissell MJ. (2007) Rap1 integrates tissue polarity, lumen formation, and tumorigenic potential in human breast epithelial cells. Cancer Res. 67(10):4759.

  8. Kenny PA, Lee GY, Myers CA, Neve RM, Semeiks JR, Spellman PT, Lorenz K, Lee EH, Barcellos-Hoff MH, Petersen OW, Gray JW, Bissell MJ. (2007) The morphologies of breast cancer cell lines in threedimensional assays correlate with their profiles of gene expression. Mol Oncol. 1(1):84.

  9. Friedland JC, Lakins JN, Kazanietz MG, Chernoff J, Boettiger D, Weaver VM. (2007) α6β4 integrin activates Rac-dependent p21-activated kinase 1 to drive NF-κB-dependent resistance to apoptosis in 3D mammary acini. J Cell Sci. 120: 3700.

  10. Debnath J, Muthuswamy SK, Brugge JS. (2003) Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in threedimensional basement membrane cultures. Methods. 30(3):256.

  11. Shirasaka Y, Kawasaki M, Sakane T, Omatsu H, Moriya Y, Nakamura T, Sakaeda T, Okumura K, Langguth P, Yamashita S. (2006) Induction of human p-glycoprotein in Caco-2 cells: development of a highly sensitive assay system for p-glycoprotein-mediated drug transport. Drug Metab. Pharmacokinet. 21(5):414.

  12. Yamashita S, Konishi K, Yamazaki Y, Taki Y, Sakane T, Sezaki, H, Furuyama Y. (2002) New and better protocols for a short-term Caco-2 cell culture system. J Pharma Sci. 91(3):669.

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