Growing Madin-Darby Canine Kidney Cells for Studying Epithelial Cell Biology
Epithelial cells display a structural and functional polar organization (Simons and Fuller, 1985). In these cells, the plasma membrane can be divided into two distinct domains, the apical membrane and the basolateral membrane, each containing different sets of proteins. The apical membrane facing a secretory or an absorptive lumen is delimited by a junctional complex from the basolateral membrane. The tight junction (zonula occludens) is the most apical member of the complex. It is found at the intersection between the apical and the lateral plasma membranes and joins each cell to its neighbors, thus limiting the diffusion of molecules between the luminal and the serosal compartments (Gumbiner, 1987). This junction also prevents the lateral diffusion of membrane proteins from one domain to another, thus maintaining their unique composition. Immediately basal to the tight junctions is the intermediate junction (zonula adherens or belt desmosomes). The other more basal junctional elements are desmosomes (maculae adherentes) and gap junctions, which attach the lateral membranes of adjacent cells to each other. The junctional complex is involved in sealing the epithelium; it prevents molecules from diffusing between adjacent cells. The basolateral membrane faces the bloodstream and is involved in cellcell contact and cell adhesion to the basement membrane.
For most studies on epithelial cell polarity, cultured cells have been used. These cells are superior to cells obtained from tissues because they can be grown under carefully controlled conditions and are easily manipulated. The cell population is homogeneous. Biosynthetic experiments using pulse-chase techniques with radioactive precursors can be accomplished at an analytical level with a short time resolution. Endocytosis and transcytosis can also be studied.
The most well-studied epithelial cell is the Madin-Darby canine kidney (MDCK) cell. This cell line is derived from normal dog kidney (McRoberts et al., 1981). An unusual feature of these cells is that while in culture they retain many differentiated properties characteristic of kidney epithelial cells. Among these are an asymmetric distribution of enzymes and vectorial transport of sodium and water from the apical to the basolateral faces. The latter gives rise to "domes" or "blisters" in confluent cultures, which are transient areas where collected fluid has forced the monolayer to separate from the substratum. Morphologically, the cells resemble a typical cuboidal epithelium with microvilli on the apical side of the cells. Two different strains of the MDCK cell are known (Richardson et al., 1981; Balcarova-St/inder et al., 1984). Strain I cells are derived from a low-passage MDCK cell stock and these cells form a tight epithelium with transepithelial resistance above 2000Ω.cm2. Strain II cells form a monolayer of lower resistance of 100-200Ω. cm2. MDCK strain II cells have been used primarily for studies of the cell biology of epithelial cells. Transcytosis is, however, studied more conveniently in MDCK strain I cells because of their high electrical resistance.
Several factors are important for optimal expression of the epithelial phenotype in vitro (Simons and Fuller, 1985). A primary consideration is the polarity of nutrient uptake. In vivo, many nutrients reach the epithelial sheet from the basolateral side, which faces the blood supply; however, when epithelial cells are cultured on glass or plastic, they are forced to feed from the apical surface, which faces the culture medium. Hence, the basolateral surface becomes isolated from the growth medium as the monolayer is sealed by the formation of tight junctions. To grow properly, the epithelial sheet must remain somewhat leaky or expose basolateral proteins responsible for the uptake of nutrients and binding of growth factors on the apical side.
These problems can be overcome simply by growing the epithelial cells on permeable supports, such as polycarbonate and nitrocellulose filters. Epithelial cells form monolayers with a higher degree of differentiation when the basolateral surface is directly accessible to the growth medium. This is evident from the morphology of the cells, their increased responsiveness to hormones, and the exclusion of basolateral proteins from their apical surfaces.
II. MATERIALS AND INSTRUMENTATION
Minimal essential medium with Earle's salt (MEM) is purchased as a powder (Cat. No. 11700-077) from Biochrom, mixed with Milli-Q-filtered H20, and sterile filtered. Glutamine (200mM, Cat. No. 25030-024), penicillin (10,000 IU / ml)-streptomycin (10,000 mg/ml) (Cat. No. 15140-122), trypsin (0.05%)-EDTA (0.02%) (Cat. No. 25300-054), and phosphate-buffered saline (PBS, Cat. No. 041-04040H) are from GIBCO-BRL. The Transwell polycarbonate filters (2.45 cm, Cat. No. 3412, and 10cm, Cat. No. 3419) are from Costar. Tissue culture flasks (75cm2, Cat. No. 156499) are from Nunc. The glass petri dishes (140mm diameter and 30mm high) for holding six 2.4-cm Transwell filters are from Schott Glasware. The laminar flow hood (Steril-Gard Hood Model VMB-600) is from Baker. The CO2 incubator (Model 3330) is from Forma Scientific. The inverted Diavert microscope is from Leitz. The electrical resistance measuring device (EVOM) is from World Precision Instruments. The centrifuge (Type 440) is from Hereaus-Christ.
A. Growing Madin-Darby Canine Kidney Cells on Plastic
MDCK I and II cells are passaged every 3-4 days up to 25 passages. One flask is usually split into five new flasks. MDCK II cells usually form domes within 2 days of splitting, whereas MDCK I cells do not blister.
B. Seeding MDCK Cells on Polycarbonate Filters
C. Transepithelial Resistance Measurement
The cell layer on the filter cannot be observed in the inverted microscope because the filters are not transparent. Transparent filters are also available commercially' but they are more expensive than the polycarbonate ones; however, either the cells in one filter can be stained or the transepithelial resistance can be measured to ensure that the layer is intact. Our experience is that when one filter in the petri dish checks out, the other filters will also be fine.
It is recommended that MDCK cells not be used for more than 20-25 passages. New stock cells should then be thawed from liquid nitrogen storage.
We use filter holders for growing MDCK cells on either 2.4- or 10-cm polycarbonate filters. It is possible to grow cells in either the six-well plate for the Transwell 3412 filter or in the petri dish supplied with the Transwell 3419 filters. Under these latter culture conditions, growth media have to be changed every day, as the cells do not get enough nutrients and do not grow to optimal density. The problems with changing the medium every day are (1) the extra work involved and (2) the considerably increased risk of contamination. Therefore, we prefer to place filter holders in petri dishes into which one can add enough growth medium to last 4 days.
Balcarova-Stander, J., Pfeiffer, S. E., Fuller, S. D., and Simons, K. (1984). Development of cell surface polarity in the epithelial Madin-Darby canine kidney (MDCK) cell line. EMBO J. 3, 2687-2694
Gumbiner, B. (1987). Structure, biochemistry and assembly of epithelial tight junctions. Am. J. Physiol. 253, C749-C758.
McRoberts, J. A., Taub, M., and Saier, M. H., Jr. (1981). The Madin- Darby canine kidney (MDCK) cell line. In "Functionally Differentiated Cell Lines" (G. Sato, ed.), pp. 117-139. A. R. Liss, New York.
Richardson, J. C. W., Scalera, V., and Simmons, N. L. (1981). Identification of two strains of MDCK cells which resemble separate nephron tubule segments. Biochim. Biophys. Acta 673, 26-36.
Simons, K., and Fuller, S. D. (1985). Cell surface polarity in epithelia. Annu. Rev. Cell. Biol. 1, 243-288.
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