Confluent monolayers

Animal cell cultures that are initiated from cells removed directly from the animal are called primary cultures (Figure 2). Primary cultures include both explant cultures (i.e., cultures initiated from small pieces of intact tissue), as well as cultures initiated from preparations of individual or dispersed cells (obtained from intact tissue by mechanical or proteolytic dismption). Nerve fiber explant cultures in blood plasma were among the earliest types of tissue cultures (Harrison, 1907). Cells grow out from such tissue explants and form a single layer of cells completely filling the tissue culture vessel surface. Such cell cultures are called confluent monolayers. Confluent monolayers can then be treated with trypsin, so as to remove the individual cells from the culture vessel surface. The resulting cell suspension is then transferred into other culture containers, so that more viable monolayer... 

Cell cultures. MDCK cells were seeded in the Transwells at a density of 2.2 x 104 cells/cm. Cells were fed by changing medium in both upper (apical) and lower (basal) compartments periodically. Confluent monolayers were obtained at 5-7 days post-inoculation, when the cell density reached 4.5-5.0 x 105 cells/cm2, and a transepithelial electrical resistance (TEER) of about 2,000 ohms cm2 was measured using an epithelial voltohmmeter (EVOM, World Precision Instruments, West Haven, CT). The amount of FBS in the cell culture medium could be decreased as the cells approached their maximum resistance, and could be maintained at that point for 2 days or longer in medium containing 1% FBS. 

Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Invest Ophthalmol Vis Sci 47, 3612-3624. 

A confluent monolayer of Madin-Darby canine kidney (MDCK) cells was grown in 96-well plates. Serial tenfold dilutions in minimal essential medium were prepared from the aliquots of allantoic fluid taken from the irradiated specimen. These dilutions were applied to MDCK cells and incubated for 48 h at 36 °C in 5% C02. The cells were then washed two times for 5 min with phosphate buffered saline (PBS) and incubated for 1 h with 100 pi of 0.5 mg/ml solution of 3-(4,5-dimethyl-thiazolyl-2) 2,5-diphenyltetrazolium bromide (MTT, ICN Biochemicals Inc., Aurora, Ohio). After lh, the colored deposit was dissolved in 100 pi DMSO, and optical density in the wells was measured on plate reader Victor 1420 (Perkin Elmer, Finland). Based on the data obtained, the infectious titer of the vims was determined as a decimal logarithm of reciprocal to the dilution of the specimen causing destruction of 50% of cells. The inhibiting action of irradiation was evaluated by decreasing the vims titer. 

The application of fullerene on the surfaces has an essential advantage in the studies with cell cultures as in this case we can obtain the maximum contact of cells with fullerene - cells adhere on the surface and colonize it as a confluent monolayer. That is the basic difference from the water-soluble complexes and micro-dispersed suspensions of fullerene C60. The pro-/antioxidant activities of fullerene were tested in chemical and biological systems. 

For the in vitro test, the fibroblasts are allowed to form a half-confluent monolayer within 24 h. Different concentrations of the test chemical are then incubated for 1 h with two sets of cells in parallel (typically on 96-well plates, 104 cells per well, passage number <100). After the incubation with the test substances, one set is irradiated with a nontoxic dose of UVA light (5 J/cm2), while the other set is kept in the dark. Twenty hours after irradiation, cell viability is evaluated by measuring the uptake of NR for 3 h. After the end of the absorption process, excess NR is removed and the cells are treated with an NR desorption solution (ethanol/acetic acid) to extract the dye taken up by the cells. Subsequently, the optical density of the NR solution is measured at 540 nm. As positive control, a test with chlorpromazine is performed. 

RPMI 2650 is the only human nasal epithelial cell line derived from a spontaneously formed tumor. This particular cell line has been mostly used for nasal metabolism studies, since it grows into a multilayer and does not form confluent monolayers (although perijunctional actin rings are present). Thus, RPMI 2650 cell monolayer is rarely used for nasal transport studies [44, 58],... 

Calu-3 (American type culture collection ATCC HTB-55) is a human bronchial epithelial cell line derived from an adenocarcinoma of the lung [59], This cell line has been shown to exhibit serous cell properties and form confluent monolayers of mixed cell phenotypes, including ciliated and secretory cell types [60], but the cilia are formed very irregularly and seem to disappear with increasing passage number (unpublished observations, C.E. and B.F.). Calu-3 cells have shown utility as a model to examine transport [61-63] and metabolism in human bronchial epithelial cells for many therapeutic compounds [64], Furthermore, they have been used in a number of particlecell interaction studies [65-67], The interactions between respiratory epithelial cells and particulates are discussed more in detail in Chap. 19. 

A sufficient plating density has also to be chosen in order to obtain confluent monolayers within the limited time of viability of primary cell cultures. 

There are several cell monolayer models that are frequently used for the evaluation of drug permeability and absorption potential (Table 18.1). For a more detailed discussion, please refer to Chap. 8. Caco-2 cells (adenocarcinoma cells derived from colon) are the most extensively characterized and frequently used of the available cell lines [5-9], A unique feature of Caco-2 cells is that they undergo spontaneous enterocyte differentiation in cell culture. Unlike intestinal enterocytes, Caco-2 cells are immortalized and replicate rapidly into confluent monolayers. When the cells reach confluency during culture on a semi-porous membrane, they start to polarize and form tight junctions, creating an ideal system for permeability and transport studies. During the past decade, use of... 

The wound assay [49] is another method of measuring endothelial cell migration. This assay is based on damaging or wounding a confluent monolayer of endothelial cells and the subsequent repair or closing of the wound by migration of endothelial cells. This assay can be carried out using different matrix components. 

Typically, continuous cell lines grow in culture only on a solid support or anchor (the surface of a petri dish, for example) and in the presence of relatively high concentrations of nutrients. Even then, they divide only as long as the culture is sparse. When the cell density increases beyond a critical point, the growth rate decreases sharply in fact, the cells of some continuous lines stop dividing altogether once they have formed a confluent monolayer. 

Cell Culture. Human neuroblastoma IMR-32 (passaged through nude mice the cells were donated by Dr. Steven E. Brooks, Kingsbrook Jewish Medical Center, Brooklyn) and mouse neuroblastoma clones NIE-115, NS-20, and N-18) (donated by Dr. Shraga Makover, Hoffmann LaRoche, Inc., Nutley, New Jersey) were maintained in our laboratory as described previously (30,31). Confluent monolayers (6 to 8 x 10 cells per 250-ml Falcon plastic flask) were harvested for enzymatic studies with phosphate-buffered saline [7.0 mM potassium phosphate/0.14 M NaCl - buffer, pH 7.2 (Pi/NaCl)] containing 0.1% EDTA. 

Table VII. In a typical experiment, normal and mutant fibroblasts (one 150 cm2 flask, each containing approximately 2 x 10 cells) were incubated for 6 days in 20 ml medium containing 10% fetal calf serum and antibiotics. On the sixth day, medium was removed, the confluent monolayers washed five times with warm PBS, and further incubated for 48 hrs in 10 ml medium containing 1% fetal calf serum, antibiotics, and 5 p Ci of [ HJ-D-galactose. Subsequently, the medium was removed, the monolayers washed with PBS, harvested, and centrifuged at 500 x g for 5 min at 4°. The cell pellet was washed thrice with 40 vol of ice cold PBS. The washed cell pellets were suspended in a small volume of water, sonicated and a suitable aliquot withdrawn for total protein and radioactivity measurements. The remainder of the cell pellets were subjected to solvent extraction for the purposes of isolation of GSLs according to previously described procedures (30).

The experiments documented in Tables 5 and 6 use confluent monolayers of pure bovine aonn endothelial ceiis thar were grown in tissue culture petri dishes, then seeded at high concentration on collagen-coated polycarbonate filters (pore diameter 0.8 im). The fillers were fitted onto millipore filter holders placed in a buffer compartment with separate access to both sides of the Biter... 

If a strip of cells is removed from a confluent monolayer of untransformed cells (e.g. 3T3 mouse embryo cells) then the cells at the edge of the wound are stimulated to synthesise DNA and divide. They quickly colonise the unoccupied area of the wound. This phenomenon known as topo inhibition (Dulbecco, 1970) is now explained by the presence in cells on the edge of the wound of an increased surface area exposed to the medium (i.e. neighbouring cells have been removed) (Stoker, 1973 Dulbecco and Elkington, 1973). 

Rinse in medium lacking serum and continue to incubate the confluent monolayer of cells in serum-free medium. 

Grow the cells for 2-3 days in 75 cm2 flasks until a sub-confluent monolayer is formed, it is advisable to change the medium on the cells 24 h before they are due to be harvested. 

The method depends on infecting a small number of cells in a confluent monolayer. The virus produced in the infected cells will move laterally to infect adjacent cells and various techniques are used to prevent further spreading. The degenerative effect on the cells spreads until a visible area of dead cells (a plaque) is apparent. Staining of the cell sheet makes the colourless plaque more easy to see. 

Set up 20 X 50 mm dishes with 2-4 X 106BHK21 03 (EMC virus) or BSC1 (SV40) cells per dish and grow for 24 h at 37°C to obtain confluent monolayers. 

Macpherson (1961) exposed confluent monolayers or cell suspension to virus and then plated the cells at low density. They then picked out transformed colonies by appearance in the absence of any selective pressure (see Fig. 2.1). The number of transformed colonies is directly related to virus dose. When primary hamster kidney cells were exposed to polyoma virus at 96 p.f.u./cell between 1 in 100 and 1 in 5000 cells was transformed. 

Incubate at 27°C until the cells have formed 80-100% confluent monolayer. 

Remove medium and floating cells from a confluent monolayer. 

If human embryo kidney (HEK) cells are to be transfected by electroporation, proceed as follows Confluent monolayers of cells are harvested by trypsinization as described above and resuspended in IX HBS at 2 x 107 cells/mL and kept on ice. Add 30 [xg of plasmid DNA to 0.5 mL of cells and transfer to a chilled, 0.4 cm gap electroporation cuvet. Pulse at 960 xF and 260 V. Replace the cuvet on ice for a further 5-10 min then transfer the cells to a 15 cm diameter Petri dish containing 15-20 mL of pre warmed complete medium. Incubate overnight at 37°C. Change the medium the next morning. 

Cell plating (Day 0) Freshly isolated human hepatocytes are isolated using a 2-step collagenase digestion procedure and plated onto collagen-coated plate. It is important that the cells are plated at a cell density that will lead to a confluent monolayer culture, as cell density is known to affect enzyme induction results. 

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