Cell layers, confluent

The presence of a transporter can be assessed by comparing basolateral-to-apical with apical-to-basolateral transport of substrates in polarized cell monolayers. If P-gp is present, then basolateral-to-apical transport is enhanced and apical-to baso-lateral transport is reduced. Transport experiments are in general performed with radioactively labeled compounds. Several studies have been performed with Caco-2 cell lines (e.g. Ref. [85]). Since Caco-2 cells express a number of different transporters, the effects measured are most probably specific for the ensemble of transporters rather than for P-gp alone. P-gp-specific transport has been assayed across confluent cell layers formed by polarized kidney epithelial cells transfected with the MDR1 gene [86], Figure 20.11 shows experimental data obtained with these cell lines. A rank order for transport called substrate quality was determined for a number of compounds [86]. The substrate quality is a qualitative estimate, but nevertheless allows an investigation of the role of the air/water (or lipid/water) partition coefficient, log Kaw, for transport as seen in Fig. 20.11(A). For most of the compounds, a linear correlation is observed between substrate quality and log Kaw- However, four compounds are not transported at all despite their distinct lipophilicity. A plot of the substrate quality as a function of the potential of a... 

Fig. 14.2. Plaques of herpes simplex virus on a BHK/C13 cell layer. A confluent layer of BHK21/C13 cells was infected with a herpes simplex virus type 2 preparation at different dilutions. On the left few plaques are visible but on the right there are many plaques. (With thanks to Dr. J.B. Clements.)...

If the teratocarcinoma cells become confluent they will begin to differentiate into many different cells types, but this process is not so dramatic as the differentiation which occurs if cells isolated by method 2 are transferred to a vessel without a feeder layer. Then the cells attach very poorly and form clumps in suspension. These clumps remain healthy and quickly differentiate (Evans, 1972) to form an outer layer of endoderm cells. The presence of endoderm can be shown by assaying for the serine protease plasminogen activator which is a marker typical of endoderm cells (Strickland et al., 1976). 

Fig. 2 Workflow for air-liquid cultivation. This scheme can be adapted to most cell lines and primary cells. Cells are propagated as submerged cultures and seeded on top of porous filters. Both compartments are loaded with medium. Filter size, pore density and pore size, filter material, and coating are factors which affect the success of cultivation. Also the density at which the cells are seeded needs to be optimized. Proliferating cells from cell lines (e.g., NCI-H441, Calu3 cells) need lower densities, ranging between 20,000 and 60,000 cells/cm2, whereas primary cells need higher cell numbers ranging between 500,000 and 800,000 cells/cm2. Once cells form a stable and almost confluent cell layer, the medium has to be removed from the apical side. Usually the medium leaks into the apical compartment 2-3 days after air-liquid interface has been established. To maintain air-liquid interface conditions the medium should be removed from the apical surface at least once a day until the air-liquid interface becomes stabilized. When air-liquid interface condition has to be established, it might be necessary to replace the basolateral medium with a medium which propagates cell differentiation...

Taken together, these studies revealed that confluent cell layers in contact to the resonator lead to a significant increase of energy dissipation from the shear oscillation [33] as we had learned already from the QCM-D experiments presented in Sect. 2.4. The impact of the cells on energy dissipation is individual and dependent on the cell type. It is important to mention in this context that different batches of a certain cell line may also cause a different QCM response within certain limits. This is not surprising for cell biologists since cells of the same kind but taken from different batches may show a certain variance in their behavior and it underlines that the QCM is capable of picking up these subtle differences. 

Taken together, these studies show that cell-substrate separation distance is not the most significant parameter when explaining the acoustic load of shear wave resonators covered by confluent cell layers. It may still have a minor impact but the QCM response is apparently dominated by other mechanisms. 

As sketched in Fig. 10 and mentioned before, the cells anchor to adhesive proteins that are immobilized on the surface. When cells are cultured for a certain time, they even produce their own adhesive proteins and secrete it into the space between membrane and substratum. Thus, we tried to address whether or not these adhesive proteins underneath the cell body may contribute to the total QCM readout of a confluent cell layer. Instead of limiting the analysis to preadsorbed layers of one or two purified proteins, we tried to study the complex extracellular material underneath the cells (extracellular matrix or ECM) by removing the cell bodies but leaving the macromolec-ular network of proteins and sugars behind on the substrate. The protocol required a combination of hypotonic stress and detergent extraction [16]. Microscopic inspection of reference substrates revealed that this procedure hfted the cell bodies effectively off the substrate. The surface was, however, still decorated with proteins as revealed by immunocytochemical staining. 

Figure 11.26 A schematic diagram of an apparatus used to obtain estimates of the passive permeability of a drug candidate across the intestinal mucosa using Caco-2 cells. A monolayer of Caco-2 cells is grown on a porous polyethylene terephthalate (PET) membrane (a so-called confluent monolayer of cells that grows only in two dimensions on such a substrate from an initial small inoculation). In the experiment the cells are submerged in Hanks s Balanced Salt Solution (HBSS) buffer (contains Na+, K+, CP, phosphate, glucose, and in some formulations also Ca +, Mg + and S04 ) the Caco-2 cell layer provides the only connection between an apical (donor) reservoir, into which the drug candidate is dosed, and a basolateral (receiver) reservoir. For the assay, aliquots are removed for analysis from the apical reservoir at Omin, and from both reservoirs at 120 min. Reproduced from Van Pelt, Rapid Commun. Mass Spectrom. 17, 1573 (2003), with permission of John Wiley Sons Ltd.

Cells should be at confluent density, ideally forming a contiguous monolayer. However, formation of cell layers is detrimental. 

Fibroblast seeding onPU Cell seeding Cell proliferation and response Static cell culture Endothelial cells Confluent and vital cell layer, decrease of inflammatory response [82]... 

Ti(C, N)-layeronPU Plasma-activated chemical vapor deposition Type of PU Cell proliferation and platelet adhesion Static cell culture static in vitro Endothelial cells human PRP Confluent cell layer, modified PCU nearly no platelet adhesion [83]... 

Heart valve scaffolds with conduit and leaflets made entirely from P3HO-3HH porous films [287] were tested in vitro imder static and pulsatile flow cell culture conditions [44,281]. A larger number of vascular cells, together with the formation of an oriented and confluent cell layer, was found on scaffolds exposed to pulsatile flow [281]. The P3HO-3HH heart valves, preseeded with vascular cells under static conditions, were tested for up to 17 weeks in the pulmonary position in a lamb model. The valves showed good functionality... 

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... 

It is assumed that the convective flow of water across the ABL, cell mono-layer, and filter owing to pressure gradients is negligible and that the cell mono-layer is uniformly confluent. When these conditions are not met, Katz and Schaeffer (1991) and Schaeffer et al. (1992) point out that mass transfer resistances of the ABL and filter [as described in Eq. (21)] cannot be used simply without exaggerating the permeability of the cell monolayer, particularly the paracellular route. An additional diffusion cell design was described by Imanidis et al. (1996). 

Plaques are essentially windows in the lawn of confluent cell growth. With bacterial viruses, plaques may be obtained when virus particles are mixed into a thin layer of host bacteria which is spread out as an agar overlay on the surface of an agar medium. During incubation of the culture, the bacteria grow and form a turbid layer which is visible... 

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