Adherent cultures

Cells in vivo exist either attached to a surface or free in suspension. Adherent cell lines originate from cells of solid tissue. Breast carcinoma cell lines (such as MCF7, T47D, and SK-BR-3) are adherent cultures, and these cells are grown on the surface of plastic flasks that have been treated to facilitate adhesion (see Fig. 6.2). Suspension culture cell lines originate from cells that exist in suspension, such as those cells present in the blood and the lymphatic system (see Fig. 6.3). 

Strategy for Adherent Cultures of Neural Stem Cells. 179... 

To overcome these limitations, we have developed culture substrates that enable the highly efficient expansion of specific cells in adherent cultures [37, 85-88]. An important characteristic of these substrates is that specifically engineered growth factors are immobilized on the surface. Extensive protein engineering techniques were used to optimize the presentation of growth factors to cells. 

Nakaji-Hirabayashi T, Kato K, Iwata H (2008) Essential role of structural integrity and firm attachment of surface-anchored epidermal growth factor in adherent culture of neural stem cells. Biomaterials 29 4403 -408... 

Brock R, Hamelers IHL, Jovin TM. Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor. Cytometry 1999 35 353-362. 

Pollard SM, Yoshikawa K, Clarke ID et al (2009) Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell 4 568-580... 

Preparation of Slides Using CaSki (or Other Adherent Cultured) Cells... 

The use of trypsin is common to detach cells from adherent cultures however, in this case it is not recommended. MCF-7 cells treated with trypsin tend to form clumps, which are difficult to breakup. The use of Accutase yields a mostly single-cell suspension that will not form clumps even after it has been washed with labeling buffer. Total cell count and viability assays will also be easier for the researcher to carry out. 

The range of culture flasks and reactor types employed is quite wide, both for suspension and adherent cultures, from small Carrel s or Roux s flasks to roller bottles. Fixed- and fluidized-bed bioreactors, air-lift reactors and even stirred and aerated tanks with capacities up to 15 m3 are common in large plants producing monoclonal antibodies (mAbs) for anticancer therapies (Adams and Weiner, 2005 Griffiths, 1988). 

High-Five cells (BTI-TN-5BI-4) are derived from Trichoplusia ni cells and are frequently employed due to their capacity to express high protein levels when compared with other insect cell lines, such as Sf-9 cells (Rhiel et al., 1997). This cell line shows high growth rates in adherent culture, and can easily be adapted to grow in suspension and in serum-free media. 

A comparison between insect and mammalian cell culture technology shows various similarities and differences. Due to the fact that insect cell lines typically derive from specific organs or non-differentiated embryonic tissues, the same cell line can proliferate in suspension or in adherent manner (in monolayer). This versatility in growth mode, which contrasts with most mammalian cell lines, increases the choices of bioreactor types and culture strategies for production. Contact inhibition shown by various mammalian cell lines is low or absent for insect cells, and they tend to aggregate in suspension or in adherent cultures. 

For adherent cultures, trypsinize cells for 5 min and pool plates within each treatment group (4-6 100-mm plates for each group should be sufficient). 

The number of commercially available NS/PC lines has been constantly increasing. The NSC lines ReNcell VM and ReNcell CX (Millipore) are derived from developing human brains, ReNcell VM from the ventral mesencephalon (VM) and ReNcell CX cells from the cortical (CX) regions of the brain. For immortalization, they were transduced with the myc transcription factor. ReNcell lines grow as adherent cultures and have the ability to differentiate into neurons and glia cells. Also hESC (EI9/derived... 

It is possible to label cells directly in suspension for intracellular antigens, but the detail of rounded cells for intracellular sites may be obscured by the cell shape, except for gross distributions, such as nuclear or cell-surface patterns. To discern the detail of intracellular distribution in suspended cells, it is best to attach them and, if possible, cause them to spread or flatten onto a substratum so that intracellular detail is visible. Otherwise, confocal microscopy is necessary. Such an approach is similar to that used for surface labeling, in which an adhesive substrate, such as poly-L-lysine (Sigma-Aldrich) or Cell-Tak (BD Biosciences), is fashioned to attach cells to the surface of a dish. The cells can then be handled the same as adherent cultured cells. For cells that do not spontaneously flatten, cyto-centrifugation is useful, although the amount of intracellular detail is still somewhat limited. However, mitotic cells that are rounded in many cultured cell lines are an example of the pos-... 

Barker, K., Fan, H., Carroll, C., Kaplan, G., Barker, J., Hellmann, W., and Cohn, Z.A. 1996, Nonadherent cultures of human monocytes kill Mycobacterium smegmatis, but adherent cultures do not. Infection Immunity 64 428-433. 

Rinse adherent cultured cells twice for 5 min at 37°C with 0.5 mL/well assay buffer. 

A more recent alternative for looking at cytotoxicity in adherent cultured cells involves the use of real-time cell electronic sensing (RT-CES) equipment. Cells are plated into special multiwell plates through which a current is passed at defined time intervals. The impedance of that current is correlated to the density of adherent cells in the well through calculation of a cell index that is measured over the duration of the experiment. The RT-CES assay has an advantage over... 

In these investigations, we have used A549 cells, derived from a human lung carcinoma, grown as adherent cultures in RPMl 1640 medium supplemented with 10% foetal bovine serum. The effect of either BLM or BCNU on cell proliferation was assessed by measuring the increase in the number of cells in cultures 3 days after drug treatment. This is expressed as percentage cell number increase , defined as... 

Adherent cultures Cultures of cells that are attached to a natural or artificial substrate. Often referred to as monolayer cultures, but may be expanded to include multilamellar cultures. 

A cell-based bioassay procedure generally includes cell growth in suspension or adherent cultures for several days/weeks. In a typical assay, cells are activated/inhibited with the therapeutic protein of interest in a multiwell assay... 

Wartelle C, Schuhmann W, Blockl A, Bedioui F (2005) Integrated compact biocompatible hydrogel-based ampenunetric sensing device for easy screening of drags involved in nitric oxide production by adherent cultured cells. Electrochim Acta 50 4988-4994... 

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