Cell culture seeding density

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. 

To evaluate cell proliferation in the hydrogel, the L929 cells were immobilized at a density of 1.0 x 105 cells/mL. As a control sample, L929 cells were seeded onto a conventional cell culture plate at a density of 0.5 x 104 cells/mL. 

We investigated the efficiency of NSC expansion on surfaces with EGF-His immobilized in the correct orientation. NSCs were obtained from neurosphere cultures prepared from fetal rat striatum harvested on embryonic day 16. NSCs were cultured for 5 days on EGF-His-immobilized substrates prepared with mixed SAMs of different COOH-thiol contents. Cells adhered and formed network structures at a density that increased with the COOH-thiol content of the surface. As a control, cells were seeded onto surfaces without immobilized EGF-His. This resulted in poor cell adhesion during the entire culture period. In addition, when EGF-His adsorbed to SAMs with 100% COOH-thiol or SAMs with NTA-derivatized COOH that lacked Ni2+ chelation, we observed poor initial cell adhesion, and the cells formed aggregates within 5 days. Interestingly, the substrate used to covalently immobilize EGF-His with the standard carbodiimide chemistry was not a suitable surface for cell adhesion and proliferation. The control experimental results contrasted markedly with results from EGF-His-chelated surfaces. 

Finally, estradiol induced a response in the luciferase activity of MVLN transfected cells. The hormone increased three-fold the basal level of enzymatic activity (Fig. 7.3.2). The luciferase activity of each experiment was normalised to the steroid-free control cultures to correct for differences in the initial seeding density. The activity induced by the antiestrogen RU 58 668 was lower than the response to the control. 

For further purification, primary cultures of PBEC are passaged at the third day of culture by gentle trypsinization at room temperature. This enzymatic treatment selectively releases endothelial cells, leaving behind contaminating cells, such as pericytes and smooth muscle cells. Usually, contamination by nonendothelial cells should be below 1-3%. Endothelial cells are then seeded at a density of 30,000-50,000 cells/cm2 on rat-tail collagen-coated cell culture inserts (Figure 17.5). 

Start a new culture with fresh media and seed cells such that the seeding density is not lower than 1 x 10 cells/ml. Cells can be centrifuged and subsequently resuspended at 1 X 10 cells/ml. 

Discard supernatant and resuspend cells in 200 pL of culture medium. Using a 200 pL Pipetman with the volume set at 180 pL, gently dissociate the pellet for 20-40 times. Dilute a 5 pL aliquot from each sample in 15 pL of Trypan blue and count in a Burker or Neubauer chamber. Seed cells at a density of 8 X 10 viable cells/cm in culture medium containing EGF and FGF2. 

Adherence to Principles of GMP in a Prechnical Developmental Process Efficient Standardized MSC Propagation Using Low Cell Seeding Density Superior MSC Proliferation Resulting from HPL-Driven as Compared to FBS-Driven Cultures... 

FIGURE 7 CFU-F Evaluation of MSCs depends on BM seeding density. An appropriate dilution of the heparinised BM aspiration is needed for accurate enumeration of the primary CFU-F frequency as indicated in this representative experiment where whole heparinized BM was seeded corresponding to the respective measured BM-MNC number per square centimeters of growth area, cultured for 11 days at 37°C/humidified atmosphere/3% 02/5% C02. Nonadherent cells were removed at day 3. CFU-F are visualized by Harris hematoxylin staining. 

The same cell cultures used in the previous experiments were employed, except the low PD culture had been grown through two more PDs to 14 rather than 12. Human primary fibroblasts were seeded onto a polystyrene surface at a predetermined density (105 cells per surface), and allowed to attach over a period of 100 mins. At six time points the flasks were gently agitated, and the supematent drawn off and the cell content counted. This simple procedure yielded a characteristic and highly reproducable attachment/time curve (Figure 2). 

Adherent cells Cells are seeded in 16 mm wells in 4- or 24-well tissue-culture plates (Nunc supplied through Gibco) and grown for 2 d to a final density of... 

After a cell type (in vitro model system) has been chosen for cytotoxicity or apoptosis assays, several key parameters must be determined to characterize assay performance (1) optimal cell seeding density, (2) volume of culture medium per well, (3) equilibration period after dispensing cells, (4) concentration of compound to be tested, (5) length of exposure of cells to the test compound, and (6) selection of appropriate assay chemistry. 

Cells are seeded in a flat-bottomed 96-microwell plate 2 to 3 days before the experiment, at a density sufficient to reach about 80 % confluence before further manipulation (10 cells/well for HIT-T15, INS-1 or RINmSF). Seed them in 6 to 8 rows, each row having 4 to 6 wells. The methods to culture insulin-secreting cells have been described in detail elsewhere (Asfari etal., 1992, Wollheim etal., 1990, Wollheim et al., 1990). 

Flasks and plates are routinely seeded at a density of 3-5 x 104 cells/cm2. This seeding density will produce confluent cultures in 3-5 d. Seeding densities of approx 1 x 104 cells/cm2 are permissible and will prolong time to confluence. Seeding densities lower than this will usually lead to colonies with... 

KB/HeLa cells are seeded in 24 well culture plates at a density of 3 X 10 cells per well and cultured overnight. 

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