Cell Culture Conditions

Certain ingredients, such as electrolytes and macromolecules, and the pH of the cell culture medium may alter the surface and physico-chemical properties of the NPs and, thereby influence the gene dehvery efficiency. In particular, presence of serum or serum proteins is often reported to lower the transfection efficiencies of several NPs. Even if the NPs are stable in the presence of serum or serum proteins, batch variations in serum quality can lead to differences in transfection efficiency. Thus, it is often advisable to test a small lot of serum from a reputable supplier in a control experiment. Once a given lot has yielded sadsfactoiy and reproducible results, sera from this lot should be used for further experiments. 

Cell culture substrate can also the influence gene delivery by affecting the occurrence of endocytosis and, thereby, the uptake of DNA-bound NPs by the cells. Hsu et al. [139] observed that the culture of MSCs on chitosan or HA-modified chitosan membranes increased the intracellular uptake of iron oxide NPs ( 5 nm) as well as naked DNA (3.3 kb, 5 mn) by more than fivefold. The increased internalization of NPs was associated with an increase in clathrin-mediated endocytosis on chitosan ( 50%) and in caveolae-mediated endocytosis on chitosan-HA ( 30-40%). In the case of naked DNA, but not iron oxide NPs, macropinocytosis also occurred on both substrates. 

Microbial contaminadOTi by bacteria, fungi and mycoplasma during in vitro cell culture are additional factors that may influence the efficiency of gene transfection by modifying the growth behaviour of the infected cells. Variation in the growth behaviour of infected cells may lead to different transfection efficiencies between replicate experiments. 

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In culture, the human colon carcinoma cell hne Caco-2 spontaneously differentiates at confluency into polarized cells with enterocyte-like characteristics. The principle of this approach consists of following the passage of the compound of interest from the apical or lumen-like sides to the basolateral or lymph-hke sides of Caco-2 cells, thus following the absorption of the compound per se. One obhgate step for fat-soluble nutrients such as carotenoids to cross the intestinal barrier is their incorporation into CMs assembled in the enterocytes. Under normal cell culture conditions, Caco-2 cells are unable to form CMs. When supplemented with taurocholate and oleic acid, Caco-2 cells were reported to assemble and secrete CMs. ... 

All of these characteristics can be under the regulation of the cell and influenced by the cell culture conditions. The age of the cell monolayer in culture can have a profound impact on the quality of the barrier. In monolayers with actively dividing cells, resistance increases with time in culture as tight junctions form (see Fig. 15, Section III.C.4). Resistance reaches a plateau, then decreases as cell viability declines (Section III.C.4). Time in culture may also be a factor in the expression of polarity, which is related to tight junction formation as well as the state of differentiation of the cells (e.g., differential gene expression). 

Over a long time period it may well not be possible to duplicate library cell culture conditions. What happens when the lot of media used in the final culture step prior to pyrolysis has been consumed Can culture media suppliers assure nutritional identity between batches Media types for growth of fastidious strains invariably include natural products such as brewer s yeast, tryptic soy, serum, egg, chocolate, and/or sheep blood. Trace components in natural products cannot be controlled to assure an infinite, invariable supply. The microtiter plate wells used here do not hold much media. Even so, the day will come when all media supplies are consumed and a change in batch is unavoidable. When that happens, if there were no effective way to compensate spectra for the resulting distortions, it would be necessary to re-culture and re-analyze replicates for every strain in the reference library. Until recently the potential for obsolescence was a major disincentive for developing PyMS spectral libraries of bacteria. Why this is no longer an insurmountable problem is discussed in the next section. 

Karl, MO, Valtink, M, Bednarz, J, and Engelmann, K, 2007. Cell culture conditions affect RPE phagocytic function. Graefes Arch Clin Exp Ophthalmol 245, 981-991. 

Under linear concentration conditions (for a P-C concentration range of 0.12-6pM) at 16h incubation and under cell culture conditions mimicking the in vivo postprandial state, the extent of absorption of all-trans P-C through Caco-2 cell monolayers was 11% a value similar to that reported from different human studies. In humans, the bioavailability of a single dose of P-C... 

PMBV and PVA can spontaneously form a hydrogel without using any crosslinkers. Even in cell culture conditions, the gelation can be confirmed. Therefore, it was possible to encapsulate cells in the PMBV/PVA hydrogel. The encapsulation method is illustrated in Fig. 6. 

We wondered why NSCs proliferated exclusively on surfaces with EGF-His ligands anchored by coordination. We focused on two aspects in particular the conformational integrity of coordinated EGF-His and the stability of coordinate bonds at the interface. Conformational information was acquired with multiple internal reflection-infrared absorption spectroscopy (MIR-IRAS) [97]. The stability of coordinate bonds was assessed by culturing NSCs on a surface with a small region of EGF-His ligands anchored by coordination. This spatially restricted EGF-His anchoring enabled an intuitive exploration of EGF-His release under cell culture conditions. 

It was known that the intracellular concentrations of the reduced and oxidized forms of the pyridine nucleotides vary in different cell types and under different cell culture conditions.(17) Harrison and Chance applied the NAD(P)H fluorescence technique and found that culture fluorescence can be related to the metabolic state of the cells. 18,19) Since then, more than a hundred papers on NAD(P)H fluorometry have been published. However, they are primarily divided into three major categories ... 

Behrens I, Kissel T (2003) Do cell culture conditions influence the carrier-mediated transport of peptides in Caco-2 cell monolayers Eur J Pharm Sci 19 433-442. 

Cell monolayers grown on permeable culture inserts form confluent mono-layers with barrier properties and can be used for drug absorption experiments. The most well-known cell line for the in vitro determination of intestinal drug permeability is the human colon adenocarcinoma Caco-2 [20, 21], The utility of the Caco-2 cell line is due to its spontaneous differentiation to enterocytes under conventional cell culture conditions upon reaching confluency on a porous membrane to resemble the intestinal epithelium. This cell model displays small intestinal carriers, brush borders, villous cell model, tight junctions, and high resistance [22], Caco-2 cells express active transport systems, brush border enzymes, and phase I and II enzymes [22-24], Permeability models... 

Kunkel J.P., Jan D.C., Butler M., and Jamieson J.C. (2000), Comparisons of the glycosylation of a monoclonal antibody produced under nominally identical cell culture conditions in two different bioreactors, Biotechnol. Prog. 16, 462 -70. 

This review is focused on the evaluation of the interaction between the artificial material and cells in cell culture conditions. In comparison with in vivo experiments, where both material and contacting cells are subjeeted to multiple and complex influences of the whole organism, the cell eulture represents a relatively simple and well-defined system. This system enable sereening of a wide range of materials and their surfaee modification in order to select the most appropriate variants for testing on laboratory animals and then for clinical studies. Therefore, the use of cell eulture eonsiderably saves these animals, and thus fulfills the ethical requirements for advanced scientific research. 

Stability and longevity of the surface coating under cell culture conditions... 

The glycosylation profile is affected by the cell culture conditions. Ideally, a product should be characterized at least once to identify the potential glycosylation sites as well as the specific carbohydrate at each site. 

Examples of small molecules, altered cell culture conditions, proteins, and genetic constructs used to probe RME. CID chemical inducer of dimerization. 

Small molecules and modified cell culture conditions that block CME... 

The virus reduction studies of the three process steps discussed here were performed with HFV-l, Bovine viral diarrhea virus (BVDV), Pseudorabies virus (PRV), Reovirus type 3 (Reo), Hepatitis A virus (HAV), and Porcine parvovirus (PPV). HIV-1 was included as a relevant enveloped virus, while BVDV and PRV were tested as specific model viruses for HCV and HBV, respectively (Table 1). Reo was chosen as a non-specific model non-enveloped virus, HAV was included as a relevant virus and PPV was used as a surrogate for human parvovirus B19. All viruses were propagated using standard cell culture conditions. " The appropriate cell lines were infected, at a low multiplicity of infection, and incubated until 4-1- cytopathic effects were observed. The infected cells were frozen and thawed three times to release virus, centrifuged at low speed to remove cell debris and the clarified supernatants were removed for use as virus spikes. 

Mammalian cell cultures producing high-value biopharmaceuticals are expensive and time-consurning to study due to their exclusive dependence on experimentation. A mathematical model has been developed that describes batch/fed-batch hybridoma suspension cultures under normal and chemically-arrested conditions, which is also used to optimise the fed-batch cultures. The optimised strategy was tested experimentally demonstrating that product concentration was closely predicted though the viable cell concentration was partly underestimated. Overall, the model has assisted in reducing the number of experiments required to determine optimal cell culture conditions. Further work is required to improve the model predictability. 

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