Cell culture extraction procedure

With respect to one of these precursors, the term, tropoelastin, has been used as a designation for a non-cross linked elastin precursor of approximately 70,000 daltons (1). Since it is currently the best characterized of the non-crosslinked elastins and is used extensively by those familiar with elastin, this term will be retained. Elastin will be used to designate the protein in its crosslinked form. This term, however, is at best operational, since elastin is only isolated from tissues or cell culture by procedures that would be offensive to most protein chemists. As a component of extracellular matrices, elastin is extremely insoluble and in close association with many other extracellular components (2). In order to remove these components, harsh treatments such as autoclaving, extraction with alkali or... 

A massive body of evidence has already been presented clearly indicating that the medicinal plants of the Pacific Rim elaborate a broad array of cytotoxic substances. Most of these have been characterized using experimental procedures designed to examine the cytotoxicity of natural products against human tumor cell lines. These procedures involve in vitro screening where the viability of cultured cells after exposure to an extract or a purified substance is measured. 

In the next part of our research on EBI-fungicides, we restricted ourselves to Pyricularia oryzae since from our point of view the In vitro results with that organism are representative and the test procedure is rather simple. The test chemical is applied in a suitable concentration to the culture medium which is then inoculated from an untreated pre-culture. After a 24-hour fermentation, the cells are separated from the culture filtrate by centrifugation, resuspended in a chloroform/methanol-mixture and homogenized using an ultraturrax treatment. After this extraction procedure, the sterol-conjugates are split to free sterols by a potassium-hydroxide treatment. Adsorption of the sterols to a Sep-pak column and step-wise desorption leads to a sterol fraction which can be analyzed directly by gas chromatography on a SE-30 capillary column. 

Cells freshly extracted and isolated from an organ or tissue and plated in a defined culture medium (e.g., PBS or L-15 media). During this procedure two parallel processes occur 1) differentiated cells of the original tissue explants usually do not divide and, with time, will successively lose some of their specialized functions (dedifferentiation) and 2) decrease of number of specialized cells (e.g., fibroblasts divide rapidly, and will eventually outnumber the specialized cells). Volume 1(14). 

One example of a miniaturized LC/MS strategy is the use of 96-well sample plates (Kaye et al., 1996) for extraction. This sample extraction procedure combines batch sample processing within a miniaturized format. Increased sensitivity and decreased volume advances have fostered a new wave of scale-down models. Experiments that were formerly performed at the bench are, instead, performed at the microliter scale in the batch mode. For example, synthetic process research was traditionally performed manually with apparatus at the milliliter level. This approach involves the testing of a range of synthetic conditions for optimum yield and minimum impurity production. Now, process research conditions are tested in microliter levels to produce information on purity and structure (Rourick et al., 1996). This strategy requires fewer reagents and accelerates the evaluation of a wider range of conditions in a shorter time. Another example includes the direct analysis of samples from cell culture experiments (Kerns et al., 1997). 

Conditioned medium can be prepared from organ-specific cells cultured in vitro, essentially as described under autocrine growth stimulation. Organ-specific cells can also be extracted in vitro, with the same procedure described for organ extracts. Conditioned medium and/or organ extracts are then tested in a growth assay on tumor cells. 

The need for sample preparation prior to the first chromatographic step is dependent upon sample type. In some situations samples may be taken directly to the first capture step. For example cell culture supernatant can be applied directly to a suitable chromatographic matrix such as Sepharose Fast Flow and may require only a minor adjustment of the pH or ionic strength. However, it is most often essential to perform some form of sample extraction and clarification procedure. 

The production of shikonins is an example of both the promise and limitations of current plant cell culture procedures in the area of natureJ products. While productivity at least 10-fold higher than in plants was obtained, this was the result of a systematic but empirical search for the optimal conditions for shikonin accumulation. In many cases it has not been possible to find the culture conditions appropriate for the expression of a secondary metabolic pathway (H, 15). About a dozen examples are known of cell cultures producing a secondary metabolite at levels equal to or higher than in the whole plant (Table I), but with the exception of shikonin none of these systems has commercial use, or is competitive with existing extraction technology. 

Hutin and co-workers (436,437) reported that usual alkaloid extraction procedures were unable to extract any alkaloid from cell cultures of P. somniferum. By refluxing with hydrochloric acid (1 M, 10 min) or formic acid (3 M, 4 min) morphine and codeine were extracted in detectable amounts. 

Metabolism of CLN in Intestinal Epithelial Cells. Caco-2 cells (5 X 10 cells/dish) were precultivated in a 55-cm tissue culture dish for 24 h. Then, c9,tll,tl3-CLN was added to Ae culture medium as 10% AmeAyl sulfoxide (DMSO) solution. The final concentration of DMSO never exceeded 0.1% (v/v). After incubation, cells were trypsinized and washed three times with phosphate buffered saline. The TL of cells was extracted by Ae Folch s procedure (23). 

Based on these electrochemical studies we developed a method for the quantitation of ajmalicine and catharanthine in cell cultures. These alkaloids were extracted from freeze-dried cells and purified by the solid-phase procedure described by Morris et al. (1985), except that ethanol was used as the extracting solvent instead of methanol. A dual-electrode coulometric cell was used in the screen mode. The potential of the first electode was set at +0.2 V (vs. Pd), which was at the base of catharanthine s voltammogram. The alkaloids were detected by the second electrode at +0.8 V, as this offered the best S/N ratio. Higher potentials led to lower S/N ratio, since the background current and noise started to increase exponentially above +0.85 V, due to the oxidation of water. The mobile phase was purified by a guard cell between the pump and injector. The guard cell operated at +0.8V. 

The nucleotide patterns of cultured animal cells and ascites tumor cells are generally less vulnerable than those of tissues however, if washing is required, a medium capable of supporting energy metabolism should be employed. If cells are to be packed by centrifugation prior to extraction, cultures or ascitic fluids may first have to be cooled. Cells are extracted in the cold some procedures employ alternate freezing and thawing in the presence of acidic extractants. Extraction of cultured mouse embryo cells with 60% methanol for 16 hours at — 20 C has been employed in the analysis of deoxyribonucleoside triphosphates 9). 

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