Serum-free conditioned media

Leprince P, Rogister B and Moonen G (1989). A colorimetric assay for the simultaneous measurement of plasminogen activators and plasminogen activator inhibitors in serum-free conditioned media from cultured cells. Ann Biochem, 177, 341-346. 

Yamaguchi, K. (2002). Peptide differential display of serum-free conditioned medium from cancer cell lines. Cancer Lett. 176, 199-203. 

The effect of crocin on the TNF-a-induced cell death of PC-12 cells is shown in Fig. (6). In serum-free GIT medium conditions, PC-12 control cell morphology remained intact at 24 h (panel A). However, the cells treated for 24 h with TNF-a (500 units/ml) appeared rounded and showed the characteristics of necrotic and/or apoptotic cells (panel B). In the combination with 10 pM crocin, PC-12 cell morphology retained intact neuronal cell morphology at 24 h (panel C). Crocin alone had no effect on the morphology of PC-12 cells (data not shown). 

The cells can then be adapted through growth in a series of progressively lower serum concentrations. To achieve the final step to serum-free condition, one of three methods can be used addition of commercial serum substitutes, addition of defined proteins or addition of only amino acids, trace elements and other small molecules to provide a protein-free medium. The last of these alternatives is the most difficult to achieve however, it may ultimately prove to be the most useful. 

This discussion provides an overview of the methods for selecting a serum lot and balanced nutrient medium as starting points for adapting a cultured cell line to serum-free conditions. The ultimate stringency of serum-free medium attained (containing serum substitutes, defined additives or protein-free) depends on the needs of the researcher and the characteristics of the cell line. Serum provides many different functions for the cell and there are many different types of cells, each of which requires a medium derived to meet its particular needs. The information provided here should enable the development of a serum-free medium appropriate for each particular case. 

There have been several investigations into the use of hormonally defined medium in order to maintain the differentiation of primary cells, as it is suspected that serum may be a factor in dedifferentiation. The application of defined medium also allows a more standardized approach to cell culture delivering greater reproducibility and transferability. For renal tubular epithelial cells defined medium supplements have been described as far back as 1982 [148], We have been, over the last number of years, successfully cultivating human renal proximal tubular cells (primaries and cell lines) in serum free hormonally defined medium containing EGF, hydrocortisone, insulin, transferrin, and sodium selenite using DMEM-Hams F12 as the base medium [36, 112, 114, 149], Both the HK-2 cell line and the RPTEC/TERT1 have been developed in serum-free conditions. 

Proceed to serum-free conditions inoculating at a high level, and maintaining them at this level (by centrifugation and concentration if necessary) until they start dividing. The use of conditioned medium is usually beneficial at this stage. 

Cells are sometimes cultured in serum-free medium. In this condition, the surface should carry substituents of serum proteins that can directly interact with cells. SAMs of alkanethiols with bioactive ligands have been used to control interactions between the material surface and cells [80-83]. Several bioactive ligands have been tested, including RGD [80], PHSRN [81], and laminin-derived peptides [82, 83]. These ligands were expected to directly interact with cell surface integrins. 

Incubation temperature and medium pH are also important regarding proteolytic activity of baculovirus infected insect cell cultures. Cruz et al. [25] have shown that the highest proteolytic activity was obtained at the normal culture conditions, 27 °C and pH 6.5. This could then be considered a drawback when the production of protease sensitive particles Hke HIV-CLPs and HIV-VLPs is envisaged [5]. The pH of Sf9 cells has been reported to reach a minimum of 5.9 in serum-free media under uncontrolled pH conditions in stirred tank reactors... 

A 50 50 mixture of fresh growth medium with one of the above conditioned media is often recommended for growth of embryonic stem cell cultures. Otherwise the protein fraction can be concentrated for addition to serum-free medium. 

If the conditioned medium is to be made serum-free, grow the cells to confluence in medium containing serum as in Subheading 2.1.4.L, then change to serum free medium and incubate up to 7 d before harvesting the medium. 

Cell growth during the time of the assay might influence the results, in the sense that the filling of the artificial wound could be contributed from both cell replication and movement. In order to avoid this problem, the assay should be conducted in serum-free or at least low-serum conditions, to limit cell growth. Alternatively, inhibitors of DNA synthesis (mitomycin-C or aphidicolin at 0.5 /Lig/ml), can be included in the culture medium, since it has been shown that active migration may depend on protein, but not DNA synthesis (Geimer and Bade, 1991 Chen et al., 1994). 

Control sample 5-20 /rl serum-free medium conditioned by HT1080 human fibrosarcoma, SK-N-BE human neuroblastoma, or other suitable cell lines. 

It is interesting also to note that the ability of serum to inhibit lipofection is an often described phenomenon (12). In cell culture systems, liposome-mediated gene transfection is usually carried out in serum-free medium or in at most 10-20% serum. The inhibitory effect of serum on transfection mediated by lipoplexes to hepatocytes (13) has been also reported. Thus, during the in vitro assessment of transfection reagent, it is important to emulate in vivo conditions by using high concentration of serum, as is has been done in this protocol. 

The conditions of cell culture are critical for chemical testing. Particular attention must be given to the medium used for the cultivation of cells. While the use of serum, usually foetal bovine serum (FBS), has long been customary to provide isolated cells with a pool of nutrients, attachment factors, and hormones, several issues are encouraging scientists to move towards serum-free hormonally defined medium [53]. In particular, the composition of FBS varies from batch to batch, rendering highly discriminative analyses such... 

For transfection, H9C2 cells (5 x 10s) are placed in six-well culture plates together with fetal calf serum in DMEM at 37°C, 5% C02, and incubated overnight. Then the cells are washed with PBS and recultured in 1 mL of serum-free medium (SFM)/liposome mixture (0.5 mL SFM + 0.5 mL PL or IL in PBS at lipid concentration of 7-10 mg/mL). Nitrogen gas is passed through the medium for 1 min, and wells are covered tightly with Parafilm and incubated for 6 h (these conditions result in mild hypoxia). Normoxic cells are kept under normal conditions for 6 h. 

Media for the production phase are usually richer in nutrient content than the seed train medium in order to maintain viability and productivity of the cells for a minimum of 1-2 weeks. For facilitating recovery and purification of the product and for cost reduction, serum is not used for the production phase. The attachment of cells to the surface of the rollerbottle wiU not be compromised by such a modification in the medium. However, gentle handling is required or the sheets of cells will detach. Upon incubation of the confluent cells with the enriched, serum-free medium, the secreted product will be harvested, leaving the adherent cells inside the bottle. Sometimes, a refeeding with fresh medium for a second production cycle is possible, on the condition that the product of the first and the second harvest will be similar in composition and quality. Since a standard 2-L rollerbottle will contain about 300 mb of medium for harvest, 1000 roUer-bottles will provide from 300 L to 600 L of supernatant. Over a one-year period, a manufacturing process based on this schedule will deliver 15 000-30000 L of... 

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