Supplement-free medium

Plasmid DNA solution prepare DNA solution e.g. luciferase reporter plasmid or eGFP plasmid, at a concentration of 12 pg DNA/ml by dilution of the stock solution with a serum- and supplement-free medium (e.g., RPMl 1640). 

For the untransfected control setup, add 300pl of serum- and supplement-free medium and 60 pi of water to well Al. For other controls and references (e.g. magnetic nanoparticle-DNA or siRNA duplexes without enhancer, enhancer-DNA or siRNA complexes without magnetic nanoparticles), substitute the omitted component(s) with medium and/or water. Incubate for 15 min at RT. 

During the incubation time, fill each of the remaining wells of columns 1, 4, 7, and 10 with 180 pi of serum- and supplement-free medium (RPMI 1640). Prepare a 1 1 dilution series, when the 15 min incubation time is over, as follows transfer 180pl, from each of Al, A4, A7, and AlO to Bl, B4, B7, and BIO, respectively, using a multichannel pipette, then mix, transfer 180 pi from the respective wells in row B to row C and so on down to row D or further to cover the desired nucleic acid dose range per well. 

Table 4. Growth Supplements for Two Cell Lines in Serum-Free Medium...

FGF), and hydrocortisone to grow serum free (see Table 4) (Hutchings and Sato, 1978). Both of these cell types can grow serum free with appropriate supplements at the same rate as that obtained in serum-supplemented medium. Both cell types can also survive over extended culture periods in hormonally defined serum-free medium. 

F9 embryonal carcinoma cells have a simple set of growth supplements which are required for growth in serum-free medium insulin, transferrin, and fibronectin (Rizzino and Sato, 1978). Fibronectin is a component of the extracellular matrix and facilitates the attachment of the cells to the culture dish. In addition, high density lipoprotein (HDL) has been observed to promote the growth of F9 cells serum-free. 

Vitamins and lipids are often required for animal cells to grow in serum-free medium. Phosphoethanolamine and ethanolamine are key additives that facilitate the growth of the mammary tumor cell line 64024 (Kano-Sueoka and Errick, 1981). In addition, ethanolamine promotes the growth of human lymphocytes and mouse hybridoma cells. Short-term cultures of human diploid lung and foreskin fibroblasts grow in medium that includes among its supplements soybean lecithin, cholesterol, sphingomyelin, and vitamin E. 

The mechanisms by which the growth supplements in serum-free medium act are still not understood. In order to achieve an understanding of the biochemical basis for the hormonal and growth factor requirements of animal cells, the basic mechanism of action of hormones and growth factors must be determined. The biochemical basis for the nutritional requirements of animal cells can only be determined when we have an understanding of the metabolism of the different types of animal cells. 

A good example of the problems encountered with the use of serum with primary cultures is illustrated by the case with cultured kidney cells. The kidney epithelial cell line MDCK grows in serum-free medium supplemented with five supplements ... 

Hutchings, S.E. Sato, G.H. (1978). Growth and maintenance of HeLa cells in serum-free medium supplemented with hormones. Proc. Natl. Acad. Sci. USA 75.901-904. 

Figure 25. A-D Immunofluorescence staining of vmculrn in vascular smooth muscle cells on day 3 after seeding on polymeric surfaces (medium supplemented with 10% fetal bovine serum). A poly(DL-lactic acid), PDLLA B block copolymer of poly(DL-lactic acid) and poly (ethylene oxide) (PEO), PDLLA-6-PEO C, E PDLLA-6-PEO with 5% GRGDSG-PEO-6-PDLLA D, F PDLLA-6-PEO with 20% GRGDSG-PEO-6-PDLLA. E, F Immunoperoxidase staining of bromodeoxyuridine (arrows) incorporated into DNA newly synthesized in vascular smooth muscle cells cultured for 3 days in serum-free medium on PDLLA-Z)-PEO with 5% (E) or 20% (F) GRGDSG-PEO-6-PDLLA. Cells counterstained with light green. Bar=100 pm [41].

Mercille et al. [141] compared TFF and VFF for the perfusion cultivation of a hybridoma cell line producing an IgM, either using serum-supplemented or protein-free medium, with and without the addition of DNAse. They observed... 

The new media that are commercially available possess many distinct characteristics some are completely free of animal-derived components, others are serum-free, sometimes requiring addition of some protein fraction or containing a discrete quantity of proteins. Another class is protein-free media, which can include components derived from animals, plants, or yeast hydrolysates. In some cases, the development of novel media is unnecessary, as it is sufficient to supplement basal medium with a few known specific components. It is emphasized that the final osmolality of the formulated medium should be compatible with cellular tolerance, as discussed in Chapter 2. 

Recently, plant hydrolysates have also been tested as additives for serum-free culture media (Donaldson and Shuler, 1998 Franek et al., 2000 Heidemann et al., 2000). Donaldson and Shuler (1998) demonstrated that a peptide obtained from soy hydrolysate (Hy-Soy ) played an important role in a serum-free medium developed for insect cells. Ikonomou et al. (2003) also showed the effectiveness of culture medium supplemented with the soy hydrolysate SE50MAF in the growth of High Five cells. 

The second category includes mesenchymal cells, such as fibroblasts (BALB/c 3T3, Swiss 3T3), adipocytes, endothelial cells, smooth thin muscle cells, and neuroectodermic cells (such as glia cells). Most of these cells need maintenance factors. Some cells, such as the NIH-3T3, can grow in a serum-free medium containing minimal medium supplemented with transferrin (25 pg/ml), insulin (10 pg/ml), EGF (100 ng/ml), bFGF (100 ng/ml), and PDGF (0.5 U/ml). 

Mercille S, Johnson M, Lemieux R, Massie B (1994), Filtration-based perfusion of hybridoma cultures in protein-free medium - reduction of membrane fouling by medium supplementation with DNAse-I, Biotechnol. Bioeng. 43 833-846. 

Development of serum-free medium has great value for large-scale biopesticide production. The latest formulations are serum-free, such as SF900II (GIBCO /Invitrogen) and EX-CELL (JRH Biosciences). Cell culture medium supplementation using yeast extract (usually from alcoholic fermentation processes), milk, or soy protein concentrates, can also be an alternative to decrease cell culture medium costs (more details can be found in Chapter 5). 

SF-1 is a serum-free medium ( x 1) supplied by Costar through Northumbria Biologi-cals Ltd. and which can be supplemented with Ewing s Sarcoma Growth Factor (ESG). 

N.B. Cells cannot be frozen in protein free medium and will require adaptation each time a batch is thawed (see 7.3). It is also not recommended to use protein free medium for cloning unless it is supplemented with factors such as ESG and/or HECS. 

A serum-free medium supplemented with insulin, transferrin, ethanolamine and selenium (ITES) allows growth of certain hy-bridomas at 17-74% the rate found with 15% FBS (Wolpe, 1984) and Cleveland et al. (1983) devised a protein-free medium for growth of myeloma cells which, with addition of BSA at 2.5 mg/ml, forms the basis of Costar s SF-1 and SF-X supplemented media. Cloning is still very difficult in serum-free media, but feeder layers can be replaced by culture supernatants from human endothelial cells (HECS Astaldi, 1983) or Ewing s sarcoma cells (ESG Ley et al., 1980) — see 5.8.5. 

Sulfur metabolism of yeast. I. Study of relative growth of five yeasts on a sulfur-free medium supplemented with small quantities of sulfur compound. Contribs. Boyce Thompson Inst., 19, 437 (1958). With D. Margolis. 

Coating of 96 multiwell plates is done with 20 /xg of proteins in 0.2 ml of PBS/well overnight at 4°C. After accurate washing of the wells with serum free medium, 5 x 10 EC in 0.2 ml of DMEM containing 10% heat inactivated FCS were plated per each well, and incubated at 37°C until very dense (usually 3-4 days). Monolayers are then rinsed with DMEM supplemented with 0.4% BSA (BSA, Sigma fraction V, treated with periodic acid to remove glycoprotein impurities) and used for the adhesion assay as described below. 

EC monolayers obtained after growth on either collagen-coated glass coverslips, or tissue culture multiwell plates as previously described (Sections 2.3.3.1 and 2.3.3.2), are rinsed several times in serum Iree medium, and then incubated 1 h at 37°C with assay medium (usually serum free culture medium supplemented with 0.5-1% BSA), before they are used as substrates for the adhesion assay. Tumor cells are detached from culture dishes either by EDTA or by trypsin treatment. In the latter case, trypsinized cells should be allowed to recover from the enzyme treatment in their growth medium for 30 min at 37°C. After rinsing in serum-free medium, labeled tumor cells are resuspended at the desired concentration in the assay medium and plated onto the EC monolayer at a cell concentration ranging between 10 and 2 x 10 /cm. Incubation is... 

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