Cell growth media

This chapter will deal predominantly with studies on mammalian cells, although some examples of studies on plant cells, fungi, and bacteria will also be included. In the context of this chapter, the term in vivo refers to the intact cell. However, we have also included a discussion of NMR measurements on extracts of cells and cell growth media, particularly NMR measurements of isotopic labeling, as these illustrate the power of the NMR technique for the investigation of metabolic processes. 

Prepare 1.5 mL of each chemokine dilution to be assessed by diluting the chemokine stock solution in chemotaxis media (chemotaxis media is a 50 50 mixture of endothelial cell growth media without FCS and RPMI-1640, plus 0.5% BSA) prewarmed to 37°C. 

Akay, G. Dawnes, S. Price, V.J Microcellular Polymers as Cell Growth Media and Novel Polymers European Patent 1,183,328, Jun 3, 2002. 

Akay G., Dawnes S., Price V.J. 2002. Microcellular polymers as cell growth media and novel polymers, European Patent, EP 1 183 328. 

Fig. 2. ADPR-transferase activity in permeabilized cells. Conditions for growing cells and adding drugs was as described for Fig. 1 except that [ H]-thymidine was not present. At the times indicated cells were recovered, permeabilized and assayed for transferase activity [5]. The cell growth media contained either 100 juAf methotrexate alone ( ) or 100 yM methotrexate and 25 nM hypo-xanthine (O)...

BEGM Bronchial epithelial cell growth medium... 

Medium 500 mL Endothelial Cell Growth Medium-2 (EGM-2) medium supplemented with SingleQuot kit Lonza... 

Currently, there are many examples of cell processing in the industrial environment using tangential flow filtration. To illustrate the breadth of microbial types which may be processed by this technology, we will discuss three applications which have been in routine operation under production conditions. The applications include cell/growth medium separations directly from fermentors (Escherichia coli and Mycoplasma species) and the concentration/washing of influenza virus used in the production of flu vaccines. 

Composition and Methods of Manufacture. Vaccine is produced from the Oka attenuated strain. Vacciae is produced in human diploid cells such as MRC-5. After growth in the cell substrate, the cells themselves are harvested into the growth medium and sonicated to release the cell-associated vims. Sucrose and buffering salts are generally in the medium to help stabiLize the vims. The vacciae is presented in a free2e-dried vial to be reconstituted with sterile distilled water before injection (27). 

Nutritional Requirements. The nutrient requirements of mammalian cells are many, varied, and complex. In addition to typical metaboHc requirements such as sugars, amino acids (qv), vitamins (qv), and minerals, cells also need growth factors and other proteins. Some of the proteins are not consumed, but play a catalytic role in the cell growth process. Historically, fetal calf semm of 1—20 vol % of the medium has been used as a rich source of all these complex protein requirements. However, the composition of semm varies from lot to lot, introducing significant variabiUty in manufacture of products from the mammalian cells. 

A hybridoma can live indefinitely in a growth medium that includes salts, glucose, glutamine, certain amino acids, and bovine serum that provides essential components that have not been identified. Serum is expensive, and its cost largely determines the economic feasibihty of a particular ciilture system. Only recently have substitutes or partial replacements for serum been found. Antibiotics are often included to prevent infection of the culture. The pH, temperature and dissolved oxygen, and carbon dioxide concentration must be closely controlled. The salt determines the osmotic pressure to preserve the integrity of the fragile cell. 

Calf kidneys, dog kidneys and rhesus monkey kidneys were treated with trypsin to give suspensions of cells. The suspensions were centrifuged and the packed cells diluted with 400 volumes (calf cells) or 200 volumes (dog cells and rhesus monkey cells) of a growth medium consisting of 5% horse serum and 0.5% lactalbumen hydrolysate in Earle s saline, with 100 units/ml each of penicillin and streptomycin. These media were used separately to produce Semliki Forest/calf interferon, Semliki Forest/dog interferon and Semliki Forest/rhesus monkey interferon. The cell-containing growth medium was dispensed into 500 ml medical flat bottles (70 ml in each). The cultures were incubated at 36°C. Confluent sheets of cells (monolayers) were formed in 5 to 6 days. The growth medium was then removed and the monolayers were washed with isotonic phosphate-buffered saline, pH 7.5. 

The production of the polymer depends on several factors such as the composition of the growth medium, the time of harvest, and the particular stage of the life-cycle of organism under consideration. Eor P. polycephalum only plasmodia are the producers of j8-poly(L-malate) neither amoebae nor spherules (specialized cell forms that can survive unfavorable environmental conditions)... 

As you might have already gathered, the majority of industrial fermentations are batch processes. In closed batch systems, the growth medium is inoculated with cells and growth and product formation is allowed to proceed until the required amount of conversion has taken place. After harvesting the culture the vessel is cleaned, sterilised and filled with fresh medium prior to inoculation. For some processes, addition of all the feedstock prior to inoculation, as is done in closed batch fermentations, is undesirable and it is preferable to incrementally add the carbon source as the fermentation proceeds. Such a process is known as fed-batch culture and the approach is often used to extend the lifetime of batch cultures and thus product yields fed-batch cultures are considered further in Section 2.7.4. 

The system is balanced for cell growth by removing old culture and replacing it with fresh medium at the same rate. 

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