Prepare Cell Culture

Table 18.1 compares the relationship between cell culture surface area and bioreactor volume in many different culture systems usually used with adherent cells. For microcarriers, this coefficient might reach 60 cm2/ml of medium for culture area prepared with 10 mg of microcarriers per milliliter. For Roux bottles, this coefficient is around 3 cm2/ml. In cultures initiated with 2 mg of microcarriers per milliliter of medium, high cell densities of even 3 X 106 cells/ml are often reached, compared with smaller cell densities from 2 to 3 X 105 cells/ml usually observed in Roux bottle systems. Another great advantage of the use of microcarrier culture systems is the possibility of preparing cell cultures with hundreds or even thousands of liters (Montagnon et al., 1984). 

Prepare cell cultures on cover slips and harvest for immunoperoxidase staining as described in steps 1-3 in Subheading 3.1.3. Note Trypsinization of cell mono-layers should not be considered since this process may remove cannabinoid extracellular domains from cell surfaces. 

The EpiOcular corneal model involves culturing normal, human-derived epidermal keratinocytes to form a stratified, squamous epithelium similar to that found in the cornea. The epidermal cells, which are cultured on specially prepared cell-culture inserts using serum-free medium, differentiate to form a multi-layered structure that closely parallels the corneal epithelium. EpiOcular is mitotically and metabolically active and releases many of the pro-inflammatory agents (cytokines) known to be important in ocular irritation and... 

Preparation of clinically important natural products using plant cell culture and synthetic chemistry 97G293. 

The C-nucleoside isostere of 9-(2-hydroxyethoxymethyl)-guanine 976, was prepared as shown in Scheme 180. It showed no activity against herpes simplex virus types I and II in cell cultures (84JHC697) (Scheme 180). 

Animal cell cultures that are initiated from cells removed directly from the animal are called primary cultures (Figure 2). Primary cultures include both explant cultures (i.e., cultures initiated from small pieces of intact tissue), as well as cultures initiated from preparations of individual or dispersed cells (obtained from intact tissue by mechanical or proteolytic dismption). Nerve fiber explant cultures in blood plasma were among the earliest types of tissue cultures (Harrison, 1907). Cells grow out from such tissue explants and form a single layer of cells completely filling the tissue culture vessel surface. Such cell cultures are called confluent monolayers. Confluent monolayers can then be treated with trypsin, so as to remove the individual cells from the culture vessel surface. The resulting cell suspension is then transferred into other culture containers, so that more viable monolayer... 

Research in this area advanced in the 1970 s as several groups reported the isolation of potent toxins from P. brevis cell cultures (2-7). To date, the structures of at least eight active neurotoxins have been elucidated (PbTx-1 through PbTx-8) (8). Early studies of toxic fractions indicated diverse pathophysiological effects in vivo as well as in a number of nerve and muscle tissue preparations (reviewed in 9-11). The site of action of two major brevetoxins, PbTx-2 and PbTx-3, has been shown to be the voltage-sensitive sodium channel (8,12). These compounds bind to a specific receptor site on the channel complex where they cause persistent activation, increased Na flux, and subsequent depolarization of excitable cells at resting... 

C03-0149. A biologist needed to prepare a solution for growing a cell culture. For the culture, he needed to make 1.5 L of solution with these concentrations [KH2 PO4] =0.55M and [K2 HPO4] =0.85M. 

C18-0120. You are doing undergraduate research for a biology professor. Your first assignment is to prepare a pH =7.50 phosphate buffer solution to be used in the isolation of DNA from a cell culture. The buffer must have a total concentration of 0.500 M. On the shelf you find the following chemicals solid NaOH concentrated HCl (12.0 M) concentrated H3 PO4 (14.7 M) KH2 PO4 and K2 HPO4. Write a quantitative detailed set of instmctions that describe how you would prepare 1.5 L of the buffer solution. 

Primary cell cultures, which are prepared directly from tissues. 

Secondary cell cultures, which can be prepared by taking cells from some types of primary culture, usually those derived from embryonic tissue, dispersing them by treatment with trypsin and inoculating some into a fresh batch of medium. A limited number of subcultures can be performed with these sorts of cells, up to a maximum of about 50 before the cells degenerate. 

Inoculation of cell cultures with virus-containing material produces characteristic changes in the cells. The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate. This effect is seen as the shrinkage or sometimes ballooning of cells and the disruption of the monolayer by death and detachment of the cells (Fig. 3.6). The replicating virus can then be identified by inoculating a series of cell cultures with mixtures of the virus and different known viral antisera. If the virus is the same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum and CPE will not be apparent in that tube. Alternatively viral antisera labelled with a fluorescent dye can be used to identify the virus in the cell culture. 

Cell cultures provide infeeted fluids that eontain little debris and can generally be satisfactorily clarified by filtration. Beeause most viral vaccines made fiom cell cultures consist of live attenuated vims, there is no inaetivation stage in their manufacture. There are, however, two important exeeptions inaetivated poliomyelitis vims vaccine is inactivated with dilute formalin or /3-propiolaotone and rabies vaccine is inactivated with /3-propiolactone. The preparation of these inaetivated vaccines also involves a concentration stage, by adsorption and elution of the vims in the case of poliomyelitis vaccine and by ultrafiltration in the case of rabies vaceine. When processing is complete the bulk materials may be stored until needed for blending into final vaccine. Because of the lability of many vimses, however, it is necessary to store most purified materials at temperatures of-70°C. 

Online detection using 4H nuclear magnetic resonance (NMR) is a detection mode that has become increasingly practical. In a recent application, cell culture supernatant was monitored on-line with 1-dimensional NMR for trehalose, P-D-pyranose, P-D-furanose, succinate, acetate and uridine.33 In stopped-flow mode, column fractions can also be analyzed by 2-D NMR. Reaction products of the preparation of the neuromuscular blocking compound atracurium besylate were separated on chiral HPLC and detected by 4H NMR.34 Ten isomeric peaks were separated on a cellulose-based phase and identified by online NMR in stopped-flow mode. 

SAMPLE PREPARATION FOR RAPID, REPRODUCIBLE CELL CULTURE... 

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