Tissue culture testing

PVDE is a nontoxic resin and may be safely used in articles intended for repeated contact with food (190). Based on studies under controked conditions, including acute oral, systemic, subchronic, and subacute contact implantation and tissue culture tests, no adverse toxicological or biological response has been found in test animals (191,192). PVDE is acceptable for use in processing and storage areas in contact with meat or poultry products prepared under federal inspection and it complies with the 3-A sanitary standards for dairy equipment. 

Biological tests. USP 24 lists two levels of biological tests—the in vitro tissue culture test and the in vivo systemic injection and intracutaneous tests. A parenteral closure must pass either type of test to meet USP requirements. 

Toxicity of nanosystems what are reasonable predictive animal or tissue culture tests ... 

Burton et al [25] investigated the sensitivity and specificity of the luminescent bacteria toxicity test (LBT) as compared with the USP mouse safety test, rabbit muscular implantation, mouse systemic injection, and the MEM elution tissue culture test. The samples included industrial plastics/medical devices and low density polyethylene containing different concentrations of toxic organic substances. The results of these comparative tests showed the LBT to be significantly more sensitive than the animal tests and slightly more sensitive than the tissue culture acute toxicity assay for the samples tested. 

For all procedures, solutions should be made to the standard required for molecular biology using molecular biology-grade and/or tissue-culture-tested reagents. All solutions should be made using sterile double-distilled or MilliQ water, and where appropriate, autoclaved or filter-sterilized. 

Initial evaluation of novel biomaterial Tissue culture test on materials Tissue culture test on materials extracts Cell growth in contact with test materials Haemolytic activity test Intramuscular implantation of material Test of osmotic fragility of erythrocytes In vitro mutagenicity test... 

Interest and need is especially high with rhinoviruses. There are so many strains of rhinoviruses that chemotherapy would appear to be the method of choice for prevention or treatment. An efficient system for plaguing most rhinovlrus strains,has been developed utilizing a special line of HeLa cells.Because of the need of a broad-spectrum agent for rhinoviruses, it has been suggested to screen in tissue culture teste with mixtures of strains of rhinoviruses. ... 

Other tests include the Ames test in which a strain of bacteria, such as Salmonella typhimurium, is mixed with rat liver and the test substance then incubated for two days. The carcinogenicity is indicated by the number of mutants induced. This test is sensitive, quick and cheap. Also tissue cultures tests in which cells from a test animal are cultured in an isotonic fluid medium and the effects of adding the test substance are observed. 

Standardization and Testing". Potency is deterrained by titration of the amount of Hve vims Hi susceptible tissue culture and is mn Hi paraUel with a U.S. standard. Both in vivo and in vitro tests are used to assess safety (17). 

Commercial use of cell and tissue culture continues to expand. Improvement of organisms through recombinant nucleic acid techniques has become commonplace. Formerly, a few laboratories were well ahead of most others, but now the methods have been perfected for routine use. Another technique that is widely practiced is culturing of cells that excrete high concentrations of just one antibody protein. The specificity of antibodies and antigens is exploited in medical testing procedures using these pure monoclonal antibodies. 

Resistance to NS3/4A inhibitors was primarily tested using the replicon system in tissue culture and closely parallels what was seen with resistance to HIV-1 PI. 

Initially, the cytotoxicity against chick embryo fibroblasts of BPA, tyrosine, tyrosine dipeptide, and the dipeptide derivatives used in the synthesis of the polymers shown in Fig. 7 were evaluated in a comparative experiment (43). The surface of standard tissue culture wells was coated with 5 mg of each test substance. Then the adhesion and proliferation of the fibroblasts was followed over a 7-day period. Among all test substances, BPA was clearly the most cytotoxic material. Monomeric tyrosine derivatives containing the ben-zyloxycarbonyl group were also cytotoxic, while tyrosine itself, tyrosine dipeptide, and most of the protected dipeptide derivatives did not noticeably interfere with cell growth and adhesion and were therefore classified on a preliminary basis as possibly "nontoxic."... 

Tissue Culture Assay. Kogure et al. (48) report a novel tissue culture assay for detecting several types of sodium channel blockers. The mouse neuroblastoma cell line ATCC CCL 131 is grown in RPMI 1640 supplemented with 13.5% fetal bovine serum and 100 pg/ml gentamycin, in an atmosphere of 5% C0 95% air at 37 C. Ninety-six well plates are seeded with 1 x 10 cells in 200 pi of medium containing 1 mM ouabain and 0.075 mM veratridine. Veratridine and ouabain cause neuroblastoma cells to round-up and die. In the presence of sodium channel blockers (e.g., TTXs or STXs), the lethal action of veratridine is obviated and cells retain normal morphology and viability. An important feature of this assay is that a positive test for sodium channel blockers results in normal cell viability. Since bacterial extracts can contain cytotoxic components, this assay offers an advantage over tests that use cell death as an endpoint. The minimum detectable level of TTX is approximately 3 nM, or approximately 1/1000 mouse unit. 

The testing of disinfectants for virucidal activity is not an easy matter. As pointed out earlier (Chapter 3), viruses are unable to grow in artificial culture media and thus some other system, usually employing living cells, must be considered. One such example is tissue culture, but not all virus types can propagate under such circumstances and so an alternative approach has to be adopted in specific instances. The principles of such methods are given below. 

Since disinfectant itself might be toxic to the tissue culture or eggs, a toxicity test must also be carried out. Here, appropriate dilutions of disinfectant are mixed with inactivated horse serum and inoculated into tissue cells or eggs (as appropriate). These are examined daily for damage. 

The hepatitis B virus (HB V) does not grow in tissue culture and an acceptable animal model has been found to be the chimpanzee. This is observed for clinical infection after inoculation with treated and untreated virus, care being taken in the test series that residual disinfectant is removed by adequate means before inoculation into the animal. 

Viral vaccines present problems of safety testing far more complex than those experienced with bacterial vaccines. With killed viral vaccines the potential hazards are those due to incomplete virus inactivation and the consequent presence of residual live virus in the preparation. The tests used to detect such live virus consist of the inoculation of susceptible tissue cultures and of susceptible animals. The cultures are examined for cytopathic effects and the animals for symptoms of disease and histological evidence of infection at autopsy. This test is of particular importance in inactivated poliomyelitis vaccine, the vaccine being injected intraspinally into monkeys. At autopsy, sections of brain and spinal cord are examined microscopically for the histological lesions indicative of proliferating poliovirus. 

Recently, a potential cytosolic component of the MEP precursor pathway, xylulose kinase, has been cloned and tested for function in an Escherichia coli complementation system. " The kinase activates exogenous xylulose in the cytoplasm. DXP is the precursor for DXS, which resides in the plastid, suggesting the activated substrate must be transported into the plastid. Another xylulose kinase homologue in Arabidopsis that contains a plastid targeting sequence was not active in the E. coli system, suggesting that it may have some other function in the plastid. Perhaps plant and bacterial tissue cultures may be fed xylulose to condition accumulation of isoprenoid metabolites. 

Knutton S, Baldwin T, Williams PH, McNeish AS Actin accumulation at sites of bacterial adhesion to tissue culture cells Basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun 1989 57 1290-1298. 

Most research into in vitro foreign protein production has been undertaken using cell suspensions. However, other forms of plant tissue culture such as hairy roots and shooty teratomas have also been tested in a number of studies (Table 2.1). The characteristics of different types of plant tissue culture and their utility for large-scale foreign protein production are outlined in the following sections. 

Compared with whole plants, there has been limited development of foreign protein expression systems specifically for use in tissue culture. Some modifications of expression constructs have resulted in improved protein accumulation or have allowed simplified protein recovery. However, in general, modified expression systems have been tested only in a restricted number of cases and have not resulted in the large increases in product yield required for plant cultures to compete with other foreign protein production vehicles. Transient expression techniques, for example using viral vectors, that have been developed for use in whole plants have not yet been applied in plant tissue culture. 

The results of empirical studies carried out to test the effects of medium additives on foreign protein accumulation in plant tissue culture are summarized below. 

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