Cytotoxicity testing extracts assay

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 methods used were acrosin proteolytic activity (APA) assay (with gelatin) and acrosin activity assay with N-a-benzoyl-DL-arginine-p-nitroanihde (BAPNA)-Triton X 100, and BAPNA assay for trypsin activity [9-11]. The antioxidant activity was tested spectrometrical with ABTS+ [12]. Cytotoxicity of extracts was determined by MTT Cell Proliferation Assay [13]. 

The extract dilution type of cell culture assay requires a solvent extraction of the biomaterial under consideration and testing of this extract, most commonly at various dilutions, for evidence of cytotoxicity and cellular interaction. This type of cell culture assay finds its most common use in providing information for regulatory compliance. As identified in the preceding Materials for Medical Devices section and in Table 1, low-molecular-weight extractables are of concern regarding biocompatibility. The extraction assay, carried out with a series of solvents that are hydrophilic and hydrophobic, permits examination of the potential cytotoxicity of extracts and the identification of materials within a biomaterial that may be cytotoxic. These types of assays ultimately permit identification and characterization of cytotoxic materials within biomaterials or the lack of cytotoxicity, as well as providing correlation with in vivo assays such as sensitization, irritation, intracutaneous (intradermal) reactivity, and other tests where the in vivo injection of extracts is required. 

The cytotoxicity tests were performed with the use of the direct contact method on the extracts obtained by an 8-d incubation of the polymer PSU and PSU/Ag composite samples, placed at the bottom of the well of a 24-well culture plate, in 2 ml of culture mediiun. The incubation was conducted at 37°C in air atmosphere with a 5% content of C02and 100% of relative air humidity. Next, 0.2 ml of ihe obtained extract and its fourfold dilution in a proper culture medium was dosed for the cultures of human osteoblasts (HTB-85 cell line, ATCC, USA) and human fibroblasts (CRL-7422 cell line, ATCC, USA) adhered to the bottom of the well of a 96-well culture plate. In the case of the control test, the extract of the examined samples was replaced by the proper volume of culture medium. The plates were incubated at 37°C in air atmosphere with a 5% content of CO and 100% of relative air humidity. The incubation time for two parallelly conducted experiments was 24 h and 48 h. The cytotoxicity of PSU and the PSU/Ag composites was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium (MTT) assay [9, 10] and by lactate dehydrogenase (LDH) activity measured with the use of a commercial cytotoxicity assay kit (Roche Diagnostics GmbH, Mannheim, Germany), [11]. Each of the indications was repeated three times. 

In vitro cytotoxicity assays using isolated cells have been applied intermittently to cyanobacterial toxicity testing over several years." Cells investigated for suitability in cyanobacterial toxin assays include primary liver cells (hepatocytes) isolated from rodents and fish, established permanent mammalian cell lines, including hepatocytes, fibroblasts and cancerous cells, and erythrocytes. Earlier work suggested that extracts from toxic cyanobacteria disrupted cells of established lines and erythrocytes," but studies with purified microcystins revealed no alterations in structure or ion transport in fibroblasts or erythrocytes,... 

Fig. 8.8 Macrophage cytotoxic assays of plant extracts. Supernatant samples were tested from Trgeneration of chloroplast transgenic line pLD-JWl (proteins were extracted in buffer containing no detergent and MTTwas added after 5 hours). pLD-JWl (extract stored for 2 days) pLD-JWl (extract stored for 7 days) -> PA 5 pg ml-1 —x— Control wild type (extract stored for 2 days) ...

Additional assays included a selectivity evaluation and cytotoxicity assay where both good selectivity and low cytotoxicity were documented. The extract was also tested on unrelated viruses and other influenza viruses. Extract A4 was the only one to maintain the activity through secondary screening, and with that as a target, purification of the active extract commenced. 

Aqueous extracts of cat s claw were tested for cytotoxicity in four in vitro bioassays using Chinese hamster ovary cells and bacterial cells (Santa Maria et al., 1997). Concentrations of 10, 20, 30, 40, 50, 75, and 100 mg/mL were used. The neutral protein assay (measures inhibition of cell growth), the total protein content assay, the tetrazolium assay (measures mitochondrial succinic dehydrogenase activity), and the microtox assay (measures inhibition of light output from a luminescent bacterium) showed no evidence of cytotoxicity. 

Although the CHCls-soluble extracts were not found to be significantly cytotoxic (ED50 >20 M,g/ml), the lupane-type triterpenoid lactones were tested in a panel of human cancer cell lines. In this case it was reported, that compounds 128 and 135 were modestly cytotoxic, while 129 and 136 were not active in the panel (Table 5). On being evaluated in the National Cancer Institute (NCI) 60-cell line human tumor panel, as well as in a newly developed hollow fiber assay, ochraceolide A has been selected for in vivo evaluation at the NCI in several murine xenograft systems. 

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