Cytotoxicity là gì

Cytotoxithành phố testing can be useful as a screening tool for pharmaceuticals before more extensive sầu toxicological testing is performed. Additionally, cytotoxiđô thị testing can be used for unique control purposes for lot release testing of raw materials or of manufactured drug products.

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Cytotoxiđô thị tests are designed lớn determine the toxicity khổng lồ cells of compounds either qualitatively or quantitatively. Qualitative sầu cytotoxicity tests offered by Pacific BioLabs are the Direct Liên hệ Test, the Indirect Contact Agar Diffusion Test, & the MEM Elution Test. In addition, Pacific BioLabs offers a very sensitive sầu quantitative cytotoxicity kiểm tra, the MTT Assay.

Standards followed

USP ISO 10993-5

Available Cytotoxicity Tests

Direct Contact

The direct liên hệ method uses L-929 mouse fibroblasts which adhere to the bottom of a petri dish or plate. The cells are grown to subconfluency (approximately 80% confluent) & the thử nghiệm article is placed directly on the cells. For liquids và extracts, a piece of sterile filter paper is saturated with the demo sample và the filter paper is placed directly on the cells. The cells & demo article are incubated together & after 24 hours the samples are examined in an inverted phase contrast microscope. The cells are given a grade from 0 to 4 corresponding lớn no reactivity lớn severe reactivity.

This chạy thử is not recommended for very low or very high mật độ trùng lặp từ khóa material. Very low density material will float in the cell growth media và very high mật độ trùng lặp từ khóa material has the potential to lớn damage the cells when the sample is placed on top of them. For low và high density material the Agarose Overlay (Agarose Diffusion) method is preferred.

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Agarose Overlay (Agar Diffusion)

Agarose overlay (also referred to as agar diffusion) is similar khổng lồ the direct tương tác method described above except that it uses agar. The agar is poured over the cells và left lớn solidify. The demo sample is then placed on the agar. The agar creates a “cushion” protecting the cells from materials that are dense and will also prohibit low density materials from floating in the growth media. The cells, agar và demo sample are incubated for more than 24 hours & then examined under a microscope to determine the severity of toxiđô thị khổng lồ the cells based on a 0-4 grading scheme.

MEM Elution

Unlượt thích the agarose diffusion và direct contact methods, the MEM Elution procedure involves applying extractables and leachables from the chạy thử article to lớn the cells. The kiểm tra article is incubated with a liquid termed the extraction vehicle. Following extraction, the test article extract is added to lớn L-929 mouse fibroblasts. The cytotoxic effects of the extract are determined after a 48 hour incubation period. Scoring is based on conditions of the cells using a 0 khổng lồ 4 grading system which is correlated the observed cytotoxic affect.

MTT Assay

The MTT assay is a quantitative cytotoxicity assay that uses a dye called 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (abbreviated to lớn MTT). MTT is a yellow water soluble chemical that is cleaved by mitochondrial succinate dehydrogenase lớn form formazan which is violet color. This reaction will only occur in health living cells. Mitochondrial succinate dehydrogenase will not cleave MTT if the cell is dead.

The MTT assay is performed using an extract of the thử nghiệm article. The extract is added khổng lồ the L-929 mouse fibroblasts at four different concentrations, undiluted, 1:2, 1:4, và 1:10. After 24 hours, MTT is added to the cells and the presence of violet formazan can be analyzed using a plate reader. The samples are compared khổng lồ controls to lớn determine the percentage of viable cells.