CYTOTOXICITY TESTS

Question 1

What’s a cytotoxicity test?

Answer 1

a mehod, used to evaluate the harmful effects of substance in cells (cell viability, metabolic activity, cell death..) to assess the toxicity of drugs..
=> Test different concentrations at the same time

Question 2

What’s cell culture in this context?

Answer 2

As general information, cell cultures are growing cells in a controlled environment outside their natural habitat “lab setting”, those are exposed to toxins ( evaluate their effects on cellular processes.
=> Cell cultures help researchers understand if a substance is harmful to cells (cytotoxicity) or how it affects them (mechanism of action: param specific for the toxicity of Xenob ( early clues if sub is toxic or not)

Question 3

Difference between cell culture and screening ?

Answer 3

Cell cultures are the cells grown in a lab for studying how substances affect them. Screening is the process of testing substances to see if they’re harmful. Cell cultures can be part of screening to understand how substances affect cells.
- (screening can be hold in short time and with limited investments of work and money and its simple and reproducible indicators of damage)

Question 4

Makers of general toxicity (aspecific)

Answer 4

• Modifications of cell membrane
• Inhibition of cell proliferation
• Metabolic and morphological alterations

Question 5

To plan a cytotoxicity test, follow these steps.

Answer 5

• Select Cell Line: Choose a relevant cell line based on the study’s objectives and the substance being tested.
• Determine Concentrations: Establish a range of concentrations of the test substance to be used in the assay.
• Choose Assay Method: Select an appropriate cytotoxicity assay method (e.g., MTT, LDH release, Trypan Blue exclusion) based on the experimental requirements and resources available.
• Include Controls: Incorporate appropriate controls, including solvent-only controls and positive/negative controls, to validate the assay and interpret results accurately.
• Experimental Design: Decide on the experimental design, including the number of replicates and experimental conditions.
  -Optimize Conditions: Optimize experimental conditions such as cell seeding density, incubation time, and assay duration to ensure reliable and reproducible results.
• Data Analysis Plan: Establish a plan for data analysis, including statistical methods to assess cytotoxicity and interpret the results effectively.
  -Consider Ethical Guidelines: Ensure compliance with ethical guidelines and regulations governing the use of cell lines and experimental procedures.

Question 6

HOW TO PLAN A CYTOTOXICITY TEST
(test control test: 7PT)

Answer 6

• Incubation conditions for adherent cells:
• at least 4 [C] (op 7-8)
• AL 4 wells/dose (op6)
• AL 3 biological replicates ( =/ passg)
• =/ incubation times
• 5% CO2
• Stock solution of Xenob & subsequent serial dilutions (filter the stock if possible)
• Choose the cytotox test ( better if more than 1)

Question 7

TYPES OF CYTOTOXICITY TESTS

Answer 7

1- Evaluate Permeability of cell membrane
2- Evaluate Cell proliferation inhibition
3- Metabolic Assays
4- Other Assays (Lipid Peroxidation)

Question 8

TESTS That evaluate the PERMEABILITY OF CELL MEMBRANE

Answer 8

1- Dye Exclusion test
2- Dye Accumulation tests
3- Release of cellular constituents in the culture medium: release of enzymes
4- Detachment of cells from the Monolayer

Question 9

What’s DYE EXCLUSION TEST ?

Answer 9

=> MAIN: distinguish btw alive and dead cells
* Principle: Measure of cell permeability; a damaged cell membrane allows to the dye that usually is excluded from the cytoplasm to enter the cell