L3 Toxicity testing in cultured cell systems Flashcards
Discuss the principles of cell culture, the main types of cultures cells, and the benefits and drawbacks of using this approach in toxicology. Understand the basic principles of cell culture. Explain how cultured cells are produced and maintained; critically evaluate their use in toxicity studies.
What are the three main types of cell culture?
Primary cultures, permanent cell lines, established cell lines
What are primary cell cultures?
Cell cultures where cells have been obtained directly from an animal.
What are 2 advantages and 2 disadvantages to primary cell cultures?
Heterogeneous and genetically more similar to tissue of origin. Reaggregate cultures can be generated: various cell types in 3D spheroidal co-cultures.
Can be difficult to reproduce and often short-lived.
May require animals at a specific stage of development (but can be produced from adult humans)
What are problems with diploid permanent cell lines?
Genetic drift due to minor DNA changes during each division. ~20 division limit on cells.
How do established cell lines overcome problems seen in diploid, permanent cell lines?
They are immortalised and have limitless replicative potential.
This can be done by viruses, mutagens spontaneously or by transfections. They are tumour like.
How is a clonal cell line obtained?
From the mitotic division of a single cell.
Give an example of each of the permanent cell line types
Diploid - Human foetal fibroblasts (MRC-5)
Established - Rat pheochromocytoma 12 (PC-12-Adh)
Clonal - Rat AT2 prostate carcinoma
What are the three emerging stem cell culture models?
Embryonic stem cells.
Adult stem cells.
Induced pluripotent stem cells (iPSCs)
How are embryonic stem cells cultured?
Grown on a layer of fibroblasts, which provide ECM and GF’s to support their growth.
How are adult stem cells stained to confirm their stemness?
Stained using labelled Ab’s to Nestin. Filament protein in cytoskeleton found in stem cells, used to confirm stemness.
What are iPSCs? How do they address ethical concerns?
induced pluripotent stem cells. Derived from human skin cells and reprogrammed to form new progenitor pluripotent stem cells. Confirmed by Nestin staining.
Address ethical concerns of having to using embryonic stem cells.
What is passage? How does it differ between adherent cell cultures and free growing cells?
Passage is regular subculturing and thereby feeding of cells in culture. It is the passage of one cell culture to a new culture/medium/replacemtn of ‘spent’ medium with new.
Adherent cell cultures requires suspension before dilution, in cells that grow free in media may just be dilution of media.
How can pH and humidity be maintained in cell culture (2 ways)
Use of HEPES buffer, otherwise CO2 in incubator.
What are advantages of cell culture?
Established cell lines are relatively quick and inexpensive to maintain and manipulate.
Direct cellular effects of toxin can be studied, and cell types can be studied in controlled environment.
What are the disadvantages of cell culture and how can they be addressed?
The cost of primary cultures, stem cells and their culture media can be prohibitive.
Lack of systemic effects (whole organism). Can be addressed by adding GFs etc.
Lack of cell:cell
interaction and the complexity of whole tissue/organ.
Can be addressed by developing co-cultures and establishing 3D.
