Basics of cell and tissue culture

Basics of cell and tissue culture
? Introduction:
o Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. But in practice it refers to the culturing of cells derived from animal cells.
o Cell culture was first successfully undertaken by Ross Harrison in 1907.
o Roux in 1885 for the first time maintained embryonic chick cells in a cell culture
? Major development’s in cell culture technology:
o First development was the use of antibiotics which inhibits the growth of contaminants.
o Second was the use of trypsin to remove adherent cells to subculture further from the culture vessel
o Third was the use of chemically defined culture medium.
? Why is cell culture used for?
o Areas where cell culture technology is currently playing a major role.
o Model systems for:
- Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies
- Toxicity testing
- Study the effects of new drugs
- Cancer research
- Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells
- Virology: cultivation of virus for vaccine production, also used to study there infectious cycle.
- Genetic Engineering
- Production of commercial proteins, large scale production of viruses for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B & measles.
- Gene therapy.
- Cells having a functional gene can be replaced to cells which are having non-functional gene

? Tissue culture:
o In vitro cultivation of organs, tissues & cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is collectively known as tissue culture.
o Different types of cell grown in culture include connective tissue elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue (liver, breast, skin, and kidney) and many different types of tumor cells.
? Primary culture:
o Cells when surgically or enzymatically removed from an organism and placed in suitable culture environment will attach and grow are called as primary culture.
o Primary cells have a finite life span.
o Primary culture contains a very heterogeneous population of cells.
o Sub culturing of primary cells leads to the generation of cell lines.
o Cell lines have limited life span, they passage several times before they become senescent.
o Cells such as macrophages and neurons do not divide in vitro so can be used as primary cultures.
o Lineage of cells originating from the primary culture is called a cell strain.
? Types of cells:
o On the basis of morphology (shape & appearance) or on their functional characteristics. They are divided into three.
o Epithelial like-attached to a substrate and appears flattened and polygonal in shape.
o Lymphoblast like- cells do not attach remain in suspension with a spherical shape.
o Fibroblast like- cells attached to an substrate appears elongated and bipolar.
? Culture media:
o Choice of media depends on the type of cell being cultured.
o Commonly used Medium is GMEM.
o Media is supplemented with antibiotics penicillin, streptomycin, etc.
o Prepared media is filtered and incubated at 4 C
? Why sub culturing?
o Once the available substrate surface is covered by cells (a confluent culture) growth slows & ceases.
o Cells to be kept in healthy & in growing state have to be sub-cultured or passaged.
o It’s the passage of cells when they reach to 80-90% confluency in flask/dishes/plates.
o Enzyme such as trypsin, dipase, collagenase in combination with EDTA breaks the cellular glue that attached the cells to the surface.
? Culturing of cells:
o Cells are cultured as anchorage dependent or independent.
o Cell lines derived from normal tissues are considered as anchorage-dependent grows only on a suitable substrate e.g. tissue cells
o Suspension cells are anchorage-independent e.g. blood cells.
Transformed cell lines either grows as monolayer or as suspension.
? Cell toxicity:
o Cytotoxicity causes inhibition of cell growth.
o Observed effect on the morphological alteration in the cell layer or cell shape.
o Characteristics of abnormal morphology are the giant cells, multinucleated cells, a granular bumpy appearance, vacuoles in the cytoplasm or nucleus.
o Cytotoxicity is determined by substituting materials such as medium, serum, supplements flasks etc. at time
? Freezing cells for storage:
o Remove the growth medium, wash the cells by PBS and remove the PBS by aspiration.
o Dislodge the cells by trypsin-versene.
o Dilute the cells with growth medium.
o Transfer the cell suspension to a 15 ml conical tube, centrifuge at 200g for 5 mts. at RT and remove the growth medium by aspiration.
o Resuspend the cells in 1-2ml of freezing medium.
o Transfer the cells to cryovials, incubate the cryovials at -80 C overnight.
o Next day transfer the cryovials to Liquid nitrogen.
? Cell viability:
o Cell viability is determined by staining the cells with trypan blue.
o As trypan blue dye is permeable to non-viable cells or death cells whereas it is impermeable to this dye.
o Stain the cells with trypan dye and load to haemocytometer and calculate % of viable cells.
% of viable cells= Nu. of unstained cells x 100 total nu. of cells
? Contaminants of cell culture:
o Cell culture contaminants of two types.
- Chemical-difficult to detect caused by endotoxins, plasticizers, metal ions or traces of disinfectants that are invisible.
- Biological-cause visible effects on the culture they are mycoplasma, yeast, bacteria or fungus or also from cross-contamination of cells from other cell lines.
? Effects of Biological Contaminations:
o They compete for nutrients with host cells.
o Secreted acidic or alkaline by-products ceses the growth of the host cells.
o Degraded arginine & purine inhibits the synthesis of histone and nucleic acid.
o They also produce H2O2 which is directly toxic to cells.
? Detection of contaminants:
o In general indicators of contamination are turbid culture media, change in growth rates, abnormally high pH, poor attachment, multi-nucleated cells, graining cellular appearance, vacuolization, inclusion bodies and cell lysis.
o Yeast, bacteria & fungi usually shows visible effect on the culture (changes in medium turbidity or pH).
o Mycoplasma detected by direct DNA staining with intercalating fluorescent substances.
o Mycoplasma also detected by enzyme immunoassay by specific antisera or monoclonal abs or by PCR amplification of mycoplasmal RNA.
o The best and the oldest way to eliminate contamination is to discard the infected cell lines directly.
? Safety aspect in cell culture:
o Possibly keep cultures free of antibiotics in order to be able to recognize the contamination.
o Never use the same media bottle for different cell lines. If caps are dropped or bottles touched unconditionally touched, replace them with new ones.
o Necks of glass bottles prefer heat at least for 60 secs at a temperature of 200 C.
o Switch on the laminar flow cabinet 20 mts prior to start working.
o Cell cultures which are frequently used should be sub cultered & stored as duplicate strains.