Techniques: Mammalian cell culture and maintenance.......

Cell culture

The cells can grown in adherent monolayer (use culture vessel wall as anchor) or in suspension

Adherent monolayer cells stop proliferating as they become confluent. Tissue-derived cells are anchorage-dependent.

Hematopoietic cells i.e those derived from blood, spleen, or bone marrow, and transformed cell lines are suspension cells.

Further cells can be clustered into primary cells, finite cultures, and continuous cell lines

Their growth behavior and nutritional requirements vary. So, optimization and subculturing is necessary

Enzymatic, chemical, or mechanical methods are used to obtain cells.

After some cycles of cell division, the primary cells undergo senescence. Primary cells are considered physiologically similar to in vivo cells

Finite cell cultures are formed after the first subculturing or passaging) of a primary cell culture. Proliferative potential of some human finite cell cultures can be extended by introduction of viral transforming genes (e.g., the SV40 transforming-antigen genes)

Continuous cell line is capable of unlimited proliferative potential. This alteration is commonly known as in vitro transformation or immortalization and frequently correlates with tumorigenicity. As these cells have acquired genetic alterations, comparing them to in vivo conditions is error-prone.

All cell cultures are considered a biohazard because of their potential to harbor an infectious agents, mostly  a virus).

Primary cell cultures are likly to have undetected viruses, the latent viruses

Work requires the use of laminar flow hood using aseptic technique

The creation of aerosols should be avoided, as it causes inhalation hazard, and cross-contamination between cultures

After the work is complete, all waste media and equipment (i.e., used flasks, pipets, etc.) should be disinfected by autoclaving or immersion in a suitable disinfectant

Before starting work, disinfect the work surface of the hood by wiping with 70% ethanol

presence of microorganisms can inhibit cell growth, kill cells, and lead to inconsistent results

cultures can be infected through poor handling, from contaminated media, reagents, and equipment, microorganisms present in incubators, refrigerators, and laminar flow hoods, as well as on the skin of the worker

Bacteria, yeasts, fungi, molds, mycoplasmas, and other cell cultures are common contaminants

The presence of an infectious agent can be detected by turbidity and a sharp change in the pH of the medium

mycoplasmal infected cell culture can be discarded by autoclaving or incineration

antibiotics as quinolone, ciprofolxacin, enrofloxacin, tiamulin and minocycline can kill mycoplasma

Only work with one cell line at a time in the hood, to avoid cross contamination

Common basal media include Eagle minimal essential medium (MEM), Dulbecco’s modified Eagle medium (DMEM), RPMI 1640, and Ham F10.

(https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/classical-media/dmem.html)

These contain a mixture of amino acids, glucose, salts, vitamins, and other nutrients, and are available either as a powder or as a liquid from various commercial suppliers.

Basal media are usually supplemented just before use with serum, L glutamine, and antibiotics and/or fungicides (Penicillin,  Kanamycin,  Gentamycin, nystatin,  Amphotericin B)to make the growth medium

Serum is a undefined material that contains growth and attachment factors, and may show considerable variation in the ability to support growth of particular cells.

Fetal calf serum (FCS) is the most frequently used serum, but for some applications, less expensive sera such as horse or calf serum can be used

L-glutamine is an unstable amino acid that converts to a form that cannot be used by cells, and should be added to medium just before use.
DMEM contains a high amount of amino acids and vitamins, as well as supplements.

Primary cells require additional supplements as collagen and fibronectin, hormones such as estrogen, and growth factors such as epidermal growth factor and nerve growth factor to attach to the cell culture vessel and proliferate.

Cell cultures should be incubated in  a water-jacketed incubator

Most cell lines grow at 37°C and 5% CO2 with saturating humidity, but some cell types require incubation at lower temperatures and/or lower CO2 concentrations.

(http://www.laboratory-journal.com/products/laboratory-apparatus/cell-culture-new-brunswick-s41i-co2-incubator-shaker)

Maintain a two-tiered frozen cell bank: a master cell bank and a working cell bank

Low passage number is maintained, which avoids genetic variation within the cell culture. Usage of cells with a low passage number (<10 splitting cycles) is recommended. Avoid senescence, transformation, maintain consistency in transfection

For cell thawing, heat a water bath to 37°C, and warm the growth medium into which the cells will be plated. Add prewarmed growth medium to a cell culture vessel.

(http://technologyinscience.blogspot.com/2008/07/animal-cell-culture-introduction-animal.html#.Wgas0GhSzIU)

Remove a vial of frozen cells from liquid nitrogen, and place in the water bath until thawed.

(https://en.wikipedia.org/wiki/Cryoconservation_of_animal_genetic_resources)

Wear protective goggles and gloves when thawing vials that have been stored in liquid nitrogen. Vials may explode when removed from liquid nitrogen.

Pipet the thawed cell suspension into the cell culture vessel containing prewarmed growth medium. Swirl the vessel gently to mix the cells with the medium. 

Incubate cells overnight under their usual growth conditions.

The next day, replace the growth medium.

Trypsinization:  using the proteolytic enzyme trypsin to detach adherent cells from the surface of a cell culture vessel

Wash cells with PBS (phosphate-buffered saline) or HBSS (Hanks balanced salt solution)

Add enough warmed 1x trypsin–EDTA solution to cover the monolayer, and rock the flask/dish 4–5 times to coat the monolayer.

Place the flask/dish in a CO2 incubator at 37°C for 1–2 min.

Remove flask/dish from incubator and tap the side of the flask/dish to assist detachment.

Once dislodged, resuspend the cells in growth medium containing serum, to inactivate trypsin activity.

Harvest the cells, either by trypsinization (adherent cell cultures) or by centrifugation at 200 x g for 5 min (suspension cell cultures)