Biological advancements have contributed to the improvement of society in various forms. Biomedical research which forms the foundation for spectrum of inventions/discoveries, has its origin in techniques like cell culture.
Cell culture involves creating an artificial environment of growing that mimics the natural one in all the characteristical features in a laboratory. So, growing the cells of animals, plants or human beings, yeast and bacteria in a lab setting, constitutes the cell culture. It is mainly employed to examine new drugs and detect infectious agents (Dictionary of Cancer Terms n.d.).
The methodology of cell culture is a bit complicated. In order to achieve the robust growth of cells, specific conditions need to be maintained.
The key process involved in handling living eukaryotic cells initially is mandatory awareness of materials and methods. It involves the use of 370 CO2 incubator, phosphate buffer saline, plastic ware, glass ware, petri dishes, trypsin/EDTA, vialsfor cryopreservation media like DMEM, Hemocytometer is provided with cover slip, DMSO and FBS for cell freezing etc(Protocol: Cell Culture 2012). Initially, cell culture begins with primary culture.
It constitutes a stage where cells are picked from tissue and multiplied in the presence of suitable atmosphere till they become full grown on the platform known as substrate, resulting in confluence (Introduction to Cell Culture 2012)
Here, cells need re-culture known as passaging or subculture achieved by the transporting them to a novel vessel with medium of growth which is fresh to enable more space for the prolonged growth. Primary culture is the important step and prerequisite for any kind of cell culture technique. A failure in proper maintenance of Primary culture could lead to total failure in the overall culture process (Introduction to Cell Culture 2012).
Maintenance of cell culture is firmly linked with safety interlinked with cross contamination issues. This is because, cell culture unit contains many particular dangerous agents linked with hand contact and modifying chemicals, solutions of corrosive nature, tissues and cells of plant, animal or human. The potential dangers are punctures occurring accidentally with needles, spills on the skin, mouth contact through pipetting or ingestion and inhaling infectious agents like sprays, exposures etc.
To overcome these problems, agencies like National Institute of Health (NIH) and Centers for Disease Control (CDC) have provided biosafety recommendations United States. It is mainly focused on four types of biosafety levels (BSL). BSL-1 is the primary option of protection to many laboratories involved in basic and clinical research.
BSL-2 is suitable for medium risk contributors that lead to severe disease in human, that vary deepening on complexity, by contact with percutaneous membranes. BSL-3 is suitable for agents of indigenous nature which have a capacity for transmission like aerosols and that lead to detrimental infections. BSL-4 is suitable for indigenous agents that carry a high risk or fatal by aerosols of infectious nature and no therapy exists (Introduction to Cell Culture 2012).
But laboratories of only high containment possess these agents. Hence, there are specific guidelines that not only ensure safety but also may be helpful to avoid all possible chances of contamination from both prokaryotic and eukaryotic sources.
These are wearing equipment of specific personnel type and replacing contaminated gloves with new ones, disposal of all wastes suspected of contamination, washing hands after contact with dangerous materials prior to the laboratory closing hours, avoiding smoking, drinking, food consumption and storage in the lab, close adherence to the institutional rules and regulations with regard to handling glassware, pipettes, scalpels and needles, lessening the development of aerosols and leakages, removing surface contamination with suitable disinfectant near the work place before and after the experiments, infectious material spills, regular cleaning of laboratory devices as well as instant reporting of the laboratory incidents that occur due to contact with infectious agents to a laboratory authority (Introduction to Cell Culture 2012).
Next, for preventing contamination from sources like sneezing, skin shedding, and spores, dust which serves as the vial constituents of aerosols and airborne particles, employing a hood of cell culture, is essential. Setting up cell culture hood relies on the location where there are a restricted outlets like windows, doors and no personnel movements.
The work place must have only necessary reagents, lab ware and protocols. One must disinfect work place, clean instrument regularly before and after use with 70% ethanol, use ultraviolet light for air and surface sterilization of hood, while using at frequent intervals, as well as maintain the hood in running conditions through the available time and switching it off when there is no work.
In the cell culture, the cell lines are the most important ones to consider (Introduction to Cell Culture 2012).
The cell lines are defined as the products of primary culture obtained by subculture.
Primary culture of the given cell lines possesses a short duration of life known as finite cell lines. When these cell lines are subjected to passaging, the resultant cells obtain a robust phenotypic and genotypic stability marked with lustrous growth potential.
As such, cell line growth is achieved in two ways. One is monolayer or adherent culture
which is achieved on substrates of artificial nature and another one is suspension culture, achieved through medium of free floating nature (Introduction to Cell Culture 2012)
Cell line contamination needs to be understood from the point of view of biological contamination in general. These may be grouped under Bacterial, Mold & Virus, Mycoplasma and yeast types of contamination. Bacterial contamination is recognized by visual observation of culture during the very initial days of infection.
The cultures appear turbid with low pH of the medium and tiny appearance of bacteria. Molds are a special category of eukaryotic microorganisms and infection; in the early stages they contribute to turbidity with visual appearance of spore clumps and thread like thin filaments under microscope.
Viruses are microscopic organisms with high multiplication potential. Infected cell lines can be identified by polymerase chain reaction (PCR), immunoassays, immunostaining and electron microscopy (Introduction to Cell Culture 2012). Mycoplasma are bacteria without cell wall. Their infection of cell lines contribute to altered metabolism of cells, low multiplication potential, suspension culture agglutination, etc.
Detection is possible though PCR, immunassays, and the most important, Hoechst 33258 – fluorescent staining. Yeasts are microorganisms of eukaryotic type and their infection contributes to turbidity, pH variation, with rounded appearance in the culture which can be microscopically observed (Introduction to Cell Culture 2012).
Very often in the cell culture unrelated cell growth could lead to contamination and cell line growth more than the expected limit. This is nothing but cross-contamination which may appear as of interspecies and intraspecies among human cell lines. Possible detection strategies may include cytogenetic analysis and DNA fingerprinting.
Earlier, by employing this approach, the investigators were able to detect nearly cross contaminated cell lines brought from hematopoietic cell lines of different source and those belonging to the original researcher.
This situation of cell line cross contamination could be attributed to constant necessity in the protocol for cell culture viability and identification. Maintenance of multiple cell lines is the contributing factor sometimes and it can be avoided by regular monitoring for specificity and identity, markers, karyotyping and immunoprofile (Drexler, Dirks, & MacLeod 1999).
To better overcome the problem of contamination, U.S. National Institutes of Health has commissioned the utility of authentication of cell line investigations. Here, a private firm Promega has come forward with PCR system in a multiplex format known as StemEliteâ„¢ ID System (Oostdikv et al. 2009).
This approach better recognizes the contamination in variety of cells like those of mouse and human by making comparison between a standard genotype and genotype developed by StemEliteâ„¢ ID System (Oostdikv et al. 2009). Even for the plant cell culture contamination detection, the strategy recommended was maintenance of cultures aseptically with regard to Hazard Analysis Critical Control Point (HACCP) by meristem explants and good laboratory practice (GLP) guidelines (Cassells & Prestwich 2009).
Pure cell lines are important for a variety of applications like Blood Factor VIII, Erythropoietin (EPO), hybridoma technology to produce monoclonal antibodies (Applications of Animal cell culture 2009). Large scale culture of cells is done in industries in order to scale up for the development of cell bank systems.
For this purpose, huge bioreactors like compact-loop bioreactor will be used that optimizes the cells in the medium by providing biological, physical and chemical factors. For cultures operated in batches, spinner flasks and Micro Carrier Beads are used for scale up (Applications of Animal cell culture 2009). This indicates that pure cell lines are very important for scale up processes in industries.
It is reasonable to mention that very often impure cell line growth may contribute to adverse reactions in the bioreactors. The impure cell lines may release unnecessary bye products that may become toxic and affect the down stream process. More probably, it may interfere with the routine biological and chemical properties offered by a bioreactor, as mentioned earlier. This may not only alter the yield of the culture but also affect the equipment with great chances of interproduct contamination.
Thus cell culture appears a vital research strategy for a variety of biomedical applications.
References
Applications of Animal cell culture, 2009. Web.
Cassells Alan C & Prestwich Barbara Doyle, 2009, ‘Contamination Detection and Elimination in Plant Cell Culture‘. Web.
Dictionary of Cancer Terms: Cell culture. Web.
Drexler, H. G., Dirks, W. G., & MacLeod, R. A. 1999, ‘False human hematopoietic cell lines: cross-contaminations and misinterpretations’, Leukemia, vol. 13 no.10, pp.1601-7.
Introduction to Cell Culture, 2012. Web.
Oostdikv K., Petterson A., Schagat T. & Storts D. 2009, Stem Cell Line Authentication and Contamination Detection. Web.