Endotoxins Detection And Measurement Biology Essay

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Cell culture known to be complex process by removal of tissue or cells from plants, animals, microbes such as bacteria and viruses, and fungi process them by growing them in specific conditions and atmospheres. In the 19th century scientist discovered the way of maintaining live cell lines taken from the animals tissue, since that days animal cell culture became world widely available as a technique for scientist [1].

Principle of tissue culture was established by Wilhelm Roux In 1885, he removed a part of the medulla oblongata dish of an embryonic chicken and preserved it in a warm saline for some days[2]. The methodology of tissue culture was established by Ross Granville Harrison, while he was published results of his research work from 1907-1910[3].

In 1950s Cell culture techniques were progressed significantly in virology research, which helped in manufacture of vaccines. Development of antibiotics helped tissue culturing to be success, as it made it easy to avoid tissue culture contaminations and improving of techniques played a key role that made cell culture widely used technique for most of the scientist.

1.1 cell culture system

There are two basic culture systems, which are used for growing cells.

Monolayer culture system; capability of the growing cells to attached to the glass surface or treated plastic flask substrate such as; T-flasks, roller bottles, multiple well plates, or culture chambers.

Suspension culture system; the cells which are grown floating (unattached) free in the medium. The usual flasks used for growing this type of cell culture are:

Magnetically rotated spinner flasks or shaken erlenmeyer flasks.

Stationary culture vessels such as bottles and T-flasks, however cells are not distributed because they are not able to attach to the substrate.

Blood stream cells are naturally live in suspension without attaching to the vessels surface and called ( anchorage-independent). Modified cell lines are able to survive in suspension cultures which allow them to grow into higher density than adherent conditions. Adherent cells need a surface, such as tissue culture plastic, which might be layered with extracellular matrix components to raise adhesion properties and supply other signal required for growth and differentiation. Most cells derived from solid tissues are adherent[4].

1.2 Types of cells culture

Cultured cells are frequently expressed based on their functional features or their morphology based on shape and appearance. Three kind of cell morphology are know:

Epithelial-like: cells that are appended to a substrate and appear compressed and many-sided in shape.

Lymphoblast-like: cells that are no appended to a substrate but in same time it settled in the suspension with the globular shape ( bloodstream cells).

Fibroblast-like: cells that are appended to a substrate and appear extended and bipolar, usually in heavy culture it is found to be forming swirls.

Culture conditions plays a key role in determining form that many cell culture are able of demonstrate multiple difference in morphologies. Cell fusion techniques was used to get hybrid cells from dissimilar parents. In 1975 scientist generated cells that have the ability to make custom modified monoclonal antibodies, which called hybridomas cells. Hybridomas cells are created by fusion two unlike although related cells[5].

1.3 Cells functional characteristics

Cultured cells can achieve their characteristics from the original origin (heart, liver, etc.) and their settle in to the culture circumstances. The characteristics of some cell can be changed or lost as a consequence of being located in artificial atmosphere. There are two markers can be used to decide if the cells are still carrying their particular functions that they carry out in vivo, its recommended to use biomedical markers and morphological or ultrastructural markers. There are 6 different known cells lines which can be described as :

Finite cell line: finally those cells discontinue dividing and demonstrate symptoms of aging.

Continuous cell lines: those cell became everlasting can be continue dividing forever.

Transformed cell lines: those cells are faster growing cells and known to grow in the suspension as they have abnormal chromosomes caused by viruses, radiation, or abuse drugs .

Diploid cell lines: those cell have ordinary amount of chromosomes.

Aneuploid cell lines: those cells have other than the ordinary amount of chromosomes.

Neoplastically transformed: those cell considered as tumor cells that have been introduced to animals [6].

1.4 Contamination and cell culture

contamination consider be a serious problem for scientists, researchers and companies to create cell-base parenteral. Such contamination problem can end an experiment to misidentified or lead to wrong outcome. Recent, studies propose 15-20% of the time researchers been a victim of contamination[7].

There are two type of cell culture contamination, biological and chemicals. Biological contamination caused by fast growing yeast, bacteria and fungi. This type of contamination changes the turbidity of the medium and have observable effects on the cell culture. On the other hand, there are other types of biological contamination which are very difficult to detect such as; mycoplasmas and viruses. Chemical contamination caused by many different agents involve metal irons, plasticizers, traces of chemical disinfectants and Endotoxins[1].

1.4.1 Endotoxins and cell cultures

There is rising facts that Endotoxins be able to produce a range of problems for researchers in cell culturing technique. Endotoxins is a mixture of lipopolysaccharide (LPS) which is a most important element of the external membrane of the majority gram negative bacteria. Bacteria excrete Endotoxins into their surrounding atmosphere in little quantity while they are actively growing, and in a big quantities after they die. LPS composed of a very extremely hydrophobic lipid group (A) covalently bound to a long complex polysaccharide tale[8] (see figure1).

1.4.2 Endotoxins detection and measurement

The initial technique for identifying Endotoxins contamination was a rabbit pyrogen investigation developed in 1940's for testing water and liquids used after injecting human. this experiment is stand on the capability of Endotoxins to cause fever. In 1970's, scientists developed in vitro assay which was very sensitive based on examination that the lysate from horseshoe crap (limulus polypbemus) amebocytes would form clot in the attendance of very low concretions of Endotoxins. Nowadays, scientists developed more advance methods to detect very low amounts of Endotoxins such as chromogenic assays[8].

1.4.3 Sources of Endotoxins in cell culture

Yadav, P. R. and Tyagi. (2005) Tissue Culture. 1st edition, Discovery Publication, Delhi

NCBI UK [online}, http://www.ncbi.nlm.nih.gov/books/bv.fcgi?db=Books&rid=mboc4.table.1516. Accessed on 30th October 2010. 

Nass, R. And Przedborski, S. (2008) Parkinson's disease : molecular and therapeutic insight from model systems. 1st edition, Elesevier publication. USA

Stacy, G. and Davis, j. (2007) medicines from animal cell culture. 1st edition, Wiley. USA

Minuth, W.W, Schumacher, K, and Strehl. (2005) Tissue engineering: essentials for daily laboratory work. 1st edition, Wiley-VCH. Germany

Ian ,R. (2010) Culture of animals cells, 6th edition, Wiley-Blacwell. USA

Cabrera, CM; Cobo, F; Nieto, A; Cortés, JL; Montes, RM; Catalina, P; Concha, A (Jun 2006). "Identity tests: determination of cell line cross-contamination". Cytotechnology 51 (2): 45-50.

Wang, X. and Qunin, P. (2010) Endotoxins: structure, function and recognition. Springer, USA