Free Cell Culture Techniques: Medicine Report Sample

Cell culture Techniques

Introduction
Cell culture involves the extraction of prokaryotic and eukaryotic cells from animal or plants and growing them in artificially controlled environment in vitro. This paper summarizes Cell culturing process including culture techniques, medium selection, detection of microbial contamination, and methods of thawing, freezing and passaging of cultured cells. The embryonic chick cells were maintained for the first time in 1885 and 1907 the first cell culture was performed by Ross Harrison. The cell culture application has been highly advantageous in biomedical research, especially for drug screening, vaccine development, cancer and genome analysis and also in the preparation of monoclonal antibody (Bihl).

Cell Culture Type

Cell cultures are categorized into two types: attached and suspension culture cells. Attached culture is suitable for all cell types. It requires frequent passaging and enzymatic (via trypsin) or mechanical stimulation for dissociation. Its growth confines to the surface and may result in limited yield. Examples of attached culture are smooth muscle cells, Cardiomyocytes, and epithelial cells. Attached culturing is used in cytology and harvesting.
Suspension culture cells include monocytes and lymphocytes. This culture is easier to passage but needs cell counting and viability determination on a daily basis to observe growth patterns. Suspension cultures do not require dissociation through enzyme. The concentration of cells in medium controls the growth. Such culture is useful in protein production (Bihl).

Cell Growth and Proliferation

Cell growth and proliferation is determined by primary culture, subculture and senescent. Primary culture demonstrates the initial growth of cells removed from an organism, under appropriate culture environment. Primary culture is a population of mixed cells with a limited life span. Subculturing or passaging is performed when the cells start confluence. Passaging develops cell lines and multiple times passing results in senescent cells. Culture phase consists of three phases; latent phase when suspended cells attach themselves to the base, exponential growth phase and senescent phase when the cells stop growing and reproducing (Bihl).
For culture passaging, the most used solution to detach the cells from the surface is a mix of trypsin, dipase, collagenase enzymes combined with EDTA. Trypsin catalyzes the peptide bond of protein and yields smaller peptides, and EDTA form coordinate bonds with a single metal ion. This reaction results in loosening of surface adherence of cells (Bihl).

Cell Culture Environment

The appropriate environment for cell culture must be aseptic at a temperature of 36-37°C. Humid atmosphere and air conditioned with 5% CO2 is also suitable. For culture medium Glutamate, glucose, growth factors or FBS (fetal bovine serum) can act as nutrition culture medium (Bihl; Chen).

Cell Culture Media

The selection of medium is a significant part that relies on the type of cells that are going to be cultured. Mostly primary media are preferred. Examples include EMEM (Eagle's Minimum Essential Medium) a mammalian cell culture media, DMEM (Dulbecco’ Modified Eagle’s medium), GMEM (Glasgow Minimum Essential Medium), RPMI1640 and HamF12. GMEM is useful in investigating the genetic factors that influence the proficiency of the cells. RPMI1640 is advantageous in culturing human normal and neoplastic leukocytes. HamF12 is beneficial in growing primary cells, like hepatocytes and prostate epithelial cells (Bihl).

Cell Contamination

Chemical, as well as biological contamination of cultures, affects the cells growth through competing with host cells for nutrition. The release of endotoxins and metal ions cause chemical contamination while yeast, mycoplasma, and bacteria are responsible for biological contamination. Its effects include aborted cell growth, blocked synthesis of nucleic acid and development of turbid media, cytotoxicity, and vacuolization and cell lysis. The factors involved in contamination may include substituting materials such as serum, medium, supplements flasks and wares (Bihl).
The best method to eliminate disease is to discard the infected cells. Cell counting is conducted to determine the cell viability and cytotoxicity using Trypan Blue. For cell culture storage, applicable techniques are incubation, freezing below 4 or -20°C, Cryostorage in liquid nitrogen (N2) freezer. Sterilization can be done with autoclaving and water bath. In autoclaving the cells go through high pressure and 121 °C temperature for 20-30 minutes. For thawing cells, water bath is given at 37°C with 70% ethanol. For Cryostorage, the cells are frozen with complete growth medium containing a cryoprotective agent (DMSO) in a liquid nitrogen storage container (Bihl; Chen).

Work cited

Http://Www.Youtube.Com/Watch?V=_Fjz-Mhv22w&Feature=Player_Detailpage. 2015. DVD.
Bihl., Ji, C. Cell Culture Training. Pharmacology and Toxicology
Chen, Ji. Cell Culture Techniques. Pharmacology and Toxicology