Biology Essay 怎么写:细胞对不同浓度生长血清的反应
Culture of mammalian cells in vitro is essential for the study of mammalian viruses and the course of their replication and virulence, as well as in cytogenetic, biochemical, and molecular research and for diagnostics1. Generally, Eukaryotic cells are more difficult to grow than prokaryotic cells due their complex media requirements and susceptibility to contamination.2 Mammalian cells must also be incubated at an appropriate temperature and gas mixture, of CO2, for most mammalian cell lines.
Usually, primary cell cultures have a limited lifespan and after a certain number of rounds of mitosis, known as the Hayflick Limit, cells senesce due to the shortening of telomeres to a critical threshold, which prevents further division of cells. Established cell lines, on the other hand, can proliferate indefinitely in the correct culture conditions, either as a result of spontaneous mutation or through deliberate modification. Such cell lines may be derived from carcinoma cells, where the activity of telomerase, the enzyme which prevents successive shortening of telomeres, is altered. Cells from the original tissue can be grown and passaged repeatedly to give rise to a relatively stable cell line. The process of passaging, or sub-culturing cells enables an individual grow cells in culture for extended periods of time, allowing their growth response to specific conditions to be assayed.1
Cells are passaged by removing cell monolayers from the surface of the primary culture vessel by trypsinization or by mechanical means and the cell suspension is then subdivided into fresh cultures. Secondary cultures are monitored for growth and changes in conditions and may be further passaged. The time between passaging cells depends on the growth rate and varies with the cell line. The growth media used contains supplemented factors, often en derived from animal blood, such as foetal calf serum. It is vital to select the tissue culture medium suitable for the type of cells to be cultured and type of treatment they are used for. Media are available commercially in liquid form or in a powdered form, or alternatively, specific media may be prepared. Contamination is a major issue in the culture of mammalian cells, and contamination almost exclusively occurs from handling of cultures, reagents and materials, thus good aseptic technique must be maintained. All materials that come into contact with cultures must be sterile and care must be taken to keep the work environment equally clean. The media of choice may then be supplemented with antibiotic and/or fungicide to prevent growth of contaminants.1, 2
The objectives of this experiment were to carry out the protocols for passaging mammalian two cell lines: HeLa and baby hamster kidney cells (BHK), monitor them by observing their appearance and collecting samples in order to assess the cells’ growth response to different serum concentrations in the culture medium. The basis of this experiment is the fact that the amount of protein released from each cell upon lysis can be used to calculate the number of cells in each culture, and thus the time taken for the cells to double.
Materials and Methods
The Experiment was carried out according to the practical procedure described in the laboratory manual, pages 11-17.Over the course of five days, two cell lines, BHK and HeLa, were each cultured in two specific media containing different concentrations of Foetal Calf Serum. Each culture was passaged four times to give a total of sixteen dishes. One culture of each cell type and serum concentration was analysed over the following four days and samples collected for analysis of protein concentration at a later date. The concentration of each culture was examined.
For step 4(c) on page 11/12For the determination of cell concentration in the original cell suspensions, the cells were diluted 1/5 in versene i.e. to give a suitable cell concentration for counting, of approximately 6 x 105 cells/ml. For step 2. On page 16, the standard protein was diluted to 0.1-0.9 mg/ml protein with increments of 0.1 mg/ml to give a dilution series in order to obtain a standard curve.
Cells were washed in media +2% serum, as per step 8; p. 13 of the laboratory manual, before re-plating the cells in appropriate medium in order to remove the concentrated (approximately 16.67%) FCS used in step 3(d) on page 11 of the laboratory manual. This concentration of serum is far higher than the optimal concentration and is only required in the removal of primary cultures from the plate. Omitting this step may result in rapid growth that may be impossible to monitor and early death of cells due to overcrowding and build-up of toxic metabolic products.
The Standard Cell pellets were washed in Phosphate Buffered Saline as per page 13, step 8 in the lab manual before freezing to remove any remaining media and to maintain the ionic concentration and thus the osmolarity of the cell pellets to prevent cell death after thawing.
Biology Essay 怎么写:细胞对不同浓度生长血清的反应