Biology Essay 怎么写:细胞对不同浓度生长血清的反应

Biology Essay 怎么写:细胞对不同浓度生长血清的反应

 Biology Essay 怎么写:细胞对不同浓度生长血清的反应

体外培养的哺乳动物细胞培养的哺乳动物病毒的研究是必要的,他们的复制和致病过程中,以及在细胞遗传学、生化和分子研究和diagnostics1。一般来说,真核细胞比原核细胞更难生长,因为他们复杂的媒体要求和对污染的敏感性。2个哺乳动物细胞也必须在适当的温度和气体混合物,二氧化碳,对大多数哺乳动物细胞系。

通常,原代细胞培养一定数量的分裂了几次以后,有一个有限的寿命,被称为海弗利克极限,细胞衰老导致端粒缩短到某一临界阈值,从而阻止细胞进一步分裂。建立细胞系,另一方面,可以无限增殖在正确的培养条件下,无论是作为一个自发突变的结果,或通过故意修改。这样的细胞系可能来自癌细胞,其中的端粒酶的活性,防止连续的端粒缩短的酶,被改变。从原来的组织细胞可以生长,经多次传代后产生一个相对稳定的细胞系。反复传代过程中,或子细胞培养使个体成长为长时间在培养细胞的生长,让他们针对具体情况进行分析。1

细胞传代通过机械手段从用胰酶消化法原代培养容器的表面或去除细胞膜和细胞悬液再细分为新文化。次生文化监测生长条件的变化,可能会进一步传代。传代细胞之间的时间取决于增长率与细胞的变化。用生长介质含有补充因素,经常遇到来自动物的血液,如胎牛血清。选择适合于细胞类型的组织培养基和用于治疗的组织培养基是至关重要的。媒体可在商业上以液体的形式或粉状的形式,或替代地,特定的媒体可以制备。污染是哺乳动物细胞培养的一个主要问题,几乎完全发生在处理文化、试剂和材料的过程中,因此必须保持良好的无菌技术。所有与文化接触的材料必须是无菌的,必须注意保持工作环境的清洁。选择的媒体可能会补充抗生素和/或杀菌剂,以防止污染物的生长,2,1

本实验的目的是执行协议的传代培养哺乳动物两种细胞系HeLa细胞和幼仓鼠肾细胞(BHK),监督他们通过观察他们的外观和采集样本以评估细胞的生长反应不同血清浓度培养基。这个实验的基础是,从每个细胞释放的蛋白质的量时,裂解可以被用来计算在每个文化中的细胞的数量,从而为细胞的时间加倍。

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

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