Cell and Tissue Culture

Transcription

1 Cell and Tissue Culture S. Swaminathan Director Centre for Nanotechnology & Advanced Biomaterials School of Chemical & Biotechnology SASTRA University Thanjavur Tamil Nadu Joint Initiative of IITs and IISc Funded by MHRD Page 1 of 8

2 Table of Contents 1. CELL AND TISSUE CULTURE Types of primary culture Cell lines Immortalized cell lines Variation in cell lines Media Dissolved gases Advantages and disadvantages of serum...8 Joint Initiative of IITs and IISc Funded by MHRD Page 2 of 8

3 1. Cell and Tissue Culture The success of tissue engineering mainly depends upon the ability of the extracellular matrix analogue to control the cell fate processes such as adhesion, proliferation, differentiation and apoptosis. Hence it is mandatory to evaluate the tissue engineered medical products in terms of cell viability, cell proliferation, adhesion, migration, and also differentiation. Therefore, isolation of cells from different sources and sub culturing are the two vital components of tissue engineered medical products. Tissue culture involves culturing of a small piece of tissue either from the plant or from the animal under controlled sterile environment. Tissue culture has several advantages compared to animal studies. They are: 1. Physicochemical factors like ph, oxygen concentration, carbon di oxide concentration can be easily controlled 2. Sample is homogeneous and the distinctive features are well described 3. Cost effective, when compared to animal studies However the main limitation is the small sample size, the concentration of cellular products secreted will be low resulting in lesser sensitivity in its detection. Moreover, the function of primary cell in culture is not same as in vivo environment. 1.1 Types of Tissue culture Cells acquired from an animal / human tissue sample are denoted as primary culture. However when cells divide, it is necessary to split the cells and subculture them by a process known as passing. After passages, cells continue to grow for generations. However, the number of generations depends on the Joint Initiative of IITs and IISc Funded by MHRD Page 3 of 8

4 cell type. For example, fibroblasts grow well in culture but cardiac myocytes die within few hours of the culture. In contrast, some cell lines grow over an extended time period and are termed immortal. Examples are the tumour cells and embryonic cells in which there is difference in gene expression compared to the primary cells. Immortalized cell lines are easily conserved for their reproducible results compared to primary cells. It is also possible to convert the primary cells into immortalized cells by transforming genetic material. 1.2 Types of primary culture Tissue culture is a term used to describe both dispersed cell culture and three dimensional organ cultures. There are three types of primary culture 1. Organ culture: The tissue biopsy is placed in a suitable culture environment where it can retain its three dimensional structural features 2. Primary Explant culture: A piece of tissue is placed in liquid solid boundary where the tissue first adheres and then proliferates in the plane of the solid substrate 3. Cell culture: The tissue is enzymatically or mechanically digested and the cells are harvested. The cells are plated on a substrate or it can be grown as a suspension. Joint Initiative of IITs and IISc Funded by MHRD Page 4 of 8

5 NPTEL Biotechnology Tissue Engineering Fig 1: 1.3 Cell lines Initially the growth of cells in primary culture is slow followed by the log phase where the cell grows exponentially and then plateaus when it reaches the Hayflick limit. Finally, the cells undergo senescence and death. The Hayflick limit of a particular cell line is approximately 50 generations and it mainly depends on the telomere length. However, telomerase activity, DNA repair mechanism and other factors also contribute to the senescence Immortalized cell lines Joint Initiative of IITs and IISc Funded by MHRD Page 5 of 8

6 Transformation of primary cell line into immortalized cell line can occur instantaneously because of mutation that results in p53 or Rb gene activity loss, enhanced performance of telomerase or impairment in senescence genes. Chemical or viral agents can also influence the transformation process. Examples of viral genes that immortalize cells are large T antigen, E6 human papilloma virus and Epstein barr virus. These genes inhibit the activity of p53, p16, Rb and hence cell cycle progression will be amplified. The following are the characteristics of immortalized cell lines: 1. Uncontrolled growth and modulation in the chromosome number; 2. Non existence of contact inhibition and adherent support; 3. Serum growth factors are not essential; 4. Undergo further transformation into tumourigenic cells Variation in cell lines When cells are cultured for a long time, it leads to genotypic as well as phenotypic drift. The genotypic drift may be due to spontaneous change in chromosomes at higher rate of proliferation. The change in the phenotype may be due to changes in the interaction of genotype with respect to the environment. For example, the phenotype of cells on collagen and fibronectin will differ due to interactions of gene with external substrate. Hence, these types of cell line instability should be avoided by optimizing the experimental conditions. 1.4 Media The cell retains its ability to proliferate only in the presence of adequate supply of nutrients. The nutrients can be supplied to the cell in the culture by media. Medium is a liquid, which provides the cells with inorganic salts and other nutrients for its in vitro maintenance. Medium formulation usually includes substances that mimic the physiological environment (ph, osmolarity), supraphysiologic (hormones, nutrients) and non physiologic (indicator dyes and antibiotics). Joint Initiative of IITs and IISc Funded by MHRD Page 6 of 8

7 1.5 Dissolved gases The next important constituent required for the cell survival is the oxygen. Delivery of gas molecules to the cells in vivo differs from in vitro where there is a presence of carrier called haemoglobin. Hence for in vitro, the supply of gas molecules plays a vital role in the maintenance of culture. Cells are usually grown in humidified incubators with 5 10% CO 2 along with sodium bicarbonate as a medium additive. This is because the increase in the carbondioxide concentration reacts with water in the medium resulting in the formation of carbonic acid, which is unstable. Therefore this carbonic acid is further disassociates to form H + ions and bicarbonate ions. The formed bicarbonate ions will tend to associate with other cations and resulting leaving thin the increase of H + ions in the medium. The increased the concentration of H + ions leads to the drop in the ph of the medium. Hence, in order to neutralize the formed H + ions and also maintain the buffering property of the medium, sodium bicarbonate will be added as a medium additive. Mostly, in the cell culture it is preferred to use medium with ph indicator (phenol red). Suppose there is any change in the ph, the colour of the medium is changed from red to yellow or orange. The next challenge in cell culture is to culture metabolically active cells like hepatocytes. This is because such type of cells continuously consumes oxygen and produce carbon dioxide. Therefore, in culture oxygen delivery is limited by two factors: 1. K, the relatively low solubility of oxygen in media at body temperature 2. Transport of oxygen from gas phase to cell surface Let us see how oxygen is transported from the gas phase to cell surface? Assume cells are plated in a petridish, which is then filled with the media and we know that cells will adhere to the base of the petriplate. In the gas phase, ambient oxygen tension will be developed in the incubator. The continuous Joint Initiative of IITs and IISc Funded by MHRD Page 7 of 8

8 consumption of oxygen by the cells leads to the reduction in the oxygen tension at the bottom as compared to gas interface, thereby leading to the development of concentration gradient. This concentration gradient will be the driving force for the diffusion of oxygen from the gas interface to the cell surface. In addition to this the amount of medium or thickness of the medium will have an effect on the cell surface oxygen tension by altering the resistance to diffusion. Hence, both gas concentration as well as media thickness will change the availability of dissolved oxygen to the cell surface. Let us see what would happen to the ph of medium at high cell density? As we are well aware that at high cell density definitely there would be increase in oxygen consumption per unit area. Obviously, it will decrease the cell surface oxygen concentration, thereby shifting the metabolism from aerobic to anaerobic. This will lead to the increased production of lactic acid and CO 2 as an end product. The acidic end products as well as increase in the CO 2 concentration will decrease the ph of the medium. 1.6 Advantages and disadvantages of serum The next important component for cell culture is serum, which is a fraction of whole blood. Usually serum from calf (bovine), fetal bovine and sometimes also human are used. The sera component in the media is about 1% to 20% by volume depending upon the cells and the type of culture. Other components such as growth factors, lipids, hormones, and adhesion factors can be added to the media. The major disadvantage of using sera in cell culture is the batch to batch variation and potential regulatory problems. Joint Initiative of IITs and IISc Funded by MHRD Page 8 of 8