Bioanalytical Techniques on Tissue Culture - Term Paper Example

BIОАNАLYTIСАL ТЕСHNIQUЕS ОN ТISSUЕ СULTURЕ A Review Paper Submitted By: NAME: INSTITUTION: COURSE: INSTRUCTOR: DATE: © 2014 ABSTRACT Tissue culture is the extraction of tissues and cells from an animal or plant so as to be grown in an artificial environment provided with the necessary conditions24, p13. It is an area of research that has grown in popularity over the years and has become very fundamental in the study of cell biology. The present review paper exhaustively addresses aseptic technique of culturing mammalian tissues and cells with the aim of understanding its application free of ‘contaminants’. The aseptic technique is selected from among others for its deep concern in fostering high levels of safety. Various methods have been proposed in conducting aseptic technique of mammalian tissue culture. In the views of 14, p11, aseptic technique can be successfully carried out by developing cultures as either ‘adherents/monolayers; suspensions or three-dimensionally grown’. These three methods of developing mammalian tissue cultures aseptically have seen their fair share of praise from a number of proponents. The present review report critically analyses these three methods as they have been applied. The review paper has been keen to conclude that aseptic technique of mammalian tissue and cell culture is the most relevant in today’s molecular and cell biology due to its insistence on ensuring that both the environment and equipment are free from contamination22, p26. This bio-safety is also ensured by application of other procedures including ‘trypsinising, freezing/thawing and sub-culturing’. The cultivated tissues and cells/organs have found their use in clinical and regenerative medicine as well as in research15, p111. Objectives To identify the various bio-techniques used in mammalian tissue and cell culture, To review one such bio-technique used in the said field, To pinpoint the key aspects of the identified bio-technique in mammalian life, To analyse the various methods applicable to the identified bio-technique, and To describe the key application areas of cultivated tissues and cells/organs Key Findings The present review paper has come up with the following key findings that: Aseptic technique of mammalian tissue and cell culture is successful in maintaining a ‘safe’ bank of tissues, cells, and organs for the perpetuation of mammalian life, The technique is enabled by three methods of culturing namely: adherent, suspension, and three-dimensionally grown, The success of aseptic technique depends on keeping the “environment, equipment, and personal ‘aura’” free from contamination, and Cultivated tissues, cells and organs through aseptic technique are applicable in a wide range of medical conditions including clinical and generative medicine, research. Key words Aseptic Technique – free from any form of contamination, abiotic or biotic Adherent/Monolayer cultures – cultures requiring a surface or substratum to attach themselves Suspension cultures – cultures which don’t require anchorage but are grown suspended in space Three-dimensionally growing cultures – cultures that grow in spheroids assuming 3D shapes i.e. length, width, and depth Trypsinising – mechanically detaching adherent cells from the surface of the culture vessel Sub-culturing – transferring primary culture cells into secondary cultures to restart fresh growths. Cells in vitro – cells in artificial environments such ‘petri-dishes, flasks, and bioreactors’23, p17. INTRODUCTION As is explained in extant literature, tissue culture is synonymous to ‘cell culture, stem culture, or organ culture’, all referring to the extraction of body segments from an organism in order to cultivate them in artificial environments16, p76. The technique is a twentieth century discovery, during which time the products were only used for study purposes alone. As time went by, tissue culture techniques evolved rapidly to what they are today: “methods of mass production of potent tissues and cells capable of giving rise to independent cell lines” 4, p33. It is in contention that tissue culture techniques have opened a very hopeful window in cell biology as well as micro-biology in the management of life. Research has variously demonstrated that the artificial cultivation of tissues and cells in cultures passes through four critical stages namely: “the adaptive stage; exponential growth phase; deceleration stage; and the death stage”11, p929. Alternatively, these four growth phases have been revised into three main phases namely: ‘Lag, Log, and Plateau’13, p16 as illustrated in Appendix 1 of this review. In each of the said growth phases, key happenings necessary for successful cultivation of tissues and cells do take place. Since the body of an adult organism keeps losing volumes of cells every hour6, p96, it becomes justifiable to develop alternative methods of cultivating standby tissues and cells to supplement the natural sources. One such alternative is clearly described in this review paper. The use of aseptic technique in cultivating mammalian tissues and cells has been crucial in ensuring that these explants necessary for maintaining life are in safe and plenty supply. According to the present review, the use of aseptic technique has emerged fundamental in ensuring the successful development of tissues and cells through three main methods of ‘adherent/monolayer cultures, suspension cultures, and three-dimensionally growing cultures’ as indicated by7, p106. The aseptic technique on the one hand ensures that the culture products remain clean without any contamination whatsoever. Because of this, aseptic production of tissues, cells and organs has underscored the importance of bio-technology in modern science. In view of the underlying features associated with tissue cell technology, the present review paper critically analyses one very important technique used in culturing mammalian tissues and cells. All the relevant methods applicable in cultivating mammalian tissues and cells in artificial setups are described at length, including all the pertinent issues in aseptic technique. The review also presents a critical analysis of the extant literature in relation to aseptic technique as is applied in mammalian tissue culture. Finally, the review paper presents a short summary of the key issues, drawing a conclusion from the entire discourse. THE TECHNIQUE Culturing of tissues and cells as it has already been established requires clear-cut procedures as illustrated by9, p51. At all times when the right methods are applied correctly, cultured cells will occupy all of the available substrate through a process referred to confluence and will soon require ‘sub-culturing or passaging’25, p115. This is the detaching of cultures from the surfaces of the primary culture vessel to create space for more cells. 5, p135 explains that subculturing is important in maintaining the cells’ mitotic index as well as preventing their death. In addition to subculturing, cultured cells need to be frozen for long-term storage. Extant literature has demonstrated that various techniques have been used in culturing mammalian tissues and cells but the Aseptic Technique emerges the most valuable for the purposes of this review paper. The technique together with the methods therein is discussed exhaustively in the sections below. Aseptic Technique This is the technique that ensures that tissue and cell cultures are kept free from contamination by microorganisms in the form of bacteria, fungi, and viruses. The technique achieves this by effectively separating the sterile cell culture from the microorganisms present in the artificial environment14, p17. Additionally, it is important to source all the requirements for the culture from ‘clean’ supplies free from any contaminants. The success of the aseptic technique is hinged on a number of elements discussed below. Sterile Work Area To ensure that there is no contamination of the work area particularly by aerosols and airborne particles, it is recommended that an ‘artificial lid’ for the cells is provided. This ‘artificial cell lid’ must ensure that cell culture is restricted from strong drafts via openings such as doors, windows, and vents4, p68. Additionally, the work surfaces should be devoid of any unnecessary items and disinfected thoroughly at all times. Good Personal Hygiene Apart from providing a sterile work area, those concerned with the tissue and cell cultures must observe personal hygiene. This can be established by constantly washing hands with detergents before and after working with cell cultures1, p929. Good personal hygiene can also help in minimizing cell contamination by dirt and dust from ones skin and clothes5, p101. This can be achieved by wearing personal protective equipment like hand gloves, face masks, overalls and gumboots. Hygienic Equipment and Apparatuses It is common knowledge that equipment and apparatus are usually clean at the source but the moment they get to the laboratory, chances of being contaminated become very high. This is usually attributed to poor handling and non-compliance to the guidelines given for each reagent, media and equipment. It is therefore recommended that all the guidelines governing the acquisition and use of laboratory equipment and apparatuses must be followed and adhered to strictly. This may be in the form of disinfection and complete sterilization as noted by4, p81. Sterile Handling Just as it is in the case of the foregoing elements, 15, p135agrees that sterile handling of all implements during the entire cell culturing process is an important undertaking. This can be achieved by using common disinfectants such 70% ethanol at all times in wiping all the equipment and apparatus as well as surfaces in the laboratory before and after performing experiments. It is also common practice to observe all the necessary rules and regulations in the laboratory such as ‘avoiding eating or drinking anything while inside the laboratory, using disposable items if possible, and sealing all containers tightly when not in use’. DISCUSSION OF THE METHODS USED IN ASEPTIC TECHNIQUE Several methods have been mentioned to be applicable in conducting Aseptic technique of cultivating mammalian tissues and cells. But for the purposes of this review, three main methods namely: adherent/monolayer cultures; suspension cultures; and three-dimensionally growing cultures are addressed. i) Monolayer Cultures Just as the name suggests, a Monolayer culture is when ‘no cell is growing on top of another but instead each cell grow side by side, touching each other on the same growth surface occasionally3, p15. Monolayer cultures are alternatively referred to as adherent cultures because they have the ability to attach themselves to the surfaces of the culture vessels. This ability is owing to the property of cell membranes which have cohesion forces for attachment to surfaces of similar molecules (cell-to-cell)8, p110 as well as adhesion forces for attachment to unlike molecules (cell-to-artificial surfaces). In this sense therefore, cells are said to “form a monolayer when they adhere not only to each other but also to the surface of culture vessels” 1, p931. Depending on temperature and time parameters, different cells are capable of forming differently arranged monolayers as is the case shown by epithelial cells5, p121 and criss-crossed fibroblasts for example. The technique employed in coming up with any form of Monolayer cultures is an intricate of other procedures including trypsinizing and sub-culturing as noted by9, p17. Trypsinizing of Monolayer cultures simply implies to the addition of trypsin to the culture in a salt solution that has no calcium or magnesium ions14, p19. This is usually done when the initial culture has fully grown until it is transferred to a secondary culture to begin fresh cultivation. This process of transferring primary culture that had reached confluence is referred to as sub-culturing or passaging2, p56. The procedure is conducted when the cells have grown fully to occupy all the available space in the vessel1, p927 and is aimed at ensuring that the cells do not lose their prolificacy and are capable of starting new cultures. These adherent cells that have reached full growth capacity can be mechanically detached from the culture vessel by repetitive pipetting of the medium to help disperse floating cell aggregates which can subsequently be used in sub-culturing new colonies18, p111. The other intricacy of forming Monolayer cultures is found resident in the ability to freeze human cells grown in adherent layers20, p36. The idea behind this freezing of human cells borne of Monolayer cultures is for the purpose of storing cell lines for future study as indicated by23, p16. 14, p58further enumerates the benefits of freezing human cells to include but not limited to: “preservation of the cells; avoidance of senescence; reduction of the risk of contamination; minimizing the effects of genetic drift as well as the purposes of long-term storage”. To ensure that freezing is not detrimental to the cells, 3 explains that a ‘cryoprotective agent’ must be applied to the culture at extremely low temperatures of about minus seventy degrees Celsius and below21, p930. A good example of a cryoprotective substance is glycerol or dimethylsulfoxide (DMSO) which conserves the cultured cells for future use. ii) Suspension cultures As opposed to the adherent cultures, Suspension cultures are those Monolayer cell lines that have undergone transformation, losing their anchorage dependence on the culture surfaces and are thus kept as suspensions22, p92. In the arguments of16, p141, HeLa cells are considered the best examples of cultures which can be cultivated and kept in suspension. The commonest source of primary suspension cultures is the normal lymphocytes which are known to be anchorage independent4, p38. Their limitation is found in the fact that they have a limited life span. In spite of this shortcoming, 3, p16 maintains that Suspension cultures have their strength in the fact that they are not expensive to maintain but only requires simple glassware like flasks. iii) Three-dimensionally growing cultures Research has variously shown that it is very easy to grow primary explants in three dimensional4, p73. Similarly, works by4 and 6expressively demonstrate that cells growing in three-dimensional can also be cultivated as suspensions of multiple shapes occurring in 3Ds. This is achieved by having several Monolayer cell lines in a stirrer flask to allow them form suspension cultures by meeting each other. By doing so, 13, p15 says that these cells adheres to one another and aggregates into far much larger spheroids within a very short time as illustrated in Appendix 2 of this review report. An alternative technique of culturing other cell lines as three-dimensional growths as observed by 17, p928 is to keep the cells in petri-dishes that do not have sticky surfaces so as to induce prolific growth. The cells will first form aggregates which are irregular in shape before being transformed into multi-cellular shapes shown in Appendix 2. This is usually after the initial irregularly-shaped aggregates are placed in self-stirring flasks for a number of weeks7, p928. APPLICATION OF ASEPTIC CULTURE IN FURTHERING KNOWLEDGE IN AGRICULTURE In recent times, the field of Agriculture particularly in the area of crop improvement has greatly benefited from advances in aseptic culturing of tissues and cells. Research has proved that an application of culturing techniques has advanced knowledge in crop propagation to very higher levels9. A case in point is the modern-day production of plants and animal clones which are ‘true-to-type’ of the involved gene component as indicated by19. While this advancement has witnessed unequalled strides in crop husbandry, it has unfortunately lagged behind in animal husbandry. That’s why accessed literature material paints a glossy picture of plant tissue culture as opposed to animal tissue culture. According to massive works by23, 12and11 on the present knowledge base on ‘plant tissue culture and micro-propagation’ assures the world of successfully and efficiently managing food and horticultural production for the next few years to come. This argument is clearly supported by a number of case studies found in extant literature. 1st Case Study The application of aseptic culture has been responsible in advancing knowledge in the development of numerous explants to propagate “The Moth Orchids” as reported by19. The conventional cultivation of Orchids is an old practice dating back as early as 551 - 479 BC16. The importance of Orchids ranges from its ‘aesthetic value, exoticism ability and sweet fragrance of its flowers to its significance in food industry as well as in medicine9. It has come to realization of researchers that the vegetative propagation of this horticultural crop has become very cumbersome and time consuming, hence losing its desired seedlings characteristics and uniformity. To overcome this setback, scientists and biotechnologists alike have invested highly in vitro culture techniques aiming at realising faster propagation of Orchid species for commercial purposes17. Through this application, regeneration of micro-propagates from tissues of mature Orchid plants ensures a constant supply of explants. The explant source is kept viable and fresh in a standard medium of common reagents and solvents. The callus from which the explants are developed is constantly sub-cultured after every thirty days to achieve prolific growth8. Apart from the mentioned uses of micro-propagation of Orchid explants, research has also reported that regeneration of dead plant tissues is possible11. Continuing research work in this area is focusing on how best different potting medium compositions can be achieved at minimal cost19. This is looked vis-à-vis against the objectives of producing mass explants of Orchids to fetch good prices both locally and internationally. 2nd Case Study Another advancement of knowledge in aseptic culture is witnessed in the micro-propagation of Tobacco crop, Nicotiana tabacum found very popular in Pakistan for its economic value15. Naturally, fresh Tobacco leaves are usually processed to obtain its nicotine which is an important commercial product worldwide. Currently, agricultural activity of growing Tobacco is conventionally is moving further from this practice towards the highly hyped ‘clonal forms’ 10. In fact, 11 reports that there are four important ‘low nicotine content’ clonal hybrid varieties of tobacco 13 that have been developed with the main objective of amassing explants to be sold to farmers and industry. Using mother plants initially provided by Pakistan Tobacco Board (PTB) 17, it has been possible to create and maintain a reliable source of micro-propagates enough to have a steady supply of commercial explants. Moreover, these micro-propagates are preserved in cheap media for prolonged storage and proliferation as noted by21. Through the use of media of different concentration, the callus intended for growing the micro-propagates is efficiently induced for prolific growth. It is in contention that an application of aseptic technique of tissue culture helps to further knowledge in crop improvement in line with extant literature23. 3rd Case Study This third case study presents research work demonstrating that an application of aseptic tissue culture has furthered knowledge in the ‘multiplication and regeneration of Potato plants using explants developed from the nodes11. This implies that apart from growing Potato plants from ‘scratch’, withered and dead plant species can also be regenerated from tissues cultured aseptically. Potato which is scientifically referred to as Solanum tuberosum is considered the most important vegetable crop the world over and occupies a large percentage of cultivated land in major countries of the world. The crop is found to yield highly and contains large volumes of nutrients and as such is capable of earning the farmers better returns25. Due to the importance attached to Potato as a stable food source, research shows that tissue culture technique is being employed at an advanced level to ensure the efficient multiplication of potato explants which are free from diseases22. The research which was used to further knowledge in this technique aimed at producing mass propagates of ‘true-to-type Potato varieties. It is reported that the research came up with three Potato varieties namely: “Desiree, Diamant and Cardinal”12. Similar to the case of Tobacco mother plants, also the initial mother plants for the Potato materials used in the said research were provided by a Company named as “Four Brothers Agri Services Pakistan”16. The Company is tirelessly working for the ‘introduction of high yielding vegetable & crop varieties in Pakistan’ as indicated by19. According to recent study findings, the development of the said three Potato varieties was initiated by first taking the “disease free potato tubers and washing them using ‘detergent and distilled water’ to remove impurities and then allowing them to sprout in the dark”16. This process is otherwise referred to as ‘Chitting’ which causes the tubers to sprout after five or so days. The “sprouts” are then used as explants for direct proliferation after they were made sterile using common detergents for close to twenty minutes20. Before propagating the now sprouted explants, they were chopped into sizeable sections for easy inoculation to produce maximum plantlets per explant. After this, the plantlets were also inoculated for maximum root sprouting before being hardened off and then supplied for cultivation21. 4th Case Study The fourth case study concerns some few instances of furthering knowledge tissue culture by presenting research work in animal improvement. It is agreeable that cultured mammalian tissues and cells in vitro have also found numerous applications in a number of fields in modern science. Considering what has been available in extant literature, cultured mammalian tissues, cells as well as organs have found their usefulness in clinical treatments where a good example is witnessed in ‘wound healing using implantation of epidermis’23, p16. Similarly, animal tissues, cells and organs cultivated in vitro have been used regularly in generative medicine to replace defective organs such as the liver, heart and kidneys. But in my views, this argument may be farfetched because such organs are very vital and nay not have artificial replacements. 25, p211 continues to purport that this application is found to come in handy in this field where non-functional tissues and organs have degenerated due to old age, disease or damage and can thus be repaired or replaced using cultured products1, p931. In fact major advances in this field have been shown by successful direct injection of bone marrow to treat bone non-union according to findings by2, p106. Patients with total loss of sections of their bones have been treated successfully using this technology. Apart from the foregoing advances, animal tissue and cell cultures have again been constantly used cancer research as observed by14, p101. In this case, cultured tissues and cells offer the best opportunity to understand clearly how malignant growth of cells occurs. This understanding then forms the basis for comprehending the modes for normal cell growth and hence the ability to combat any anomaly22, p126. Moreover, mammalian tissues and cells have been used in immune intervention where they are used to boost the immunity of young ones whose mothers are incapable of doing so naturally5, p118. Their general application is also witnessed by “treating bone marrow and organ-transplant rejection; inflammatory and autoimmune diseases; and systemic collagen abnormalities” where they ‘down-regulate the active reactions of immune cells as noted by21, p929. Finally, animal tissue and cell culture have been instrumental in correcting certain genetic defects in ‘neonates’ according to study findings by13, p17. In essence, therapeutic application of cultured tissues and cells is deemed critical in leading to development of ‘models’ that can be used in research work to study cellular activities and be able to understand common genetic disorders as well as in testing therapeutic drugs. This advancement in science will definitely lead to major strides in arresting common genetic ailments that usually show up later in life due to advanced age. All the applications of mammalian tissues and cells produced in vitro mentioned in this discourse offer one major advantage of being consistent in producing results even from a batch of cloned cells. CONCLUSION Throughout this review report, it has emerged that aseptic technique of mammalian tissue and cell culture stands out as the most reliable and safe method of cultivating cells and organs for perpetuation of life. This is attributed to the technique’s ability to ensure cultured products are free from any contamination and can be safely infused into living organisms. To maintain the quality of these products from aseptic technique, it is important to take precautions at the earliest stage possible to reduce the chances of contamination. This in fact is the basis of aseptic technique and has been demonstrated throughout the review to be fundamental in all other techniques mentioned. The starting point of ensuring that no contamination of cultures occurs is by first sterilizing any equipment and apparatuses used in the laboratory as well as all surfaces in contact cultures5, p181. As a standard measure also, good personal hygiene must be observed at all times to avoid further contamination in the laboratory. This can be achieved by wearing protective clothing as well as cleaning culture equipment and vials common disinfectants such as 70% ethanol1, p928. To ensure that there is full growth of cultures throughout the entire culturing period, it is advisable to allow the cells reach at least 75% to 100% confluence before selecting them for subculturing. Letting the cells become overgrown is detrimental to their growth rate and may also hinder successful passaging from the surfaces of the culture vessel. On the other hand, the review report has also indicated that cells should not be sub-cultured before reaching confluence because they will take long to establish secondary re-growths. It is in contention that cells grown in culture are bound to undergo metamorphosis in terms of morphology and genetic constitution. These changes are also known to adversely affect the ability to reproduce reliable laboratory results and must therefore be avoided at all costs. This can also be forestalled through transformation of cells to prevent them from undergoing senescence and eventually dying4, p67. According to5, p121, cells that have not been transformed but have been passaged indefinitely are bound to undergo senescence with eventual death. Finally, the review has illustrated that cultured cell lines can be preserved for storage for future use. One major method of this preservation is through freezing and thawing as explained by4, p97. According to this explanation, freezing of the cells should be very gradual ensure their potency. The other method of preserving cell lines for storage to be used in the future is by a cryopreservative agent such as glycerol and dimethylsulfoxide (DMSO). Cryopreserved cell lines are protected against any further adverse changes and can be used to provide a backup in case of contamination. REFERENCES [1] Ahmadi A, Azadfar D, Mofidabadi AJ. Study of inter-generic hybridization possibility between Salix aegyptica and Populus caspica to achieve new hybrids. Int. J. Plant Prod. 2010, 4(2): 143-147. [2] Basu SK, Datta M, Sharma M, Kumar A. Haploid plant production technology in wheat and some selected higher plants. Aust. J. Crop Sci. 2011, 5(9): 1087-1093. [3] Chatterjee R. “Cell biology. Cases of mistaken identity”. Science, 2007; 315 (5814): 928–31. [4] Dunham JH, Guthmiller P. “Doing good science: Authenticating cell line identity”. Cell Notes, 2008, 22: 15–17. 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Present status and scope of floriculture developed through different biological tools. Res J. of Agri. Sci. 2010, 1(4): 306-314. [24] Varley P. Perspectives on cell culture for biopharmaceutical development. ESACT Annual Meeting, UK, 2013. [25] Wagiah ME, Alam G, Wiryowidagdo S, Attia K. Imporved production of the indole alkaloid cathin-6-one from cell suspension cultures of Brucea javanica.Merr. Sci. Technol. J. 2008, 1: 1-6. APPENDICES Appendix 1: Growth Cycle in an Adherent/Monolayer Culture Figure: Growth curve for cells grown in culture. Cells should be sub-cultured while still in the exponential phase. Source: Adopted by Davis JM. (2002) from Mammalian Cell Culture Lecture Handout Appendix 2: Multi-cell Spheroids Figure: The diameter of the larger spheroids is about 350 μm Source: Adopted by Freshney RI. (1993) from Methods in Animal Cell Culture Read More