PLANT AND ANIMAL TISSUE CULTURE

STB2093

UNIVERSITI MALAYSIA SARAWAK
FACULTY OF RESOURCE SCIENCE & TECHNOLOGY

STB2093 PLANT & ANIMAL TISSUE CULTURE
The Advantages and Limitation of Tissue Culture towards Medical Advancement in the Future

Lecturer: Dr. Lee Kui Soon Date of Submission: 9 April 2010 Group Members Sabatini Anak Jihob Julianne Anak Phillip Attan Nurul Ain Bt Abdul Mattin Dwenovel Dennis John Matric Number 22218 21169 22017 22817

Advantages & Limitations of Tissue Culture towards Medical Advancement in the Future.

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TABLE OF CONTENTS
Introduction Advantages & Limitations
 Organ Culture  Monoclonal Antibody  Cell Culture Vaccine  HeLa Cell Line Culture

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4 6 9 11 13 14

Conclusion References

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TISSUE CULTURE; AN INTRODUCTION
Tissue culture can be defined as the growth of tissue or cell separate from the organism. It is also known as techniques of keeping tissues alive and growing in an appropriate culture medium. Growing tissues of living organism outside the body is made possible in an appropriate culture medium, containing mixture of nutrient either in solid or liquid form.

In terms of medical application, tissue culture uses are currently increasing. Cell cultures have an extensive application in toxicology studies for initial-stage screening of compounds in drug discovery. Other than that, organ culture is also made possible by tissue culture techniques which give an opportunity to create organs that is similar and can be implanted into the body with less rejection.

There are a lot more application of tissue culture that benefits the medical field. However, we are going to emphasize more on the four main applications in medical which are organ culture, monoclonal antibody culture, cell culture vaccine and also HeLa cell line culture. These applications of tissue culture have their own advantages and also limitation toward the future medical advancement. Further details will be discussed later in this writings.

Figure 1: Animal tissue culture must be equipped with hood to eliminate contaminations.

Advantages & Limitations of Tissue Culture towards Medical Advancement in the Future.

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ORGAN CULTURE
Organ culture is a development from tissue culture methods of research, where the organ culture is accurately able to function as an original organ in various states and conditions. By the use of the actual in vitro organ itself either half or the whole organ, on the other words the culture of complete living organs (explants) of animals and plants outside the body in a suitable culture medium. To make this organ culture successful the animal organs must be small enough to allow the nutrients in the culture medium to penetrate all the cells. The main objective of this organ culture is to maintain the structure of the tissue and direct it towards normal development. In this technique, it is essential that the tissue is never being disrupted or damaged. It thus requires careful handling; it is because this will bring to the success of this technique. In such cultures, new growth is in the form of differentiated structures, for example are glandular structures in case of glands, small bronchi in case of lung tissues, and others. In tissues lined with one or the other type of epithelium, the epithelium differentiates in a pattern similar to that in the concerned organs in vivo. The cultured organs retain their physiological features, which mean their hormone dependent organs remain hormone dependent, and endocrine organ go on secreting the specific hormones.

Figure 2: A 1/4 scale replica of artist Stelarc’s ear grown using human (Stelarc’s) cells. It is cultured in a rotating micro-gravity bioreactor which allows the cells to grow in a 3D structure.

In addition, the morphogenesis in cultured foetal tissues is more or less comparable to that in vivo. In case of organ cultures, outgrowth of isolated cells from the periphery of explants is minimized by manipulating the culture conditions. The media used for a growing organ culture are generally the same as those used for tissue culture. The techniques for organ
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culture can be classified into two. First is employing a solid medium and secondly, those employing liquid medium. As the limitation for this organ culture, organ cultures are often not comparable to those from whole animals‘ studies, for example in studies on drug action, since the drugs are metabolized in vivo but not in vitro. Other than that, organ cultures can be maintained only for a few months. However, it may be desirable to study the effects of certain factors for several months. In such cases, the organs treated in vitro may be transplanted into suitable host animals, for example are nude mice. In the advantage of organ culture is that if this organ culture can be done perfectly it can help to save many lives by providing suitable organ for those who are in need. With this limitation and advantages being the factor in medical advancement, there is still space for improvement but still taking the ethics and social responsibility and move along for the future.

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MONOCLONAL ANTIBODIES
In general, antibodies are protein produced by an individual in response to the present of foreign molecule in the body. Antibodies protect the body by its binding to antigen and destroys invading organism. However, such antibodies are not restricted in the specificity to pathogens but also can form huge variety of antigen that can even destroy novel structures (David J. King, 1998). Therefore, in 1975 Kohler and Milstein come out with a method to produce antibodies of predefined specificity namely the monoclonal antibody (mAb).

Figure 3: Kohler & Milstein won the 1984 Nobel Prize in physiology or medicine for discovering a way to mass-produce monoclonal antibodies.

Monoclonal antibodies are produced by a culture of cells in which all the cell derived from a single cloned cell in a single population (Heddy Zola, 1987). The cloning of monoclonal antibodies is a matter of culturing cells so that they start and remain separate from each other. The monoclonal antibodies have

contributed a lot in medical advancement. These antibodies are very useful in several diagnostic tests to detect small amount of drugs, toxins or hormones. For example, the use of monoclonal antibody to human chorionic gonadotropin kits (HCG) in

pregnancy test

(Biotech,

1989).

Another diagnostic uses of monoclonal antibodies is the diagnosis of AIDS by the ELISA test. In addition, monoclonal antibodies are also
Figure 4

used

in

radioimmunodetection

and

radioimmunotherapy of cancer and some
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other method can even target only the cell membranes of cancerous cell (Chaudhari et al., 1994). Plus, there is a new cancer drug created based on monoclonal antibody technology namely Ritoxin, which has been approved by the FDA in November 1997 (Orrs, 1997). Besides, monoclonal antibodies can also be used to treat viral diseases which are traditionally considered as "untreatable". In fact, there is some evidence to suggest that these antibodies may lead to the cure of AIDS (P/S/L, 1997) in the future. Other than that, monoclonal antibodies can also be used to classify strains of a single pathogen. For example, Neisseria gonorrhoeae can be typed using monoclonal antibodies (Wang et al, 1977). These monoclonal antibodies act typically by either harness the patient's own immune system to fight disease or suppress an errant immune system. Therefore, the ability to culture these monoclonal antibodies had created a new possible route for disease treatment. It is no doubt that further progress and research in monoclonal antibodies culture can lead to future medical advancement. However, there are also some limitations. Monoclonal antibodies are often considered as having the potential to revolutionize cancer therapy. However there are three major limitations to these which are the lack of uptake in solid tumour deposit, the immunogenicity of the reagents causing problems with repeated administration and non-specific uptake of monoclonal antibodies into normal organs (J T Kemshead and K Hopkins, 1993).

Figure 5: Monoclonal antibody drugs like Rituxan, shown in blue, provide targeted treatment by locking on to specific molecules on immune cells.

Other than that, some diseases tend to be more refractory to monoclonal antibody therapy (Davis TA, White CA, Grillo-Lopez AJ, et al., 1999). This problem may be worse with antibodies that rely on the recruitment of immune system cells to kill tumor cells (Dyer MJS, 2001). Another limitation of currently available monoclonal antibody therapy is that patients may develop immune reactions to murine or other nonhuman components of monoclonal
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antibodies, decreasing their circulatory half-life and limiting their overall effectiveness. There are no FDA-approved monoclonal antibodies that containonly human protein sequences. Conversely, monoclonal antibody therapy may result in severe suppression of the immune system. While potentially beneficial in autoimmune disease and organ transplantation, this immunosuppression may reduce the ability of a patient to combat infections. This drawback may compound the risk of infections in patients who are already neutropenic. Additionally, bone marrow suppression and theoretically even secondary malignancies such as acute leukemias may result from the use of monoclonal antibodies linked to radioisotopes (Kaminski MS, Estes J, Zasadny KR, et al., 2000).

Figure 6: The use of monoclonal antibodies provides a new alternative for disease treatments but these antibodies still have their limitation that can be improved in the future.

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CELL CULTURE VACCINE
Nowadays there are mass productions of vaccine using biotechnology technique which is the cell culture technique. An alternative way of producing flu vaccine is based on cell or tissue cultures. This method of production was first described in the mid-nineties and is still in its experimental stage; yet all major players in the vaccines industry have embarked on its development.

Figure 8: Highly sterile and careful handling in cell culture vaccine should be maintained at all time.

Mammalian kidney cells are preferably used for these cell cultures. The virus is injected into these cells, which multiply as the virus does in them, before the cells‘ outer walls are removed, harvested, purified and inactivated. This process resembles a biotechnological fermentation, in which you move from small liter jars to huge fermenters during production. Other types of cell-culture vaccines are; Human Diploid Cell Vaccine (HDCV), Purified Chick Embryo Cell Culture Vaccine (PCECV), Rabies Vaccine Adsorbed (RAV), Purified Vero Cell Rabies Vaccine (PVRV) and BHK-Rabies Vaccine.

Leading manufacturers of vaccines and antiviral drugs are working hard to develop new and novel methods of preparing seasonal influenza vaccines, as well as pandemic vaccine candidates (Rappuoli R., 2006). The cell-culture process for influenza vaccines offers high potential as an alternative method.

Animal cell culture has the capability to offer a predictable, rapid and responsive method for production of well-tolerated and effective vaccines, with low levels of adverse. This cell culture has major potential as the cell-culture materials can be stored, so the production process can be initiated at any time. In addition, production can be scaled up in response to increased vaccine demand. Virus strain change continuously, hence the cell culture-based production is more rapid in developing new vaccines which is better compared to the egg
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based production. Cell culture-based vaccines are produced in well-defined and wellinvestigated cell substrates, which are free of external contaminating agents such as egg proteins so they can be use by people with egg allergies. Cell culture vaccine also eliminates the need for embryonated chicken eggs from managed, biosecure flocks (Rappuoli R., 2006).

Figure 9: The variety of vaccines will increase every season.

Despite the advantages that this cell culture based-vaccines yield, there are still issues and concern rise about this technique. Cell culture-based vaccines are often derived from mammalian cells and have the safety risk of passing viruses or other contaminants into those immunized. Some cell lines are based on African green monkey (Vero) kidney cells and human retinal cells. Others come from Madin-Darby Canine Kidney epithelial cell lines (MDCK), derived from a healthy female cocker spaniel in 1958 and widely used in vaccine research. While some lines of MDCK cells are not tumorigenic, others are highly tumorigenic, according to the briefing materials prepared by the Food and Drug Administration (Agres T.2006).

Figure 10: The two methods used to produce Vaccines Advantages & Limitations of Tissue Culture towards Medical Advancement in the Future. 10

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HeLa CELL LINE CULTURE
A HeLa cell is a cell type in an immortal cell line used in scientific research. It s one of the oldest and most commonly used human cell lines (Rahbari R, et al.). The line was derived from cervical cancer cells taken from a patient named Henrietta Lacks, who eventually died of her cancer on October 4, 1951. The cell line was found to be remarkably durable and prolific as illustrated by its contamination of many other cell lines used in research (CapesDavis A, et al.). The cells continue to divide rapidly and proliferately and the cells never ‗died‘.

Figure 11: Human (HeLa) cell line on SEM, they are the cell in an immortal cell line used in scientific research

HeLa cells are termed ―immortal‖ in that they can divide an unlimited number of times in a laboratory cell culture plate as long as fundamental cell survival conditions are met (i.e. being maintained and sustained in a suitable environment). There are many strains of HeLa cells as they continue to evolve by being grown in cell cultures, but all HeLa cells are descended from the same tumour cells removed from Mrs. Lacks. It has been estimated that the total number of HeLa cells that have been propagated in cell culture far exceeds the total number of cells that were in Henrietta Lacks' body (Sharrer T et al.).

Figure 12: The HeLa cell was first known to develop in Henrietta Lack’s body.

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HeLa cells line cultures has been contributing in medical fields, long time ago and have several advantages that eventually leads to the future medical advancement. Historically, HeLa cells were used by Jonas Salk to test the first polio vaccine in the 1950's. Since that time HeLa cells have been used for research into cancer, AIDS, the effects of radiation and toxic substances, gene mapping, and countless other scientific pursuits (Smith, Van et al.). These cells proliferate abnormally rapidly, even compared to other cancer cells. Other than that, HeLa cells have an active version of telomerase during cell division, which prevents the incremental shortening of telomeres that is implicated in aging and eventual cell death. In this way, HeLa cells circumvent the Hayflick Limit, which is the limited number of cell divisions that most normal cells can later undergo before dying out in cell culture. However, this HeLa cell line has its limitation in terms of its application. Because of their adaptation to growth in tissue culture plates, HeLa cells are sometimes difficult to control. They have proven to be a persistent laboratory "weed" that contaminates other cell cultures in the same laboratory, interfering with biological research and forcing researchers to declare many results invalid. The degree of HeLa cell contamination among other cell types is unknown because few researchers test the identity or purity of already-established cell lines. It has been demonstrated that a substantial fraction of in vitro cell lines — approximately 10%, maybe 20% — are contaminated with HeLa cells. Stanley Gartler in 1967 and Walter NelsonRees in 1975 were the first to publish on the contamination of various cell lines by HeLa (Masters JR et al.)

Figure 13: Staining of HeLa cells (blue) with Hoechst 33258

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CONCLUSION
Animal tissue culture role in medical filed are enormous. Tissue cultures are often used for the analysis of the cells themselves, the assessment of the cell's response to chemicals, or as a tool to produce cellular-derived protein products that really helps in medical advancement. As discussed before, animal tissue culture provides a way to produce monoclonal antibody that makes it possible to produce antibody that have specificity restricted to certain pathogen. This discovery leads to the possibility of curing various diseases such as AIDS and cancer. Other application of animal tissue culture gives a predictable, rapid and responsive method for production of well-tolerated and effective vaccines, with low levels of adverse. Other than that, the use of HeLa cell line culture in medical research will leads to a better understanding of toxic substances, gene mapping and various diseases. Although cell culture seems to come with many advantages towards medical advancement, there are also possible limitations to this. Tissue culture is having the prone of introducing contamination of the cell lines and instability of the continuous cell lines due to chromosomal instability. Other than that, there are still differences between in vitro and in vivo system during drug testing and organ transplantation which make it not totally reliable. Therefore, it can be concluded that the animal tissue culture had contributed a lot to the future medical advancement. However, there are still some boundaries to achieve unlimited medical advancement in the future. Perhaps, these limitations of animal tissue culture can be overcome in the future, thus providing the world with the latest and most advanced tissue culture applications.

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REFERENCES
Dyer MJS. Mechanisms of action of CAMPATH-1 antibodies. CLL. 2001;6:2-4. Davis TA, White CA, Grillo-Lopez AJ, et al. Single-agent monoclonal antibody efficacy in bulky non-Hodgkin's lymphoma. J Clin Oncol. 1999;17:1851-1857 Kaminski MS, Estes J, Zasadny KR, et al. Radioimmunotherapy with iodine 131 tositumomab for relapsed or refractory B-cell non-Hodgkin lymphoma. Blood. 2000;96:1259-1266. J T Kemshead and K Hopkins (1993) - Uses and limitations of monoclonal antibodies (MoAbs) in the treatment of malignant disease: a review. Journal of the Royal Society of Medicine, retrieved on April 3, 2010 from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1293954/?page=1 Steven L. McCune, MD,PhD; Jon P. Gockerman, MD; David A. Rizzieri, MD - Monoclonal Antibody Therapy in the Treatment of Non-Hodgkin Lymphoma. JAMA. 2001;286:1149-1152, retrieved on April 3, 2010 from, http://jama.ama-assn.org/cgi/content/full/286/10/1149 Heddy Zola - Monoclonal antibodies: A manual of techniques. Retrieved on April 3, 2010 from,http://books.google.com/books?id=HanZBZgkeagC&printsec=frontcover&dq=monoclo nal+antibodies&hl=en&ei=HAG2S4uMD4LGrAedoMDiAg&sa=X&oi=book_result&ct=res ult&resnum=1&ved=0CDwQ6AEwAA#v=onepage&q=&f=false David J. King - Applications and engineering of monoclonal antibodies. Retrieved on April 3 from,http://books.google.com/books?id=LwdLBp3LzGAC&printsec=frontcover&dq=monocl onal+antibodies&hl=en&ei=8P21S6HsEJO1rAf16cS3DQ&sa=X&oi=book_result&ct=result &resnum=4&ved=0CEsQ6AEwAw#v=onepage&q=&f=false A. El-Zein, S. Nehme, V. Ghoraib, S. Hasbani and B. Toth. Preparation of Fowl Pox Vaccine on Chicken-Embryo-Dermis Cell Culture. Avian Diseases, Vol. 18, No. 4 (Oct. – Dec., 1974), pp. 495-506. American Association of Avian Pathologists, Inc. Retrieved on 30th March 2010 from JSTOR website: http://www.jstor.org/stable/ Rino Rappuoli (2006). Cell-Culture-Based Vaccine Production: Technological Options. Retrieved on 1st April 2010 from http://www.nae.edu/Publications/TheBridge/Archives/V-363EngineeringandVaccineProductionforanInfluenzaPandemic/Cell-CultureBasedVaccineProductionTechnologicalOptions.aspx Cell-Culture Based Vaccine. Retrieved http://www.ifpma.org/influenza/index.aspx?57 on 1st April 2010 from

GlaxoSmithKline - Backgrounder: Egg-based vs. cell-based influenza vaccine production. Retrieved on 1st April 2010 from http://www.gsk.com/press_archive/press2005/flu_backgrounder.pdf Manjula Shenoy – Animal Biotechnology. Retrieved on April 8, 2010 from http://books.google.com/books?id=3d7UnzvaDREC&pg=PA9&dq=disadvantages+of+animal +tissue+culture&hl=en&ei=mI--SyAIYmmrQeMk9jmCQ&sa=X&oi=book_result&ct=result&resnum=4&ved=0CEQQ6AEwAw#v =onepage&q=disadvantages&f=false Rahbari R. (2009) – A novel L1 retrotransposon marker for HeLa cell line identification. Biotechniques. 2009 April; 46(4): 277–284. doi: 10.2144/000113089. Advantages & Limitations of Tissue Culture towards Medical Advancement in the Future. 14