By Monica Lestari
A Thesis submitted to the Faculty of LIFE SCIENCES
In Partial Fulfillment of the Requirements for BACHELOR OF SCIENCES
WITH A MAJOR IN BIOMEDICAL ENGINEERING
Swiss German University EduTown BSDCity
Tangerang 15339 INDONESIA www.sgu.ac.id
July 2011
STATEMENT BY THE AUTHOR
I hereby declare that this submission is my own work and to the best of my knowledge, contains no material previously published or written by another person, nor material which to a substantial extent has been accepted for the award of any other degree or diploma at any educational institution, except where due acknowledgement is made in the thesis.
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Monica Lestari Date
Approved by:
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Dr. rer. nat. Maruli Pandjaitan, Advisor Date
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Dr. drh. Joko Pamungkas, M.Sc., Co-Advisor Date
______________________________________ _________________
Chairman of the Examination Steering Committee Date
ABSTRACT
STUDY OF EFFICACIOUSNESS OF BROMELAIN ON MAMMALIAN CELL CULTURE
By
Monica Lestari
SWISS GERMAN UNIVERSITY Bumi Serpong Damai
Dr. rer. nat. Maruli Pandjaitan, Advisor Dr. drh. Joko Pamungkas, M.Sc., Co-Advisor
Bromelain, a protease enzyme, is found in the stem and fruit of pineapple. It has been used in medical as therapeutic agents and supplements, and also known to have immunomodulatory function. However, as protease, it is concerned that bromelain will affect the mammalian cells that have proteins on their surfaces. The objective of this research was to find out the effect of bromelain on mammalian cells. In order to do so, mammalian cell culture was utilized with MT-2 cells (a T cell lymphocyte) and bromelain concentrations in culture medium were 25mg/ml, 20mg/ml, 10mg/ml, 5mg/ml, and 1 mg/ml, and the evaluation of cells was using MTT assay (three MTT assays). The high concentrations resulted in inhibition more than 50%. The concentration of 5 mg/ml from second and third assay had inhibition around 50% and 1 mg/ml had no inhibition and in turn had very good viability. Therefore, this research showed that bromelain concentration of 1 mg/ml to below 5 mg/ml (specific activity 3.001 U/mg) was safe for the MT-2 cells.
DEDICATION
I dedicate this thesis to those who make me who I am today and helped me through the time of study at Swiss German University.
ACKNOWLEDGMENTS
The author wishes to thank God for His blessings so that the author has been able to complete this thesis work.
The author would like to express gratitude to Dr. rer. nat. Maruli Pandjaitan as the advisor, as well as Dr. drh. Joko Pamungkas, M.Sc., as co-advisor for their advice, time, attention, support, and guidance from the beginning until this thesis is finished.
The author would also express gratitude to Rector of Swiss German University, Dean of Faculty of Life Sciences, and the staffs during the study of the author in the university, and Director of Pusat Studi Satwa Primata – Institut Pertanian Bogor (PSSP-IPB) for their facility during this research.
The author would like to express gratitude and appreciation to Dr.drh. Diah Iskandriati as the Head of the Microbiology and Immunology Laboratory at PSSP- IPB for permitting author to do the research there and her advice to the author, Silmi Mariya S.Si., M.Si, for her guidance and advice with the cell culture during the time the author working at the lab, and the staffs of Microbiology and Immunology Laboratory for their supports.
The author would also thank Prof. Dr. Philipp Wiedemann, Dr. Jeff Wilkesman, and Ariane Tomsche, for their support and sharing the knowledge of cell culture during the author‘s 6th semester internship at Labor für Molekular- und Zellbiologie Hochschule Mannheim, Germany.
The author is thankful for the understanding family, for encouraging and helping the author during thesis work. Last but not least, to the loving and supporting friends at SGU, thank you for sharing the good and bad times and supporting each other during
TABLE OF CONTENTS
STATEMENT BY THE AUTHOR ...2
ABSTRACT ...3
DEDICATION ...4
ACKNOWLEDGMENTS ...5
TABLE OF CONTENTS ...6
LIST OF TABLES ...8
LIST OF FIGURES ...9
LIST OF APPENDICES ...10
CHAPTER 1 – INTRODUCTION ...11
1.1 Background ...11
1.2 Research Scope ...11
1.3 Objective of the Study ...11
1.4 Significance of Study ...11
1.5 Methodology ...11
1.6 Organizational of Thesis ...11
CHAPTER 2 – LITERATURE REVIEW ...13
2.1 Mammalian Cell Culture ...13
2.1.1 MT-2 Cells ...17
2.2 Human Immunodeficiency Virus (HIV) ...18
2.3 Bromelain ...20
2.4 Principles of MTT Assay ...22
CHAPTER 3 – METHODOLOGY ...24
3.1 Time and Venue ...24
3.2 Materials ...24
3.2.1 Materials for Cell Culture and MTT Assay ...24
3.2.2 Materials for Enzyme Analysis ...24
3.3 Equipments ...25
3.3.1Equipments for Cell Culture and MTT Assay ...25
3.3.2 Equipments for Enzyme Analysis ...25
3.4 Overall Procedure ...26
3.5 Preparation of Cells ...26
3.6 Preparation of Bromelain ...28
3.7 Addition of Bromelain to Cells ...29
3.8 Addition of MTT Reagent and Optical Density Reading ...29
3.9.2 Measurement of Protein Content of the Enzyme ...31
CHAPTER 4 – RESULT & DISCUSSION...33
4.1 MTT Assay Results ...33
4.2 Enzyme Analysis ...41
4.2.1 Volume Activity of the Enzyme ...41
4.2.2 Protein Content of Enzyme ...42
4.2.3 Specific Activity of Enzyme ...43
CHAPTER 5 – CONCLUSION AND RECOMMENDATION ...45
5.1 Conclusion ...45
5.2 Recommendation ...45
GLOSSARY ...46
REFERENCES ...47
APPENDICES ...51
CURRICULUM VITAE ...61
