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Jaco Botha BIOMEDICIN 10th Semester, Medicine with Industrial Specialization Effect of Substrate on Proliferation and Differentiation of Stem Cells
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Jaco Botha
BIOMEDICIN 10th Semester, Medicine with Industrial Specialization
Effect of Substrate on Proliferation and
Differentiation of Stem Cells
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Medicine with Industrial Specialization
Biomedicine
Department of Health Science and Technology
Fredrik Bajers Vej 7
9220 Aalborg
Denmark
Title: Effect of Substrate on Proliferation and Differentiation of Stem Cells
Keywords: Limbal Stem Cell Deficiency (LSCD), Limbal Epithelial Stem Cells (LESCs),
Human Adipose-Derived Stem Cells (ASCs), Collagen Cell Carrier (CCC), Collagen type I,
Amniotic Membrane, Adhesion, Proliferation.
Period: 01.09.12 – 31.05.13
Group: 805 Synopsis:
________________________
Jaco Botha
Supervisors: Vladimir Zachar
Edition size: 3
Number of pages: 56
Appendix: 0
Finished: 31.05.2013
See abstract on page 6.
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Abstract
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Preface
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Table of Contents
1 INTRODUCTION ....................................................................................................................................................... 11 1.1 ANATOMY OF THE CORNEA .............................................................................................................................. 12 1.2 LIMBAL EPITHELIAL STEM CELLS AND THE LIMBAL NICHE ................................................................... 15 1.3 LIMBAL STEM CELL DEFICIENCY .................................................................................................................... 18 1.4 CULTURED LIMBAL EPITHELIUM .................................................................................................................... 19 1.5 SUBSTRATES FOR CLET ...................................................................................................................................... 21 2 AIM .............................................................................................................................................................................. 23 3 METHODS .................................................................................................................................................................. 24 3.1 CELL CULTURES AND MEDIUM ........................................................................................................................ 24
3.1.1 NIH/3T3 MURINE FIBROBLAST CELL CULTURES ......................................................................................................24 3.1.2 Γ-IRRADIATION OF NIH/3T3 FIBROBLASTS ................................................................................................................24 3.1.3 GREEN FLUORESCENT PROTEIN (GFP) EXPRESSING HUMAN ADIPOSE-DERIVED STEM CELLS (ASCS) ...................25 3.1.3 LIMBAL EPITHELIAL CELL MEDIUM FOR 3T3 FEEDER SYSTEM ................................................................................25 3.1.4 SERUM- AND FEEDER-FREE LIMBAL EPITHELIAL CELL MEDIUM..............................................................................25 3.1.5 ISOLATION OF HUMAN LIMBAL EPITHELIAL CELLS FROM CORNEOSCLERAL RINGS ................................................25
3.2 SUBSTRATES .......................................................................................................................................................... 26 3.2.1 PREPARATION OF AMNIOTIC MEMBRANE ...................................................................................................................26 3.2.2 PRODUCTION OF ACELLULAR AMNIOTIC MEMBRANE (AAM) ...................................................................................26 3.2.3 PRODUCTION OF EPITHELIALLY DENUDED AMNIOTIC MEMBRANE (DAM) ...............................................................27 3.2.4 PRODUCTION OF PLASTIC COMPRESSED COLLAGEN MEMBRANES ............................................................................27 3.2.5 PREPARATION OF SUBSTRATES FOR CELL CULTURES ................................................................................................28 3.2.6 CELL GROWTH ASSAY ................................................................................................................................................28 3.2.7 POLY-(N-ISOPROPYLACRYLAMIDE) (PIPAA) .............................................................................................................28
3.3 FIXATION AND STAINING OF CELLS ................................................................................................................ 28 3.4 MICROSCOPY ........................................................................................................................................................ 29 3.5 DATA ANALYSIS .................................................................................................................................................... 29 4 RESULTS .................................................................................................................................................................... 30 4.1 PRODUCTION OF ACELLULAR AND EPITHELIALLY DENUDED AMNIOTIC MEMBRANE .................... 31 4.2 PRODUCTION OF PLASTIC COMPRESSED COLLAGEN MEMBRANES ...................................................... 32 4.3 CELL GROWTH ASSAY ........................................................................................................................................ 37 4.4 GROWTH AND DETACHMENT OF CELLS FROM PIPAA ............................................................................... 37 4.5 ISOLATION OF LIMBAL EPITHELIAL CELLS FROM CORNEOSCLERAL RINGS ..................................... 40 5 DISCUSSION .............................................................................................................................................................. 42 5.1 HUMAN AMNIOTIC MEMBRANE ....................................................................................................................... 42 5.2 PLASTIC COMPRESSED COLLAGEN MEMBRANES ....................................................................................... 43 5.3 ATTACHMENT, PROLIFERATION AND MORPHOLOGY OF CELLS ............................................................ 44
5.3.1 AMNIOTIC MEMBRANE................................................................................................................................................44 5.3.2 PLASTIC COMPRESSED COLLAGEN AND CCC™ ........................................................................................................44 5.3.3 PIPAA .........................................................................................................................................................................45
5.3 ISOLATION OF LECS FROM CORNEOSCLERAL RINGS ................................................................................ 45 5.3.1 DISPASE .......................................................................................................................................................................45 5.3.2 COLLAGENASE.............................................................................................................................................................46 5.3.3 LEC CULTURES AND VIABILITY OF CORNEOSCLERAL RINGS ....................................................................................46
6 CONCLUSION ............................................................................................................................................................ 48 7 PERSPECTIVES ......................................................................................................................................................... 49 8 BIBLIOGRAPHY ........................................................................................................................................................ 50
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1 Introduction
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1.1 Anatomy of the Cornea
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1.2 Limbal Epithelial Stem Cells and the Limbal Niche
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1.3 Limbal Stem Cell Deficiency
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1.4 Cultured Limbal Epithelium
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1.5 Substrates for CLET
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2 Aim
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3 Methods
3.1 Cell Cultures and Medium
3.1.1 NIH/3T3 Murine Fibroblast Cell Cultures
3.1.2 γ-Irradiation of NIH/3T3 Fibroblasts
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3.1.3 Green Fluorescent Protein (GFP) Expressing Human Adipose-derived Stem
Cells (ASCs)
3.1.3 Limbal Epithelial Cell Medium for 3T3 Feeder System
3.1.4 Serum- and Feeder-Free Limbal Epithelial Cell Medium
3.1.5 Isolation of Human Limbal Epithelial Cells from Corneoscleral Rings
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3.2 Substrates
3.2.1 Preparation of Amniotic Membrane
3.2.2 Production of Acellular Amniotic Membrane (aAM)
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3.2.3 Production of Epithelially Denuded Amniotic Membrane (dAM)
3.2.4 Production of Plastic Compressed Collagen Membranes
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3.2.5 Preparation of Substrates for Cell Cultures
3.2.6 Cell Growth Assay
3.2.7 Poly-(N-isopropylacrylamide) (PIPAA)
3.3 Fixation and Staining of Cells
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3.4 Microscopy
3.5 Data Analysis
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4 Results
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4.1 Production of Acellular and Epithelially Denuded Amniotic
Membrane
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4.2 Production of Plastic Compressed Collagen Membranes
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4.3 Cell Growth Assay
4.4 Growth and Detachment of Cells from PIPAA
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4.5 Isolation of Limbal Epithelial Cells from Corneoscleral Rings
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5 Discussion
5.1 Human Amniotic Membrane
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5.2 Plastic Compressed Collagen Membranes
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5.3 Attachment, Proliferation and Morphology of Cells
5.3.1 Amniotic Membrane
5.3.2 Plastic Compressed Collagen and CCC™
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5.3.3 PIPAA
5.3 Isolation of LECs from Corneoscleral Rings
5.3.1 Dispase
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5.3.2 Collagenase
5.3.3 LEC Cultures and Viability of Corneoscleral Rings
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6 Conclusion
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7 Perspectives
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