Mechanical stimulation of cells [Elektronische Ressource] : dynamic behavior of cells on cyclical stretched substrates / presented by Simon Jungbauer

Simon Arbeit

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Dissertation


Submitted to the
Combined Faculties for the Natural Sciences
and
for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences














Presented by

Simon Jungbauer
Born in Heilbronn, Germany




Oral examination: 02.06.2008





Mechanical stimulation of cells:
Dynamic behavior of cells on cyclical stretched
substrates


















Referees:
Prof. Dr. G. Elisabeth Pollerberg
Prof. Dr. Joachim P. Spatz

Table of Contents
Table of Contents

TABLE OF CONTENTS 1
SUMMARY 4
ZUSAMMENFASSUNG 6
1. INTRODUCTION 8
1.1 Influence of forces on tissue engineering and disease 9
1.2 Mechanical forces in cell biology 12
1.3 How do cells sense force? 14
1.4 Experimental methods for mechanical stimulation of cells 19
1.5 Stretching of cells on elastic substrates 23
1.6 Models describing cells subjected to cyclical stretch 27
1.6.1 Cells as a mechanical dipole 27
1.6.2 Interpretation of stress fiber organization under conditions of cyclic stretch 28
1.6.3 Other models 29
1.7 Objectives of the study 30
2. MATERIALS AND METHODS 31
2.1 Development of a stretching machine for live cell imaging 31
2.1.1 The experimental set-up 32
2.1.2 The stretching chamber 36
2.1.3 Calibration and specification of the stretching system 38
2.1.4 Life-cell imaging during the stretching experiments 40
2.1.5 Software used to control the system 42
2.2 Cell culture 45
2.2.1 Materials and chemicals 45
2.2.1.1 Buffers, chemicals, and media 45
2.2.1.2 Lab materials 46
2.2.1.3 General lab equipment 46
2.2.2 Cultured cell types 47
2.2.3 Maintenance of fibroblasts in culture 48
2.2.4 Primary cell cultures (human fibroblasts) 48
2.2.5 Human mesenchymal stem cells (hMSC) 49
2.2.6 Fusion proteins and transfection of cells 49
2.2.7 Experimental conditions 49
2.2.8 Immunofluorescence staining procedure 50
2.2.9 Scanning electron microscopy (SEM) 50
2.3 Image analyses and evaluation routine 52
2.3.1 Analyzing the phase contrast images 52
2.3.2 Evaluating the raw data 53
2.3.2.1 Morphological parameters 53
1 Table of Contents
2.3.2.2 Orientation of the cells 54
2.3.2.3. Apolar order parameter and dose-response curve 55
2.4 Parameters of the stretching experiments 57
2.5 Structuring the PDMS surface by micro-contact printing (µCP) 58
2.5.1 Fabrication of the master substrates by photolithography 58
2.5.2 Use of the master substrates as PDMS molds 60
3. RESULTS AND DISCUSSIONS 62
3.1 Characterization and calibration of the stretching system 62
Discussion 65
3.2 Phenotype and morphology of cells during cyclical stretch 66
3.2.1 Scanning electron microscopy images 67
3.2.2 Temporal change of cell elongation and cell area 68
Discussion 70
3.3 Reorientation dynamics of cells in dependence of the stretching frequency 71
3.3.1 Reorientation dynamics of sub-confluent REF52 cells 71
3.3.2 Reorientation dynamics of sub-confluent HDF1 cells 73
3.3.3 Characteristic regimes in frequency dependent dynamic reorientation 75
3.3.4 Influence of cell density on the dynamics of cell reorientation. 77
3.3.5 Change of the maximum orientation <cos2 > with frequency 79 MAX
3.3.6 Lag time of the cellular reorientation process 80
3.3.7 Cellular response during cyclical stretch with different stretching rates 81
3.3.8 Discussion 85
3.4 Reorientation dynamics of single cells in dependence of the stretching amplitude 90
Discussion 93
3.5 Comparison of reorientation dynamics of cells from young and old donors 95
Discussion 97
3.6 Change of strain direction 99
Discussion 101
3.7 The reorganization of focal adhesions as a result of cyclical stretch 102
Discussion 106
3.8 Cell division during cyclical stretch 107
Discussion 109
3.9 Summary of the results 110
4. CONCLUSIONS AND OUTLOOK 111
BIBLIOGRAPHY 116
APPENDIX : ADDITIONAL EXPERIMENTS 126
A.1. Dynamic behavior of human mesenchymal stem cells during cyclical stretch 126
Discussion 127
A.2 Influence of the surrounding temperature on cellular reorientation during cyclical stretch 128
Discussion 129
2 Table of Contents
A.3 Stretching of human fibroblasts on micropatterned substrates 130
Discussion 132
A.4 Actin filaments and the extracellular matrix staining 133
Discussion 137
Abbreviations 138
Supplementary Materials 139
ACKNOWLEDGEMENTS 140
3 Summary
SUMMARY

Besides the biochemical factors in the environment, physical factors can also influence
biological processes in tissues or in single cells. For, example the mechanical stimulation of
cells can regulate their proliferation, apoptosis or the expression of genes within them.
Previous studies concerning the influence of cyclical strain on cells adhering to flexible
substrates showed that the cells attempt to reorient themselves to be perpendicular to the
stretch direction. This behavior has been described qualitatively, but no systematic,
quantitative studies of this phenomenon have yet been undertaken. Furthermore, the cells
were only observed prior to and following stretch. Studies of cellular dynamics during the
cyclical stretch are lacking.
In the present study, our aim was to both observe and quantitatively describe the dynamics of
the cellular reaction by means of a biophysical model. We therefore developed a new
stretching system which allows live-cell observations during the stretching experiments.
The behavior of different cell types was investigated, according to a variety of different
parameters such as stretching frequency, stretching amplitude, or cell density. As a model
system, we used two types of fibroblasts: rat embryonic fibroblasts (REF52) and primary
human fibroblasts (HDF) taken from donors of various ages.
We observed that the perpendicular reorientation of the cells occurs at an exponential rate
over time. Accordingly, we employed a simple mathematical model to determine how long it
characteristically took for the cells to reorient themselves in response to the various
mechanical parameters.
Our results demonstrated a previously unknown characteristic biphasic cellular behavior
which depended on the stretching frequency. Both REF52 and HDF fibroblasts were found to
reorient faster, until a certain threshold frequency was reached. In this regime the
characteristic reorientation time decreased by a power law, as the frequency increased
n(characteristic time ~ f ). Above this threshold frequency, the characteristic time ceased to
decrease. When the cells were stretched with higher frequencies than this threshold frequency,
a saturation of the characteristic time was reached. All tested cell types displayed this biphasic
behavior. Cell-specific differences, however, were observed in the reaction kinetics and in the
threshold frequencies. The REF52 cells already began to react at a frequency which is
approximately 10 times lower compared to the HDF1 cells, in general they reoriented
themselves faster than the HDF1 cells at all frequencies. Furthermore, we demonstrated that
older HDF cells reoriented themselves faster than young HDF cells.
When we increased the cell density to a confluent cell layer, we also observed a power law
dependent decrease in the characteristic reorientation time, when the frequency increased.
Compared with the single cells, however, a plateau of saturation of the characteristic
reorientation time could not be observed. Furthermore, the confluent cells reacted
approximately twice as fast as the single cells. Activation of cell-cell contacts involved in
mechanotransduction in addition to focal contacts may constitute one possible explanation for
this observation.
When the stretching amplitude was varied, the characteristic reorientation time was found to
decrease, along with an increase in amplitude. However, in contrast to the frequency variation,
in this case we observed a linear decrease.
The different reaction characteristics resulting from variations in the stretch frequency and the
stretch amplitude (power law-dependent and linear) suggested that the inserted energy, the
reorientation process depends on can not be described as a simple product of frequency and
amplitude.
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