Luận án: Ảnh hưởng gradient nhiệt lên hành vi tế bào bằng mảng dây vi của Truong Ka My Dang

Fakultät für Elektrotechnik und Informationstechnik Professur für Neuroelektronik Microwire arrays for studying the influence of temperature gradients on the cellular behavior Truong Ka My Dang Vollständiger Abdruck der von der Fakultät für Elektrotechnik und Informationstechnik der Technischen Universität München zur Erlangung des akademischen Grades eines Doktor-Ingenieurs (Dr.Christian Jirauschek Prüfer der Dissertation: 1. Oliver Hayden Die Dissertation wurde am 13.2018 bei der Technischen Universität München eingereicht und durch die Fakultät für Elektrotechnik und Informationstechnik am 03. A BSTRACT Temperature plays an important role in regulating the biological function of cellular development. Over the past few decades, the development of microfabrication technology has enabled highly localized temperature control at the scale of a few microns.

This allows the study of thermal influences on biological microsystems. In this context, the present thesis investigates the changes of cellular behavior depending on temperature gradients using microwire arrays. In addition, the thesis also introduces a sacrificial layer approach to fabricate microchannels for studying neu- ronal guidance. The integration of the microwire array into microchan- nels presented in this thesis proposes a novel method for governing neu- ronal networks under the influences of temperature gradients.

The first part of this thesis presents the application of microwire arrays to study the effects of localized temperature gradients on signal propaga- tion in cardiac cell networks. In order to investigate the effects of temper- ature on cellular networks, a monolayer of cardiomyocytes is cultured onto the chip. The localized temperature is induced by using a power supply connected to microwire arrays. A calcium imaging method is performed to evaluate changes in signal propagation under local heat stimulation.

Calcium signals are recorded using a high speed and low light camera. Also in this part, the velocity of the calcium signal prop- agation in cellular networks is locally increased upon heat stimulation. Additionally, this part presents a relocation of the pacemaker cell and a deformation of the calcium wavefront caused by an increase of the local temperature. In the second part, the growth of neurons is investigated under the in- fluence of temperature gradients.

In this study, the PC12 neuron-like cells are cultured on the microwire array chip. The effects of temperature gra- dients on neurons are examined, focusing on the speed and direction of neurite outgrowth. The growth of neurites under local heat stimulation is recorded using a time-lapse imaging method. The images are then ana- lyzed to estimate the development of neurons within temperature gradi- ents.

The result indicates that the growth speed of neurons increases with heat stimulation. Moreover, the evidence that temperature gradients af- fect the growth direction of neurites is observed in a long-term period experiment (24 h after heating). The last part of the thesis presents a promising fabrication approach, based on standard clean room fabrication and sacrificial layer etching, for combining microwire arrays with a set of perpendicular axon-guiding mi- crochannels. This approach enables the positioning of neurites, as well as control over their polarity.

In particular, an array of asymmetrical mi- crochannels with wide inlets and narrow outlets is fabricated to allow neurites to grow in one direction. The experiment with neurites from PC12 cells demonstrates that neurites can grow from the entrance to the exit of the microchannels. Furthermore, the fabrication of microchannels, integrated with microwire arrays, allows the study of temperature gradi- ents effects on the formation of functional neuronal networks. Overall, this thesis focuses on investigating thermal impacts on cellu- lar behavior using the localized heating generated by microwire arrays.

This work demonstrates the important role of microscopic heating in reg- ulating signal propagation, as well as the growing properties of cellu- lar networks. Specifically, the temperature highly confined in a small area governs the calcium signal propagation of cardiomyocytes and the growth of neurites towards the heat source. Other than that, concerning the technical aspects, the fabrication of thin microwire enables us to con- trol high temperature in a microscopic area. Furthermore, the fabrication of sacrificial microchannels contributes benefits for neuronal guidance, ii such as reducing a number of fabrication steps and improving the adhe- sion between microchannels and the chip’s surface.

iii iv C ONTENTS 1 Introduction 1 2 Fundamentals & Theory 5 2.1 The cell structure .3 Action potentials and signal propagation .4 Signal propagation in cardiac cells .5 TRPV channels as temperature sensors .1 HL-1 cells: A cardiomyocyte cell line .2 PC12 cells as a model for neurons .3 Devices and fabrication techniques .2 Microfluidic channels for cell culture .3 Fabrication technologies for microfluidic channels .2 Fluorescence lifetime imaging (FLIM). 35 3 Heat stimulation for modulating signal propagation in HL-1 cell networks 37 C ONTENTS 3.3 Materials and methods .1 Fabrication of microwire array chips .2 Culture of HL-1 cells on the microwire array chip .3 Thermal stimulation and Ca imaging .4 Cross-correlation analysis .4 Results and discussions .1 Effect of localized heat stimulation on Ca2+ signal propagation .2 Effect of localized heat stimulation on the pacemaker position .5 Conclusions and outlook. 51 4 Heat activation and guidance of neurite outgrowth 55 4.3 Materials and methods .1 PC12 cell culture on chips .2 Heat stimulation Setup .3 Thermal stimulation and imaging .4 Image processing using the optical flow method .4 Results and discussion .1 Effect of temperature gradients on cellular growth .2 Influence of temperature gradients on the direction of cellular growth .3 Temperature distribution on microwire .5 Conclusions and outlook. 70 5 Fabrication of microchannel structures for neuronal guidance to- wards heat activation 73 5.3 Materials and Methods .1 Fabrication of PDMS microchannels .2 Fabrication of aligned SU-8 microchannel .4 Fluorescence imaging of the neurite length in mi- crochannels .4 Results and discussion .1 PDMS microchannels and its drawbacks .2 Microwire array and SU-8 microchannel structures 82 5.3 Neurite outgrowth into SU-8 microchannels .5 Conclusion and Outlook.

87 6 Conclusions and outlook 89 6.1 Summary of the Thesis. 91 Appendix 95 A Geometry and parameters for temperature simulation 95 B Protocol for bonding PDMS to a polyimide surface 97 C Protocol for HL-1 cell culture 99 D Protocol for PC12 cell culture 103 E Protocol for preparing dyes 107 E.2 Calcein-AM/Ethidium Homodimer(EthD). 107 Author’s list of publications 127 Conference presentations 129 Acknowledgment 131 vii C ONTENTS viii C HAPTER 1 I NTRODUCTION Temperature is an important physical parameter affecting every life pro- cess. In mammals, temperature is well known as an essential vital sign that indicates sickness or health.

Additionally, temperature plays a cru- cial role in regulating a variety of mechanisms in biological systems. It has been demonstrated that cells decrease their growth rate under low temperature conditions [1, 2]. Conversely, the proliferation rate of cells rises in response to an increase of ambient temperature [3]. In line with this, the previous study of Kang et al.

[4] shows that temperature has a comprehensive influence on intracellular calcium dynamics, which ma- nipulates most biochemical processes such as neural transmission, mus- cle contraction, and heart beat. In fact, many studies have demonstrated that temperature has a profound effect on living systems such as birth length [5], life span [6] and aging-associated diseases [7]. Together with temperature itself, temperature gradients also play a sig- nificant role in regulating the living systems. Spatial and temporal vari- ation of temperature induces changes in the nature of chemical reactions and alters the configuration of atoms that build up nucleic acids, pro- teins, lipids and other biomolecules [8].

A spatial temperature gradient has been demonstrated to be a guidance cue for the migration of organ- isms. Many studies indicate that thermotaxis can cause an organism to move up or down according to a gradient temperature [9–11]. I NTRODUCTION iment with Caenorhabditis elegans, a transparent nematode (roundworm), has shown that Caenorhabditis elegans migrates to a certain temperature when subjected to a spatial temperature gradient [12]. While the temper- ature sensitivity of organisms is well known, it has been not well under- stood how individual cells respond to different temperatures.

The study of temperature sensitivity at a cellular level has been a challenging engi- neering problem, possibly due to the lack of microscopic systems which can produce temperature at microscales. Over the past few decades, microtechnology has been developed as a new approach for studies in biophysics and physical chemistry [13–15]. Microtechnology employs fabrication methods (e. photolithography) to create microsystems on chips, such as lab-on-a-chip or micro-total- analysis-systems.

As a result of the development of microelectromechan- ical systems (MEMS) and microfluidic devices, scientists are now able to investigate and manipulate biological systems at a cellular level [16– 19]. MEMS offer the ability to precisely manipulate cells on microscales, which makes it possible to study cellular interactions with physical and chemical microenvironments surrounding them [17, 20]. These interac- tions offer deep insights into the fundamental behaviors of biological mi- crosystems, such as pacemaker cells, calcium concentrations, neuronal outgrowth, cellular viability, and cellular locomotion. This thesis aims to study cellular networks regulating and responding to local heating generated by microwire array chips.

Particularly, the in- fluence of localized temperature gradients on calcium signal propagation in cardiomyocytes will be investigated. A rise of local temperature will cause an increase in the propagation speed of these signals. This results in a deformation of the calcium wave in the cardiac networks. Addition- ally, the local temperature will be used to stimulate cells in the heated zone.

This stimulation causes the cells to become more active. This re- sults in the relocation of pacemaker cells to the heated area. Moreover, the effects of temperature gradients on neuron-like cells will be exam- ined in this work. Temperature gradients could accelerate the speed of neurite outgrowth and be a guidance cue for neurites.

To access cellular 2 behavior in response to localized heating, a fabrication approach which combines microheaters and microfluidic channels for neurite guidance will be introduced in this thesis. The local heating with high spatial accuracy induced from microwire array chips provides an effective approach to study heat effects at cellu- lar levels. Studying the influence of heat on living cells at a microscale creates a pathway to understanding how cells are sensed by the adja- cent cells and communicate with each other in response to temperature changes. The thesis contains six chapters.

In the introduction, the motivation for the research is presented. The second chapter takes up the fundamentals and methods. This chapter describes the basic concepts of biology and physics which are used in the following experiments and studies. The third chapter presents the study of local heat stimulation for modulating signal propagation in cardiomyocyte-like cell (HL-1 cells) networks.

The study of the effect of heat activation on the guidance of neurite outgrowth will be conducted in the fourth chapter. The next chapter describes the fabrication of microchannels perpendicular to microwire arrays for study- ing the effects of thermal gradients on neurite outgrowth and neuronal guidance. The last chapter presents the conclusions and outlook, which suggests possible applications of microwire arrays in studies of neuronal regeneration, cell migration, and dopamine releases. I NTRODUCTION 4 C HAPTER 2 F UNDAMENTALS & T HEORY 2.1 T HE CELL STRUCTURE The cell is the basic unit of biological activity.

The name “cell” was suggested by Robert Hooke in 1665. The meaning is derived from the Latin cella which means storeroom or chamber [21]. There are two dif- ferent types of cells, prokaryotic cells and eukaryotic cells. The structure of prokaryotic cells is very simple, whereas the structure of eukaryotic cells is more complex.

The cells from plants and animals are all eukary- otic cells. Since the cells used in this work are cardiomyocyte-like cells and neuron-like cells, only the structure of eukaryotic cells is described in detail. The size of eukaryotic cells is between 1 and 100 µm. The structure of eukaryotes is shown in Figure 2.

Surrounding the cell is the plasma membrane, which protects and separates the cell from the external envi- ronment. The cell membrane contains many biomolecules such as pro- teins.