细胞电刺激系统的研制及其对心肌细胞行为和功能的作用研究

摘要

Cardiovascular diseases (CVDs) are currently the number one killer worldwide.Heart transplantation being the only current therapeutically, because of low number of donors, needs an urgent alternative replacement.The interdisciplinary field of Tissue Engineering along with the evolving field of Regenerative Medicine has proposed the engineering of cardiac tissue with cells that can be potentially harvested from the patients' stem cells or differentiated myocardium.
　　In order to induce cardiac cells to differentiate and proliferate one has to take into account the complexity of the native environment.As for cardiac cells, the integration of electrical cues has been shown to be advantageous for the improvement of cell morphology and physiology, especially for cardiomyocytes.
　　In this work we developed an electrical stimulation system that is simple to fabricate, inexpensive and reliable in the traditional laboratory setting.We also used this system to investigate the effects of electrical stimulation on the morphology and physiology of cardiomyocytes.
　　Chapter 1
　　In this chapter a survey of the approaches of cardiac tissue engineering towards the realization of a replacement therapy for diseased cardiac tissue, focusing on conductive scaffolds and electrical stimulation is given.
　　Before even stimulating cells of a given tissue kind for differentiation and proliferation, a proper cell source has to be harvested.Mature cardiac cells is not considered in the prospects of final tissue engineering therapies because they tend dedifferentiate and because harvesting large amounts of them from biopsies of the heart is impossible.Mature cardiac cells of animals, however, have been used as models to do research in this area.Embryonic stem cells have been particularly attractive due to their pluripotency, yet, ethical debates on their use have made it impossible for research to include them in long term studies.Induced pluripotent cells have been shown to be able to differentiate into many cells types, including cardiac cells.
　　The extracellular matrix not only provides structural support to give the tissue its characteristic mechanical properties but also is involved in signaling that is essential for cell function.It has been shown that conductive scaffolds have positive effects in the enhanced growth and contractile ability of cardiomyocytes.In this work we give a survey of different material types and fabrication methods to synthesize conductive scaffolds and hydrogels.
　　Conductive scaffold may well enhance the proliferation of cardiac cells, yet electrical field must be present in order for this to be observed.Electrical stimulation has been shown to enhance cardiac cells', especially cardiomyocytes', morphology and physiology.It has also been shown that electrical stimulation promotes cardiac differentiation of cells derived from embryonic stem cells.
　　Chapter 2
　　The integration of electrical cues has shown to be advantageous for the improvement of cell morphology and physiology, especially for cardiomyocytes. Many research teams are already investigating ways that would allow the development of cardiac tissue in vitro, such as adequate bioreactors and materials.In this study an electrical stimulation platform that is simple to fabricate, inexpensive and reliable in the traditional laboratory setting is proposed.The electrical stimulation platform base was manufactured from polycarbonate (PC) and has a printed circuit board (PCB) that can easily couple carbon electrode bioreactors to a module that allows electrical stimulation and monitoring.With this system the laboratory worker will be able to electrically stimulate cells while observing the potential drop across the parallel-placed bioreactors and thus monitoring electrical stimulation quasi-real-time. The stimulation platform is also designed in such a way as to reduce contamination of the stimulated cells inside the incubator.H9c2 cells were cultured in small drops for 2 hours and then covered with cell medium and electrically stimulated for 2 days while monitoring the stimulation.Light Micrographs show that cells grow well in stimulated and non-stimulated cultures.
　　Chapter 3
　　The very new discipline of tissue engineering along with new biological techniques has made the creation of engineered cardiac tissue that contracts possible.In order to characterize the functionality of this type of tissue many have relied on traditional patch clamp that analyses cell electrophysiology but this his technique is both expensive and laborious.Development in computer science has allowed the advancement of digital image processing.In this chapter we developed image processing tools to analyze the contractile ability of cells from recorded digit l videos.We use the zeroth and first order moment of digital images to calculate the contracted area of beating cells or cell groups and the centroid displacement, respectively.We designed a beating phantom to consider the ideal case of a beating cell or group cells and were able to demonstrate the beating characteristics of it using both contracted area and centroid displacement.We also showed that the contracted area routine can be used to observe the beating ability of a group cardiomyocytes cultured on polystyrene substrate.The centroid displacement routine did not show any meaningful representation of beating because of the nature of the video, yet another video of paced cells that were stimulated with pulsatile electric field demonstrated that this routine has the potential for extracting information about beating cells.
　　Chapter 4
　　Studies on electrical stimulation of cardiac cells have demonstrated that cells elongate and orient better along both electric field directions and topographical cues. Others have also demonstrated that with the integration of electrical stimulation and conductive scaffolds cardiomyocytes' contraction ability can also be enhanced.In this study, the calcium sensitive dye Fluo-AM is used to assess intracellular calcium of cardiomyoctes that have been stimulated with electricity.Also two types of scaffolds, microgrooved Gelatin/graphene oxide (GO) and electrospun poly (vinylidene fluoride) (PVDF), were compared for their electrical coupling by using calcium fluorescent dies to test their calcium transients.Also a digital video analysis of beating cardiomyocytes cultured on Gelatin/GO scaffolds with and without microgrooves was made by using the centroid displacement algorithm described in Chapter 3.We show that electrical stimulation along with topographical cues enhances the elongation and alignment of cells.Also, by observing intracellular calcium transients and using video optical analysis we have determine that electrical stimulation enhance the contraction ability of these cells
　　Chapter 5
　　In the last chapter we give a brief survey of the trend that current research in cardiac engineering is taking.We discussed that the most promising clinical application is on the use of decellularized hearts.Also we mentioned that heart-on-chip has potential in the drug screening field.

目录

声明

Abstract

Table of Contents

Chapter 1 Introduction

1．1 Overview and Motivation

1．2 Tissue Engineering

1．3 The Prospects of Tissue Engineering

1．4 Cardiac Tissue Engineering

1．5 Approaches to Cardiac Tissue Engineering

1．6 Cell Sources

1．6．1 Mature Cells

1．6．2 Stem Cells,Progenitor CellS and Induced Pluripotent Stem(iPS)Cells

1．7 Seaffolds

1．7．1 Conductive Scaffolds

1．8 Biophysical signals

1．9 Electrical Stimulation of Engineered Cardiac Tissue

1．10 Electrical stimulation promotes cardiac differentiation

Chapter 2 A simple and versatile electrical stimulation system for cardiomyocytes

2．1 Introduction

2．2 Materials and Methods

2．2．1 Electrical Stimulation Platform

2．2．2 Electrode Blocks

2．2．3 Electrical Stimulation and Monitoring

2．2．4 Cell Culture

2．3 Results and Discussion

2．3．1 Clean Electrical Stimulation Setup

2．3．2 Cell Morphology

2．3．4 Cell Electrical Stimulation and Monitoring

2．3．5 Inexpensive and Reliable Electrical Stimulation System

2．4 Conclusion

CHAPTER 3 Assessment tools for cardiomyocyte contraction using image processing：moments based measurements

3．1 Introduction

3．2 Methods and Materials

3．2．1 Software

3．2．2 Phantom Design

3．2．3 Beating Cardiomyocytes Imaging

3．2．4 Programs

3．3 Algorithm Description

3．3．1 Moments

4．2．2 Digital Video Processing

3．4 Comparison between Zeroth Order Moment and Centroid(with First 0rder Moment)

3．4．1 Phantom Video Analysis

4．4．2 Beating Cells Video Analysis

4．5 Discussion

4．6 Conclusion

CHAPTER 4 Effects of electrical stimulation on cardiac cells

4．1 Introduction

4．2 Materials

4．2．1 Reagents

4．2．2 Equipment/Apparatus Manufacturer

4．2．3 Software

4．3 Methods

4．3．1 Modified Tyrode's Solution

4．3．2 Hanks Buffered Salt Solution(HBSS)

4．3．3 Phosphate-buffered saline(PBS)

4．3．4 Polydimethylsiloxane(PDMS)Stamps

4．3．5 Gelatin and Gelatin/Graphene Oxide (GO)Scaffolds

4．3．6 Poly(vinylidene fluoride)(PVDF)Scaffolds

4．3．7 H9c2 Cell Culture

4．3．8 Ventricular Cardiomyoctes Culture

4．3．9 Electrical Stimulation of Cells

4．3．10 Cover Slip Cleaning

4．3．12 Cell Viability

4．3．13 Live/dead Stain

4．3．14 Intracellular Calcium Measurements in H9c2 Cells

4．3．15 Intracellular Calcium Measurements in Ventricular Cardiomyocytes

4．4 Results

4．4．1 Cell Morphology

4．4．2 Cell viability

4．4．3 Calcium Fluorescence in H9c2 Cells

4．4．4 Calcium Transients in Cardiomyocytes

4．4．5 Beating measurements

4．5 Discussion

4．6 Conclusion

Chapter 5 Trends in Cardiac Tissue Engineering

5．1 Introduction

5．2 Deceilurization and Re-perfusion of Cadaveric Hearts

5．3 Heart on Chip

References

Acknowledgements