Bioengineering in Cell and Tissue Research (eBook)

Editorial Board 5
Introduction 6
Contents 15
Part I Reporter Genes in Cell Based ultra High Throughput Screening 29
1 Reporter Genes in Cell Based ultra High Throughput Screening 29
1.1 Introduction 29
1.2 From Gene to Target 30
1.3 Screening Assay Classes 30
1.4 Reporter Gene Classes 31
1.5 Flash-Light Reporter Genes 32
1.6 Glow-Light Reporter Genes 34
1.7 Coelenterazine Dependent Luciferases 34
1.8 Luciferin Dependent Luciferases 36
1.9 Non-Luciferase Glow-Light Reporter Genes 37
1.10 Fluorescent Proteins 38
1.11 Cell Based Assay Formats in ultra High Throughput Screening (uHTS) 40
1.12 Reporter Genes in uHTS 42
1.13 Photoprotein Readouts and Cell-Based Assay Development 43
1.14 Multiplexing Reporter Gene Readouts 44
1.15 Ultra High Throughput Screening 45
References 47
2 Gene Arrays for Gene Discovery 49
2.1 Introduction 49
2.2 Data Processing 2.2.1 Challenges in Data Acquisition and Processing 51
2.2.2 Data Preprocessing: An Overview 51
2.3 Gene Discovery by Gene Clustering 53
2.4 TGF-ß1 Signaling in Dendritic Cells Assessed by Gene Expression Profiling 55
2.4.1 Linking Gene Expression Data to Knowledge-based Databases 58
2.5 Conclusions 58
References 61
3 Physical Modulation of Cellular Information Networks 63
3.1 Introduction 63
3.2 Cellular Responses to Physical Stimulation 64
3.2.1 Electrical Potential 65
3.2.2 Hydrostatic Pressure 68
3.2.3 Shear Stress 69
3.2.4 Heat Shock 70
3.3 Electrically Controlled Proliferation Under Constant Potential Application 3.3.1 Electrical Potential- Controlled Cell Culture System 71
3.3.2 Cell Viability Under Constant Potential Application 71
3.3.3 Electrical Modulation of Cellular Proliferation Rate 73
3.4 Modulated Proliferation Under Extreme Hydrostatic Pressure 73
3.5 Electrically Modulated Gene Expression Under Alternative Potential Application 3.5.1 Electrically Stimulated Nerve Growth Factor Production 76
3.5.2 Electrically Induced Differentiation of PC12 Cells 78
3.6 Cellular Engineering to Enhance Responses to Physical Stimulation 80
3.7 Concluding Remarks 83
References 85
Part II Cell and Tissue Imaging 88
4 Fluorescence Live-Cell Imaging: Principles and Applications in Mechanobiology 89
4.1 Introduction 89
4.2 Fluorescence Proteins 90
4.3 Fluorescence Microscopy 93
4.4 Applications in Mechanobiology 95
4.5 Perspective in Cardiovascular Physiology and Diseases 102
References 104
5 Optical Coherence Tomography (OCT) – An Emerging Technology for Three- Dimensional Imaging of Biological Tissues 108
5.1 Introduction 108
5.2 Optical Coherence Tomography (OCT) 110
5.3 Applications in Tissue Engineering 118
5.4 Conclusion 121
References 122
6 Ultrasonic Strain Imaging and Reconstructive Elastography for Biological Tissue 126
6.1 Introduction 126
6.2 Ultrasound Elastography 128
6.3 Reconstructive Ultrasound Elastography 137
6.4 Medical Results of Elastography 146
6.5 Results of an Intravascular Ultrasound Study 147
6.6 Summary and Conclusion 150
References 152
Part III Regenerative Medicine and Nanoengineering 156
7 Aspects of Embryonic Stem Cell Derived Somatic Cell Therapy of Degenerative Diseases 157
7.1 Introduction 157
7.2 Rationale for the Cardiac Tissue Engineering 158
7.3 Embryonic Stem Cells as an Unlimited Source for Cardiomyocytes 159
7.4 Therapeutical Cloning of Embryonic Stem Cells 162
7.5 Stem Cell Derived Cardiomyocytes 165
7.6 Cardiac Tissue Slices 167
7.7 Bioartificial Heart Tissue Based on Biomaterials 169
7.8 Scaffolds for Cardiac Tissue Engineering 170
7.9 The Ideal Cell 173
7.10 Preparation of Cells for In-Vitro Tissue Engineering: Cell Permeable Cre/ loxP System 174
7.11 Outlook 176
References 178
8 Collagen Fabrication for the Cell-based Implants in Regenerative Medicine 180
8.1 Regenerative Medicine 180
8.2 The Cell-Based Implants 181
8.3 Requirements of Materials for the Cell-Based Implants 182
8.4 Biomaterials in the Cell-Hybridization 183
8.5 Characteristics of Collagen 184
8.6 Fabrication of Collagen 186
8.7 Collagen in the Cell-based Implants 191
8.7.1 Skin Regeneration 191
8.7.2 Bone Reconstruction 196
8.7.3 Esophagus Replacement 198
8.7.4 Wound Healing promoting Anti-Adhesive Matrix 203
8.7.5 Liver Regeneration 205
8.8 Discussion 208
References 211
9 Tissue Engineering – Combining Cells and Biomaterials into Functional Tissues 213
9.1 Introduction 213
9.2 The Cells 214
9.3 The Material 222
References 229
10 Micro and Nano Patterning for Cell and Tissue Engineering 235
10.1 Overview 235
10.2 Regulation of Cell Functions by Matrix Patterning 236
10.3 Topographic Regulation of Cell Functions 241
10.4 Engineering 3D Environments with Micro Features 242
10.5 Nano Patterning for Cell and Tissue Engineering 245
10.6 Perspective 247
References 248
11 Integrative Nanobioengineering: Novel Bioelectronic Tools for Real Time Pharmaceutical High Content Screening in Living Cells and Tissues 249
11.1 Introduction 249
11.2 Real Time Monitoring and High Content Screening 250
11.3 Outlook and Future Aspects 263
References 264
Part IV Soft Materials in Technology and Biology – Characteristics, Properties, and Parameter Identification 268
12 Soft Materials in Technology and Biology – Characteristics, Properties, and Parameter Identification 268
12.1 Introduction 268
12.2 Material Description 270
12.3 Basics of Continuum Mechanics 287
12.4 Basics of Material Theory 293
12.5 Material Laws for Technical and Biological Polymers 309
12.6 Volume Change in Biopolymers 318
12.7 Summary and Outlook 325
References 327
13 Modeling Cellular Adaptation to Mechanical Stress 330
13.1 Introduction 330
13.2 A Brief Review of Stretch-Induced Cell Remodeling 331
13.3 Measurements, Modeling, and Mechanotransduction 335
13.4 A New Approach for the Study of the Mechanobiology of Cell Stretching 345
13.5 Illustrative Examples 354
13.6 Closure 357
References 359
14 How Strong is the Beating of Cardiac Myocytes? – The CellDrum Solution 362
14.1 Introduction 362
14.2 The CellDrum Technique 365
14.3 Preparation of Samples 367
References 378
15 Mechanical Homeostasis of Cardiovascular Tissue 381
15.1 Introduction 381
15.2 Shear Stress and Scaling Laws of Vascular System 382
15.3 Stress and Strain 384
15.4 Intramural Stress and Strain 386
15.5 Perturbation of Mechanical Homeostasis 388
15.6 Limitations, Implications and Future Directions 394
References 397
16 The Role of Macromolecules in Stabilization and De- Stabilization of Biofluids 402
16.1 Introduction 402
16.2 The Effects of Macromolecules on the Stability of Colloids 405
16.3 Macromolecular Depletion at Biological Interfaces 406
16.4 Cell–Cell Interactions Mediated by Macromolecular Depletion 410
16.5 Stabilization of Bio-Fluids via Macromolecules 415
16.6 Destabilization of Bio-Fluids via Macromolecular Binding 417
16.7 Conclusion & Outlook
References 420
17 Hemoglobin Senses Body Temperature 424
17.1 Instead of an Introduction 424
17.2 Physiological Aspects of Thermoregulation in the Body 427
17.3 Red Blood Cells 429
17.4 Temperature Transition in RBC Passage Through Micropipettes 429
17.5 The Molecular Mechanism of the Micropipette Passage Transition 430
17.6 Hemoglobin Viscosity Transition 432
17.7 Circular Dichroism Transition in Diluted Hb Solutions 433
17.8 A RBC Volume Transition Revealed with Micropipette Studies 435
17.9 Micropipette Passage Transition in D2O Buffer 437
17.10 NMR T1 Relaxation Time Transition of RBCs in Autologous Plasma 438
17.11 Colloid Osmotic Pressure Transition of RBC Suspended in Plasma 441
17.12 The Temperature Transition Effect so Far 442
17.13 Strange coevals – Ornithorhynchus anatinus and Tachyglossus aculeatus 443
17.14 Hb Temperature Transition of Species with Body Temperatures Different from 37 C 444
17.15 Molecular Structural Mechanism of the Temperature Transitions 447
17.16 Physics Meets Physiology 449
References 452
Part V Bioengineering in Clinical Applications 457
18 Nitric Oxide in the Vascular System: Meet a Challenge 458
18.1 Nitric Oxide: NO 458
18.2 NO in Vascular Biology 458
18.3 Key Questions 460
18.4 Assessment of NO Mediated Vasoactivity 460
18.5 From the In-Vivo and Ex-Vivo Detection of NO Effects to Biochemical Assessment of NO 462
18.6 On the Road to a Potential Sensitive Marker for NO Formation: Is Nitrite a Candidate? 463
18.7 More Information About NO Interactions in the Blood 465
18.8 Intravascular Sources of NO 466
18.9 The Potential Relevance of RBC NOS Activity 466
18.10 Outlook 469
References 472
19 Vascular Endothelial Responses to Disturbed Flow: Pathologic Implications for Atherosclerosis 476
19.1 Introduction 476
19.2 Endothelial Dysfunction is a Marker of Atherosclerotic Risk 477
19.3 Correlation Between Lesion Locations and Disturbed Flow Regions of the Arterial Tree 478
19.4 In Vitro Studies on the Effects of Disturbed Flow on ECs 480
19.5 In Vivo Studies on the Effects of Disturbed Flow on ECs 490
19.6 Summary and Conclusions 492
References 495
20 Why is Sepsis an Ongoing Clinical Challenge? Lipopolysaccharide Effects on Red Blood Cell Volume 503
20.1 Introduction 504
20.2 Physiopathological Events During Sepsis 505
20.3 Markers in Clinical Diagnosis of Sepsis 505
20.4 Microcirculation and Sepsis 506
20.5 Therapy 506
20.6 Activated Protein C 507
20.7 Red Blood Cell Behaviour During Sepsis 507
20.8 New Perspective 508
References 513
21 Bioengineering of Inflammation and Cell Activation: Autodigestion in Shock 515
21.1 Introduction 516
21.2 Inflammation in Shock and Multi-Organ Failure 517
21.3 The Pancreas as a Source of Cellular Activating Factors and the Role of Serine Proteases 518
21.4 Blockade on Pancreatic Digestive Enzymes in the Lumen of the Intestine 519
21.5 What Mechanisms Prevent Auto-digestion? 521
21.6 Triggers of Shock Increase Intestinal Wall Permeability 521
21.7 Intestine as Source of Inflammatory Mediators in Shock 522
21.8 Characterization of Protease-Derived Shock Factors 522
21.9 Cytotoxic Factors Derived from the Intestine 524
21.10 Removal or Blockade of Intestinal Cytotoxic Mediators 525
21.11 Conclusions 526
References 528
22 Percutaneous Vertebroplasty: A Review of Two Intraoperative Complications 531
22.1 Introduction 531
22.2 Vertebroplasty: Minimally Invasive and Cost- Effective Solution 532
22.3 Extravertebral Biomechanics: Excessive Delivery Pressure 533
22.4 Intravertebral Biomechanics: Risk of Extravasation 535
22.5 Injectable Biomaterials 537
22.6 Discussion 538
References 540
Part VI Plant and Microbial Bioengineering 543
23 Molecular Crowding: AWay to Deal with Crowding in Photosynthetic Membranes 544
23.1 In the Crowd 544
23.2 Macromolecular Crowding 546
23.3 Photosynthesis in a Crowded Environment 553
23.4 Crowding Effects in Photosynthetic Membranes 559
23.5 Summary and Outlook 571
References 575
24 Higher Plants as Bioreactors. Gene Technology with C3- Type Plants to Optimize CO2 Fixation for Production of Biomass and Bio- Energy 580
24.1 Introduction 580
24.2 The C3 and C4 CO2 Fixation Mechanisms 582
24.3 The Metabolism of Glycolate in Escherichia coli and in Some Green Algae 586
24.4 Increased Biomass Production in Transgenic Arabidopsis Plants Containing the E. coli Glycolate Pathway in the Chloroplasts 24.4.1 The Strategy to Improve CO2 Fixation and Hence Photosynthesis 588
24.4.2 Establishment of the E. coli Glycolate Pathway in Arabidopsis Chloroplasts 589
24.5 Analysis of the GT-DEF Transgenic Plants. DNA, RNA, Proteins, Physiology, Growth and Production of Biomass 593
24.6 Summary 596
References 598
25 Controlling Microbial Adhesion: A Surface Engineering Approach 600
25.1 The LostWorld of Sessile Microorganisms 600
25.2 Biotechnological Potential of Adhered Microorganisms and Its Limitations 603
25.3 Physicochemical Aspects of Microbial Adhesion 606
25.4 Biological Aspects of Microbial Adhesion 608
25.5 Surface Conditioning as a Tool FacilitatingMicrobial Adhesion 611
References 620
26 Air Purification Technology by Means of Cluster Ions Generated by Plasma Discharge at Atmospheric Pressure 623
26.1 Ion Generating Device 623
26.2 Characteristics of Positive and Negative Ions 624
26.3 Effect of Removing Airborne Bacteria 625
26.4 Effect of Removing Floating Fungi (Mould) 628
26.5 Effect of Deactivating Floating Viruses 629
26.6 Virus Deactivation Model Using Cluster Ions 631
26.7 Allergen Deactivation Effect 632
26.8 Conclusion 635
References 636
27 Astrobiology 637
27.1 Introduction 637
27.2 Origin and History of Life on Earth 638
27.3 Impact Scenario and Interplanetary Transport of Life 642
27.4 Strategies of Life to Adapt to Extreme Environments 643
27.5 Signatures of Life 645
27.6 Criteria for Habitability 647
27.7 Planets and Moons in Our Solar System That are of Interest to Astrobiology 649
27.8 Planetary Protection 654
27.9 Search for Life Beyond the Solar System 657
27.10 Outlook 658
References 660
Authors 662
Index 676

Chapter 1 Reporter Genes in Cell Based ultra High Throughput Screening (p. 3-4)

Stefan Golz

Bayer Healthcare AG, Institute for Target Research, 42096Wuppertal, Germany, stefan.golz@bayerhealthcare.com

Abstract Pharma research in most organizations is organized in discrete phases together building a "value chain" along which discovery programs process to fi- nally drug candidates for clinical testing. The process envisioned to identify targetspecific modulators lacking several side effects. Following a technical assessment of the targets "drugability", the probability to identify small molecule modulators, and technical feasibility target-specific assays are developed to probe the corporate compound collection for meanful leads. "High-Throughput-Screening" (HTS) started roughly one decade ago with the introduction of laboratory automation to handle the different assay steps typically performed in microtiter plates. Today a large arsenal of screening technologies is available for researchers in industry and academia to set up uHTS or HTS assays. Here the use of reporter genes offer an alternative for following signal transduction pathways from receptors at the cell surface to nuclear gene transcription in living cells.

1.1 Introduction

The modern drug research process has reversed the classical pharmacological strategy. Today, research programs are initiated based on biological evidence suggesting a particular gene or gene product to be a meaningful target for small molecule drugs useful for therapies. The process envisioned to identify target-specific modulators lacking several side effects. Also, it allows setting up a linear drug discovery process starting from target identification to finally delivering molecules for clinical development. One central element is lead discovery through high-throughput screening of comprehensive corporate compound collections. Pharma research in most organizations is organized in discrete phases together building a "value chain" along which discovery programs process to finally drug candidated for clinical testing (Hüser et al. 2006).

This pipeline is fueled by targets suggested from external or in-house generated data suggesting a gene or gene product to be disease relevant. Today a large arsenal of technologies is available for researchers in industry and academia to generate data in support of a functional link between a given gene and a disease state.

1.2 From Gene to Target

Following a technical assessment of the targets "drugability" (Hopkins and Groom, 2002), the probability to identify small molecule modulators, and technical feasibility target-specific assays are developed to probe the corporate compound collection for meanful leads. Lead discovery in the pharmaceutical industry today still depends largely on experimental screening of compound collections. To this end, the industry has invested heavily in expanding their compound files and established appropiate screening capabilities to handle large numbers of compounds within a reasonable period of time. "High-Troughput-Screening" (HTS) started roughly one decade ago with the introduction of laboratory automation to handle the different assay steps typically performed in microtiter plates. HTS technologies during the last decade have witnessed remarkable developments. Assay technologies have advanced to provide a large variety of various cell-based and biochemical test formats for a large spectrum of disease relevant target classes (Walters and Namchuk, 2003). In parallel, further miniaturization of assays volumes and parallelization of processing have further increased the test throughput. The ultra-high-throughput is required to fully exploit big compound files of >,1million compounds and is performed entirely in 1536-well plates with assay volumes between 5 – 10 ìl. This assay carrier together with fully-automated robotic systems allow for testing in excess of 200,000 compounds per day. The comprehensive substance collection, together with sophisticated screening technologies, have resulted in a clear advantages in lead discovery especially for poorly druggable targets with a poor track record in the past.