||Preliminary Development of an Integrated Microchip for Multipurpose Caenorhabditis elegans Studies
||Department of BioMedical Engineering
Caenorhabditis (C.) elegans
Ultraviolet (UV) light
Pluronic (P) F-127
Despite prolonging the life of humans, the number of people suffering from neurodegenerative diseases and cancer increases significantly. Therefore, postponing aging to decrease the incidence of diseases and provide better prognosis is important. Given its high similarity with humans in terms of genes, the multicellular organism Caenorhabditis elegans is the simplest model animal widely used in many research fields, such as neurology, genetic engineering, developmental biology, and pharmaceutics. However, research on the nematode still requires painstaking operations, thus reducing the throughput and efficiency. To address the problem, an integrated microchip was proposed in this study as a solution. This study is divided into three parts, namely, worm exercise, cancer screening, and immobilization.
Regardless of whether microsurgery, fluorescent imaging, or physiological observation is used in this tiny organism, immobilization is an essential step. However, minimal work has been performed. Hence, immobilization is an essential function fabricated on microchips. An immobilization technique based on the combined use of an optoelectric device and a 20% w/v thermos-reversible hydrogel solution, Pluronic F-127, was developed first. Second, the optoelectric device was coated with a photoconductive layer to allow the local circuit channels to be rapidly switched by optical illumination. After simultaneously applying light and electric fields under optimal conditions, the hydrogel reached gelation within 4 s, and the immobilized C. elegans appeared to resume its full locomotion within 1 s after the light was switched off. The gelation region and location could be manipulated by changing the laser size and illuminated region. According to the assessments, worms should not be exposed to the hydrogel environment for more than 3 h. Given the thermo-reversible property of PF-127, the sample was also conserved in the entire experiment. Aside from C. elegans, this technique can be applied to other microorganisms.
Exercise not only makes an organism more energetic during the aging process; it can also postpone the occurrence of degenerative diseases. Short-wavelength light elicits a photophobic, movement-reversal response from C. elegans. Rather than using the electrotaxis method, ultraviolet light was introduced to keep C. elegans swimming to achieve the effect of exercise. Therefore, the design of the channel was simplified significantly. After a 4 min delay in the droplet, worms that received stimuli of 5 s UV light every 2 min for 8 min accomplished 20 min of continuous exercise. Cancer is another issue linked with human aging. We placed C. elegans in the supernatant of Caco-2 and HeLa cell line culture medium separately to examine the biomechanical performance of C. elegans. Worms in the cancer cell culture medium tended to swim oddly instead of the normal forward movement. The body bend frequency and unit kinetic power decreased significantly. The results contribute to the highly developed chemosensory system of C. elegans. Therefore, using C. elegans as a sensor for cancer screening might help in the early diagnosis and successful treatment of the disease.
The proposed integrated chip can be subsequently performed for multipurpose analyses. Thus, the mechanism by which C. elegans reacts to the secretion of cancer cells and its relationship with exercise, antioxidants, and aging must be determined. The results are expected to provide information on the treatment of degenerative diseases and cancer in higher animal forms.
LIST OF TABLES VIII
LIST OF FIGURES IX
CHAPTER 1 INTRODUCTION 1
1.1 Background 1
1.2 Motivation and Purpose 3
1.3 Caenorhabditis (C.) elegans 4
1.4 Pluronic F-127 5
CHAPTER 2 MATERIALS AND METHODS 7
2.1 Flow Chart of Research Process 7
2.2 Basics of C. elegans 8
2.2.1 Strains and Growth Condition of C. elegans 8
126.96.36.199 Wild-Type Worm 8
188.8.131.52 Transgenic Worm 8
2.2.2 C. elegans Culture Media Protocols 9
2.2.3 Age-Synchronized Method 9
2.2.4 Growth Assay 10
2.2.5 Progeny Assay 10
2.2.6 Lifespan Assay 10
2.2.7 Statistical Analysis 11
2.3 Integrated Microchip 11
2.3.1 Fabrication of the Integrated Microchip 11
2.4 Worm Immobilization 12
2.4.1 Preparation of PF-127 12
2.4.2 Immobilization Device and Operation 13
2.4.3 Image Processing and Analysis 14
184.108.40.206 Kymogram of Body Curvature 15
220.127.116.11 Gait Correlation Diagram 16
18.104.22.168 Trajectories 17
2.4.4 Water Bathing Technique 18
2.4.5 Stress Response Assay 18
2.4.6 Temperature Measurement 19
2.5 Exercise 20
2.5.1 Photophobic Behavior of C. elegans 20
2.5.2 Ultraviolet Light Stimulation 21
2.5.3 Measurement of the Worm’s Body Bends 21
2.6 Cancer Screening 22
2.6.1 Locomotive Gaits of C. elegans 22
2.6.2 Preparation of Culture Media from Cancer Cells 23
2.6.3 Behavior Analysis of C. elegans 24
2.6.4 Measurement of Kinetic Power 24
CHAPTER 3 RESULTS AND DISCUSSION 26
3.1 Integrated Microchip 26
3.1.1 Operation 26
3.2 Worm Immobilization 27
3.2.1 Effect of Long Exposure to PF-127 27
3.2.2 Exposure Time Evaluation 27
3.2.3 Assessment of Worm Immobilization 30
3.2.4 Addressable Immobilization 32
3.2.5 Long-term Imaging of Worm Senescence Process 34
3.3 Exercise 36
3.3.1 Effect of the Stimulation by Ultraviolet Light 37
3.3.2 Reproducibility of Photophobic Behavior 38
3.4 Cancer Screening 40
3.4.1 Gait Evaluation 40
3.4.2 Kinetic Power 42
CHAPTER 4 CONCLUSION 44
CHAPTER 5 PROSPECTS 46
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