Improvement of In Vitro Fertilization (IVF) Technology through Microfluidics.

Despite advances in in vitro manipulation of pre-implantation embryos, there is still a lag in the quality of embryos produced in vitro leading to lower pregnancy rates compared to embryos produced in vivo. Reducing the incidence of high-order multiple pregnancies while maintaining the overall in vitro fertilization (IVF) success rate is a holy grail of human IVF and would be greatly assisted by the ability to produce and identify the highest quality embryos. A promising new technology, microfluidics, does exist and is becoming increasingly studied. A challenge of studying embryo on microfluidic device is that preimplantation mouse embryos are highly sensitive cells and their development is affected greatly by osmolality shifts as will occur in devices with thin poly(dimethylsiloxane) (PDMS) membranes even in typical humidified cell culture incubators. Here we characterized and resolved evaporation mediated osmolity shifts that constrained microfluidic cell culture in Poly(dimethylsiloxane) devices. Next, we developed a dynamic microfunnel embryo culture system would enhance outcomes by better mimicking the fluid mechanical stimulation and chemical agitation embryos experience in vivo from ciliary currents and oviductal contractions. Using a mouse embryo model, average cell counts for blastocysts after 96 hours of culture in dynamic microfunnel conditions increased 70% over that of conventional static cultures. Importantly, the dynamic microfunnel cultures significantly improved embryo implantation and ongoing pregnancy rates over static culture to a level that approached that of in utero-derived preimplantation embryos. Lastly, we reported a new computerized microfluidic real time embryo culture and assay device that can perform automated periodic analyses of embryo metabolism over 24 hrs. Biochemical methods for embryo analysis based on measurement of metabolic rates do exist, but are not practical for clinical use because of difficulties in manipulating precise amounts of sample and reagents at the sub-microliter scale. The convenient, non-vasive, reliable, and automated nature of these assays open the way for development of practical single embryo biochemical analysis systems. Collectively, these results confirm that microfluidic technology can be used to properly mimic a broad range of the embryo environments seen in physiology and to assess embryo viability for in vitro fertilization clinics.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/61666/1/yunsheo_1.pd

Microfluidic Reduction of Osmotic Stress in Oocyte and Zygote Vitrification.

Microfluidic cryoprotectant exchange enables vitrification of murine zygotes with superior morphology as indicated by a smoother cell surface and higher developmental competence compared to conventional methods. Bovine oocyte vitrification also benefit as evidenced by higher lipid retention. Experimental observations and mathematical analysis demonstrate that the microfluidic advantage arise predominantly from eliminating high shrinkage rates associated with abrupt and uneven exposure to vitrification solutions that readily occur in current manual protocols. The microfluidic cryoprotectant exchange method described has immediate applications for improving animal and human oocyte, zygote, and embryo cryopreservation. On a fundamental level, the clear demonstration that at the same minimum cell volume, cell shrinkage rate affects sub-lethal damage should be broadly useful for cryobiology.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/107056/1/davlai_1.pd

AN ION SELECTIVE MICROGRIPPER SENSOR DEVICE

This thesis presents the design, fabrication, characterisation and testing of a chemically modified electrothermally actuated microgripper. The chemical modification involves the integration of a potentiometric ion selective electrode (ISE) onto a bare electrode fabricated within the tip of the microgripper. This microgripper sensor device is intended for use in the application of detecting, in real time, the movement of key ions that are involved in intercellular communication from a mechanically stressed cell. An optimised fabrication route for the specifically designed microgrippers, which have tip dimensions of 10 – 60 µm, is described in detail. The fabrication route delivers a high yield (95%) of operational unmodified devices. An 1800 ± 20 µm2 bare gold electrode that is fabricated at the tip of the microgripper is modified into an all solid state ISE that uses PEDOT as the ion-to-electron solid contact. Suitable ionophores that selectively detect K+, Na+ and Ca2+ are used to fabricate potassium, sodium and calcium ion selective microgripper sensor devices. The quality control and testing characteristics that follow the guidelines defined by IUPAC are performed to ascertain the sensitivity, selectivity and stability of the microgripper sensor devices. Good selectivity is achieved, with limits of detection of 2.4 x 10-4 M, 1.8 x 10-4 M and 2.0 x 10-5 M for the K+, Na+ and Ca2+ devices respectively. Proof of concept experiments of the real life testing of the K+ ISE device used to mechanically stress mouse oocytes gave preliminary measurements that indicate that stress signalling occurs via a switch on mechanism, and that there is a small increase in K+ concentration as applied stress increases. Due to the high systematic error within the calibration process the magnitude of this concentration increase is unknown. The Na+ and Ca2+ ISE devices suffer from interference and sensitivity restrictions respectively so a signal response vs. applied cell stress relationship of these ions is currently unobtainable

Lab-on-a-chip technologies for manipulation and imaging of C. elegans worms and embryos

The nematode Caenorhabditis elegans is an attractive model organism, owing notably to its short life cycle, genetic tractability, and optical transparency facilitating microscopic observation. This thesis deals with the realization of technological tools for the manipulation of worms and for studying thereby biologically relevant questions. The novel microfluidic devices that were developed are: #1 A microfluidic approach for size-dependent sorting of C. elegans nematodes on-chip. We take advantage of the external pressure-deformable profile of polydimethylsiloxane (PDMS) transfer channels that connect two on-chip worm chambers. The pressure-controlled effective cross-section of these channels creates adjustable filter structures that can be easily tuned for a specific worm sorting experiment, without changing the design parameters of the device itself. Considering that our sorting device is merely based on geometrical parameters and operated by simple fluidic and pressure control, we believe that it has strong potential for further use in advanced, automated, microfluidic C. elegans-based assay platforms. #2 A microfluidic device for studying signaling via diffusive secreted compounds between two specific C. elegans populations over prolonged durations. In particular, we designed a microfluidic assay to investigate the biological process of male-induced demise, i.e. lifespan shortening, in C. elegans hermaphrodites in the presence of a physically separated male population. For this purpose, male and hermaphrodite worm populations were confined in adjacent microchambers on the chip, whereas molecules secreted by males could be exchanged between both populations by periodically activating controlled fluidic transfer of ÎŒl-volume aliquots of male-conditioned medium. For male-conditioned hermaphrodites, we observe a reduction in mean lifespan of 4 days compared to non-conditioned on-chip culture. #3 Development of two reversible C. elegans immobilization methods for imaging applications. The first immobilization method takes advantage of a biocompatible and temperature-responsive hydrogel-microbead matrix. Our gel-based immobilization technique does not require a specific chip design and enables fast and reversible immobilization, thereby allowing successive imaging of the same single worm or of small worm populations at all development stages for several days. The second immobilization method takes advantage of the elastic properties of PDMS. We present two distinct microdevices, namely a micropillar array and a serpentine microchannel, for on-chip feeding and high-resolution imaging studies, respectively. Both devices consist of size-tunable PDMS structures that allow the same chips to be used for immobilization of worms at all development stages. Our microfluidic approach provides appropriate physiological conditions for long-term studies and enables worm recovery after the experiment. #4 A fully integrated microfluidic approach for the exploration of C. elegans early embryogenesis including the possibility of testing small-molecule inhibitors with increased throughput and versatility. Here, up to 100 embryos can be immobilized in parallel for simultaneous high-resolution time-lapse imaging of embryonic development from the 1-cell stage to hatching. We demonstrate time-controlled and reversible drug delivery to on-chip immobilized embryos, which is of relevance for biochemical and pharmacological assays

Microfluidic tools for metabolomics

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008.Includes bibliographical references (p. 153-160).A primary challenge in embryology is to understand the factors that govern the development of preimplantation (PI) embryos and how these factors relate to embryo viability in the field of in vitro fertilization (IVF). This is particularly important as clinical policy moving towards single embryo transfer (SET) has gained awareness to manage unprecedented numbers of multiple births, such as twins and triplets, resulting from artificial reproductive techniques. Conditions that correlate with developmental potential of candidate embryos are disputed in the field, however, as the requisite data is difficult to obtain.The metabolic profiles of embryos during in vitro culture have been suggested as a key indicator of developmental potential, and approaches have been clinically implemented to select transfer candidates which make the most efficient use of nutrients. Existing microdroplet analysis techniques are accurate and suitable for non-invasive assessment of single embryos. Unfortunately, the process of determining metabolite levels in nanoliters of culture media through fluorometric assays is low-throughput and requires specialized expertise, hindering widespread clinical use of these methods. The goal of this thesis is to develop microfluidics-based approaches for improving metabolic analysis of PI embryos and mammalian cells. This challenge necessitates two competencies: methods for automating chemical assays and methods for supporting cell cultures, which can be integrated with analysis. Contributions include a standalone platform for determining the metabolite use of single embryos. Profiles may be acquired automatically, which reduces significant technician hours and improves repeatability. Techniques are developed for performing embryo culture in the smallest culture volumes to date in microfabricated environments. Microfluidic approaches have enabled culture that outperforms the current state of art approach based on cell count measurements.(cont.) An integrated system is introduced, merging analysis and culture competencies to perform metabolic profiling of separate cultures of mammalian cells in parallel. Finally, new paradigms in microfluidic design are presented based on the concept of vertically integrated architectures, suitable for overcoming density limitations of microfluidic assays. A scalable analysis platform for refining embryo selection has been long warranted and would enable pursuit of the difficult questions relating metabolism and embryo viability as the clinical movement towards SET continues.by John Paul Urbanski.Ph.D

Micromachines for Dielectrophoresis

An outstanding compilation that reflects the state-of-the art on Dielectrophoresis (DEP) in 2020. Contributions include: - A novel mathematical framework to analyze particle dynamics inside a circular arc microchannel using computational modeling. - A fundamental study of the passive focusing of particles in ratchet microchannels using direct-current DEP. - A novel molecular version of the Clausius-Mossotti factor that bridges the gap between theory and experiments in DEP of proteins. - The use of titanium electrodes to rapidly enrich T. brucei parasites towards a diagnostic assay. - Leveraging induced-charge electrophoresis (ICEP) to control the direction and speed of Janus particles. - An integrated device for the isolation, retrieval, and off-chip recovery of single cells. - Feasibility of using well-established CMOS processes to fabricate DEP devices. - The use of an exponential function to drive electrowetting displays to reduce flicker and improve the static display performance. - A novel waveform to drive electrophoretic displays with improved display quality and reduced flicker intensity. - Review of how combining electrode structures, single or multiple field magnitudes and/or frequencies, as well as variations in the media suspending the particles can improve the sensitivity of DEP-based particle separations. - Improvement of dielectrophoretic particle chromatography (DPC) of latex particles by exploiting differences in both their DEP mobility and their crossover frequencies

Study of the influence of a DC electric field on the development of the embryo of the nematode Caenorhabditis elegans

Tese de mestrado em Engenharia Biomédica e Biofísica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2012A bioelectricidade pode influenciar a polarização de uma célula, responsável pela divisão assimétrica, pelo que tem um impacto significativo no desenvolvimento de tecidos ao contribuir para controlar a migração e orientação de células e também a proliferação, diferenciação e apoptose celulares. Estas propriedades da bioelectricidade em tecidos biológicos tornam-na numa ferramenta importante em engenharia de tecidos e medicina regenerativa, pois permite controlar várias respostas fisiológicas a estímulos eléctricos e usá-las para fins benéficos. O nematóide Caenorhabditis elegans é um organismo primitivo, mas cuja fisiologia partilha várias características da biologia humana. Estas características, juntamente com a sua simplicidade de cultivar em grandes populações e de ser conveniente para análise e manipulação genética, tornam-no num dos organismosmodelo mais vastamente utilizados em investigação científica, existindo actualmente uma enorme quantidade de informação disponível sobre este nematóide. O embrião do C. elegans representa um modelo ideal para o estudo da divisão celular e da embriogénese, visto que o seu desenvolvimento é altamente reprodutível e facilmente observável. Além disso, o padrão de desenvolvimento do embrião é praticamente invariável, tornando possível construir um diagrama que representa toda a sua linhagem celular, o que facilita imensamente a análise da embriogénese. A motivação deste projecto surge do conhecimento de que vários seres vivos, como a hidra ou a salamandra, possuem uma grande capacidade de regeneração que está grandemente relacionada com a bioelectricidade. Compreender e controlar os seus mecanismos será certamente um grande avanço na área de engenharia de tecidos e terá como consequência o surgimento de diversas potenciais aplicações. Este projecto tem como objectivo aplicar a aplicação de um campo eléctrico DC num organismo vivo, de forma a induzir uma resposta por parte dele. Devido às suas características, o embrião do nematóide C. elegans foi escolhido como organismo modelo para a aplicação deste estímulo eléctrico. A principal assumpção é que o campo eléctrico aplicado ao longo do eixo antero-posterior do embrião do nematóide C. elegans, induza uma acumulação de carga oposta nas suas extremidades anterior e posterior com o intuito de alterar o potencial de membrana, onde se levantou a hipótese de poderem induzir respostas celulares diferentes em cada extremidade, causando alterações na embriogénese. A ideia surge de uma analogia à aplicação de um gradiente de temperatura ao longo desse mesmo eixo, sabendo-se actualmente que induz uma resposta relativamente à duração de ciclos celulares, visto que é fortemente dependente da temperatura. Através dos resultados, pretende-se também avaliar a utilidade deste embrião como um organismo-modelo para o estudo do impacto de campos eléctricos DC em tecido vivo, visto que outros embriões, como de anfíbios ou pintos, apresentam grandes distúrbios no seu desenvolvimento quando estimulados com campos eléctricos contínuos, com consequências catastróficas no organismo final. A tecnologia dos micro-sistemas permite controlar eficazmente o ambiente celular de uma forma bastante precisa. Assim, um sistema microfluídico é usado como plataforma para estudar o impacto da aplicação do campo eléctrico ao embrião. Este é colocado no interior de um microcanal contendo PBS (tampão fosfato-salino), um bom electrólito e ao mesmo tempo biocompatível para o seu desenvolvimento. Recorrendo a eléctrodos de platina, é aplicada uma diferença de potencial nas extremidades do canal, o que torna possível a existência de uma corrente eléctrica que deverá resultar numa acumulação de carga oposta nas extremidades do embrião, visto que a sua resistividade deverá ser superior à do PBS. A largura e altura do canal são semelhantes à espessura do embrião, o que garante que o campo eléctrico passa através do embrião ou que seja desviado por ele, no caso de este ser isolante. Tendo em conta que o campo eléctrico propaga-se através do canal em direcção ao embrião, é necessário saber qual a sua magnitude à volta dele. Além disso, interacções físicas como o efeito de Joule e reacções redox entre os eléctrodos e o electrólito podem alterar a temperatura e o pH no interior do canal e comprometer o desenvolvimento do embrião de forma indesejada. Assim é também efectuada uma caracterização física detalhada do sistema microfluídico, com o intuíto de saber quais os principais fenómenos físicos que podem ocorrer no interior do canal durante a aplicação do campo eléctrico. Esta caracterização permite tomar as medidas necessárias para minimizar estes efeitos secundários que possam perturbar o normal desenvolvimento do embrião. A caracterização física do sistema consiste essencialmente numa análise das interacções dos eléctrodos de platina com uma solução de cloreto de sódio, o principal constituinte do PBS e em determinar a relação entre a corrente eléctrica no canal e a diferença de potencial aplicada. Para isso são efectuadas medições experimentais e simulações recorrendo ao software de elementos finitos COMSOL Multiphysics. O procedimento experimental para a análise do impacto do campo eléctrico no embrião começa com a recolha de um embrião de um nematóide hermafrodita adulto, proveniente de uma cultura em pratos de agarose semeados com Escherichia coli como fonte de alimento. O embrião escolhido deve estar no início do desenvolvimento, de preferência após a meiose, de forma a poder ser estimulado o mais cedo possível. Sabese que perturbar os eventos iniciais da embriogénese pode ter consequências em todo o restante desenvolvimento, visto que este depende grandemente das primeiras divisões celulares. Recolhido o embrião, este é inserido no microcanal, onde é fixado e permanece durante todo o ensaio experimental. De seguida, o campo eléctrico é aplicado através da aplicação de uma diferença de potencial nos eléctrodos em contacto com as extremidades do canal. Pontes salinas de agarose são utilizadas em algumas experiencias para evitar a contaminação do electrólito com produtos das reacções químicas que ocorrem na superfície dos eléctrodos. O desenvolvimento do embrião é observado através de microscópios invertidos equipados com contraste de fase. Imagens de time-lapse são capturadas e posteriormente analisadas recorrendo ao software ImageJ. Eventos da embriogénese seleccionados para análise são comparados para embriões em experiencias de controlo e embriões estimulados com o campo eléctrico. Métodos de análise estatística são efectuados para auxiliar a comparação. Parâmetros a observar e comparar são os tempos de ocorrência dos eventos seleccionados e orientação e posição de células no embrião. O comportamento da minhoca juvenil após o nascimento também pode fornecer informação relativamente à embriogénese, pelo que também é analisado. A caracterização do chip revelou que uma tensão de 5V é apropriada para criar um campo eléctrico com dimensões fisiológicas (cerca de 0.3mV/μm), não aumentando a temperatura para valores fora dos limites de desenvolvimento normal do embrião. Alterações do pH foram minimizadas através da utilização das pontes salinas inseridas nas entradas do canal. A comparação entre embriões de controlo e embriões estimulados revela que não existem grandes diferenças entre os eventos da embriogénese de embriões de controlo e embriões estimulados pelo campo eléctrico, o que sugere que o embrião resiste ao campo eléctrico, talvez devido à barreira de permeabilidade presente na sua carapaça. Pequenas diferenças nos tempos embriológicos entre embriões de controlo e estimulados sugerem que o campo eléctrico possa ter efeitos a longo prazo na embriogénese, retardando os seus eventos mais tardios. As diferenças são demasiado pequenas para se poder tirar alguma conclusão, contudo, os resultados sugerem que deveria ser feita uma análise mais detalhada ao ciclo celular de certas células do início da embriogénese, o que não foi possível com o equipamento disponível. Comparando estes resultados com os de outros estudos com embriões, como por exemplo em anfíbios ou pintos, conclui-se que o impacto do um campo eléctrico DC no desenvolvimento do embrião do nematóide C. elegans não é tão significativo como nesses modelos, o que os torna preferíveis para estudos com embriões intactos. No entanto, existe ainda a possibilidade de estudos a nível de células isoladas extraídas do embrião e também não é de descartar a hipótese de aplicar um campo eléctrico AC ou de tentar remover a carapaça do embrião em futuras investigações.Bioelectricity has an impact on the development of tissues because it can influence cell polarization, essential for asymmetric cell division. This feature may be an important tool for tissue engineering and regenerative medicine applications. The nematode Caenorhabditis elegans is a primitive organism, but whose physiology shares several characteristics of human biology. Its embryo is an ideal model for the study of cell division and embryogenesis, since its development is almost invariant and highly reproducible and readily observable. This project has the objective of studying the impact of a DC electric field in the C. elegans embryo development and to assert if this organism is a good model for further research in this field. To accomplish it, a DC electric field is applied in a microchannel filled with PBS, where to the embryo is confined. Embryogenesis is studied by analysing selected development stages, which are compared for control and electric field experiments. To optimize the application of the electric field to the embryo and minimize other physical phenomena which can disturb embryogenesis, a detailed physical characterization of the microfluidic system is also performed. The results show that there are no big differences in the events of embryogenesis, suggesting that the embryo resists to the electrode field, perhaps due its eggshell. Nevertheless, small differences in embryological times suggest that the electric field can have long term effects on embryogenesis, and that a more detailed analysis should be performed. In summary, the embryo nematode C. elegans is not as suited to study the impact of DC electric fields in embryogenesis as other embryo models, such as amphibians. However, there is still the possibility of studies of single isolated embryo cells. Besides, it is not discarded the possibility of applying an AC electric field or attempting to remove the eggshell in future studies

Custom-Designed Biohybrid Micromotor for Potential Disease Treatment

Micromotors are recognized as promising candidates for untethered micromanipulation and targeted cargo transport. Their future application is, however, hindered by the low efficiency of drug encapsulation and their poor adaptability in physiological conditions. To address these challenges, one potential solution is to incorporate micromotors with biological materials as the combination of functional biological entities and smart artificial parts represents a manipulable and biologically friendly approach. This dissertation focuses on the development of custom-designed micromotors combined with sperm and their potential applications on targeted diseases treatment. By means of 2D and 3D lithography methods, microstructures with complex configurations can be fabricated for specific demands. Bovine and human sperm are both for the first time explored as drug carriers thanks to their high encapsulation efficiency of hydrophilic drugs, their powerful self-propulsion and their improved drug-uptake relying on the somatic-cell fusion ability. The hybrid micromotors containing drug loaded sperm and constructed artificial enhancements can be self-propelled by the sperm flagella and remotely guided and released to the target at high precision by employing weak external magnetic fields. As a result, micromotors based on both bovine and human sperm show significant anticancer effect. The application here can be further broadened to other biological environments, in particular to the blood stream, showing the potential on the treatment of blood diseases like blood clotting. Finally, to enhance the treatment efficiency, in particular to control sperm number and drug dose, three strategies are demonstrated to transport swarms of sperm. This research paves the way for the precision medicine based on engineered sperm-based micromotors