Combining inertial and deterministic lateral displacement (DLD) microfluidic sorting to isolate m... more Combining inertial and deterministic lateral displacement (DLD) microfluidic sorting to isolate mesenchymal stem cells (MSCs) from human adipose tissue for regenerative medicine applications.
All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell throug... more All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsion...
2009 IEEE/LEOS International Conference on Optical MEMS and Nanophotonics, 2009
In this presentation, we will report on our research progress on the optofluidic microscope (OFM)... more In this presentation, we will report on our research progress on the optofluidic microscope (OFM). The OFM method uniquely combines optics and microfluidic technology to enable low cost and highly compact microscope systems. The OFM does not use any lens elements and yet is capable of resolution comparable to a conventional microscope. We demonstrate the use of the microscope for C. elegans, pollens and Giardia micro-organisms with sub-micron resolution. OFM devices can potentially be made at low cost and massive ...
changes in nucleosomes. Our immediate goal is to measure the structural changes in single nucleos... more changes in nucleosomes. Our immediate goal is to measure the structural changes in single nucleosomes and nucleosome arrays in response to transcription factor (TF) binding. Initial experiments using the nucleosomecaliper construct with GAL4-VP16, a hybrid transcription factor capable of highly-efficient transcription activation, shows that the angular distribution broadens as a result of increasing TF concentrations. Our measurements revealed a dissociation constant in the range of 1-10nM, which agrees with bulk measurements. These results demonstrate the potential of a DNA origami device probing chromatin structure and function in vitro, in particular, the ability to measure structural changes in the range of 10-100nm.
Micropipette aspiration is arguably the most classical technique in mechanical measure-ments and ... more Micropipette aspiration is arguably the most classical technique in mechanical measure-ments and manipulations of single cells. Despite its simplicity, micropipette aspiration has been applied to a variety of experimental systems that span different length scales to study cell mechanics, nanoscale molecular mechanisms in single cells, bleb growth, and nucleus dynamics, to name a few. Enabled by micro/nanotechnology, several novel microfluidic devices have been developed recently with better accuracy, sensitivity, and throughput. Further technical advancements of microfluidics-based micropipette aspira-tion would have broad applications in both fundamental cell mechanics studies and for disease diagnostics. [DOI: 10.1115/1.4029936]
Abstract Micro-fluidic devices have recently received significant attention since many applicatio... more Abstract Micro-fluidic devices have recently received significant attention since many applications in biotechnology involve manipulation of small objects, eg DNA or protein molecules, in liquid media. Microchannel liquid flow requires large pressure to be driven. Hence, surface-driven flows such as electroosmotic flow, taking advantage of surface charge, are becoming the popular choice. Typical negative surface charge results in a plug flow in the direction of the applied electric field. Since both the velocity and the length ...
Metastatic cancer cells migrate through constricted spaces and experience significant compressive... more Metastatic cancer cells migrate through constricted spaces and experience significant compressive stress, but mechanisms enabling migration in confined geometries remain unclear. Cancer cell migration within confined 3-dimensional (3D) microfluidic channels has been shown to be distinct from 2D cell migration. However, whether 3D confined migration can be manipulated by mechanosensory components has not been examined in detail. In this work, we exogenously introduced a mechanosensitive channel of large conductance (MscL) into metastatic breast cancer cells MDA-MB-231. We discovered that inducing expression of a gain-of-function G22S mutant of MscL in MDA-MB-231 cells significantly reduced spontaneous lung metastasis without affecting the growth of orthotopic tumor implants. To further investigate the effects of G22S MscL on cell migration, we designed a microfluidic device with channels of various cross-sections ranging from a 2D planar environment to narrow 3D constrictions. Both MscL G22S and control breast cancer cells migrated progressively slower in more constricted environments. Migration of cells expressing MscL G22S did not differ from control cells, even though MscL was activated in cells in constricted channels of 3 lm width. Interestingly, we found MscL expressing cells to be more frequently "stuck" at the entrance of the 3 lm channels and failed to migrate into the microchannel. Our work demonstrates the possibility of engineering mechanotransduction for controlling confined cell migration.
Isolation of pure populations of mesenchymal stem cells from bone marrow aspirate is a critical n... more Isolation of pure populations of mesenchymal stem cells from bone marrow aspirate is a critical need in regenerative medicine such as orthopedic and cartilage reconstruction with important clinical and therapeutic implications.
Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hyper... more Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hypertrophic cardiomyopathy (HCM). Haploinsufficiency of full-length MYBPC3 and disruption of proteostasis have both been proposed as central to HCM disease pathogenesis. Discriminating the relative contributions of these 2 mechanisms requires fundamental knowledge of how turnover of WT and mutant MYBPC3 proteins is regulated. We expressed several disease-causing mutations in MYBPC3 in primary neonatal rat ventricular cardiomyocytes. In contrast to WT MYBPC3, mutant proteins showed reduced expression and failed to localize to the sarcomere. In an unbiased coimmunoprecipitation/mass spectrometry screen, we identified HSP70-family chaperones as interactors of both WT and mutant MYBPC3. Heat shock cognate 70 kDa (HSC70) was the most abundant chaperone interactor. Knockdown of HSC70 significantly slowed degradation of both WT and mutant MYBPC3, while pharmacologic activation of HSC70 and HSP70 ac...
Directed evolution has long been a key strategy to generate enzymes with desired properties like ... more Directed evolution has long been a key strategy to generate enzymes with desired properties like high selectivity, but experimental barriers and analytical costs of screening enormous mutant libraries have limited such efforts. Here, we describe an ultrahigh-throughput dual-channel microfluidic droplet screening system that can be used to screen up to ~10enzyme variants per day. As an example case, we use the system to engineer the enantioselectivity of an esterase to preferentially produce desired enantiomers of profens, an important class of anti-inflammatory drugs. Using two types of screening working modes over the course of five rounds of directed evolution, we identify (from among 5 million mutants) a variant with 700-fold improved enantioselectivity for the desired (S)-profens. We thus demonstrate that this screening platform can be used to rapidly generate enzymes with desired enzymatic properties like enantiospecificity, chemospecificity, and regiospecificity.
Various micro-engineered tools or platforms have been developed recently for cell mechanics studi... more Various micro-engineered tools or platforms have been developed recently for cell mechanics studies based on acoustic, magnetic, and optical actuations. Compared with other techniques for single cell manipulations, microfluidics has the advantages with simple working principles and device implementations. In this work, we develop a multi-layer microfluidic pipette aspiration device integrated with pneumatically actuated microfluidic control valves. This configuration enables decoupling of cell trapping and aspiration, and hence causes less mechanical perturbation on trapped single cells before aspiration. A high trapping efficiency is achieved by the microfluidic channel design based on fluid resistance model and deterministic microfluidics. Compared to conventional micropipette aspiration, the suction pressure applied on the aspirating cells is highly stable due to the viscous nature of low Reynolds number flow. As a proof-of-concept of this novel microfluidic technology, we built ...
Page 1. Optofluidic Microscopy: Chip-scale imaging cell cytometry Guoan Zheng, Seung Ah Lee, Sean... more Page 1. Optofluidic Microscopy: Chip-scale imaging cell cytometry Guoan Zheng, Seung Ah Lee, Sean Pang, Chao Han, Lapman Lee, Changhuei Yang Electrical Engineering Department, California institute of Technology, Pasadena CA 91125 Abstract We will discuss our recent work on chip-scale microscopy, including fluorescence and laser-scanning imaging techniques. The optofluidic microscope (OFM) represents a significant departure from the conventional microscope design in several significant ways. ...
The completion of my thesis is based on support and help from many individuals. First, I would li... more The completion of my thesis is based on support and help from many individuals. First, I would like to express my gratitude to my PhD advisor Prof. Changhuei Yang for offering me a chance to work on an emerging research field of optofluidics and participate in the development of optofluidic microscopy (OFM), which leads to many successful results. His guidance has led the OFM project from an elegant engineering idea to reality. His enthusiasm and creativity in conducting academic research is forever young, motivating us to pursue excellence in our projects. I thank him for introducing me to biophotonics and allowing me to play a role in contributing to the field. I want to thank Prof. Yu-Chong Tai for being my thesis committee chair. His pioneering work in MEMS has always been my motivation to pursue something ‘big ’ in the world of ‘small’. You will find yourself learning something new every time you interact with him, not only in science but also in life. I want to thank Prof. Chi...
Journal of nanotechnology in engineering and medicine, 2014
Micropipette aspiration is arguably the most classical technique in mechanical measurements and m... more Micropipette aspiration is arguably the most classical technique in mechanical measurements and manipulations of single cells. Despite its simplicity, micropipette aspiration has been applied to a variety of experimental systems that span different length scales to study cell mechanics, nanoscale molecular mechanisms in single cells, bleb growth, and nucleus dynamics, to name a few. Enabled by micro/nanotechnology, several novel microfluidic devices have been developed recently with better accuracy, sensitivity, and throughput. Further technical advancements of microfluidics-based micropipette aspiration would have broad applications in both fundamental cell mechanics studies and for disease diagnostics.
Combining inertial and deterministic lateral displacement (DLD) microfluidic sorting to isolate m... more Combining inertial and deterministic lateral displacement (DLD) microfluidic sorting to isolate mesenchymal stem cells (MSCs) from human adipose tissue for regenerative medicine applications.
All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell throug... more All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsion...
2009 IEEE/LEOS International Conference on Optical MEMS and Nanophotonics, 2009
In this presentation, we will report on our research progress on the optofluidic microscope (OFM)... more In this presentation, we will report on our research progress on the optofluidic microscope (OFM). The OFM method uniquely combines optics and microfluidic technology to enable low cost and highly compact microscope systems. The OFM does not use any lens elements and yet is capable of resolution comparable to a conventional microscope. We demonstrate the use of the microscope for C. elegans, pollens and Giardia micro-organisms with sub-micron resolution. OFM devices can potentially be made at low cost and massive ...
changes in nucleosomes. Our immediate goal is to measure the structural changes in single nucleos... more changes in nucleosomes. Our immediate goal is to measure the structural changes in single nucleosomes and nucleosome arrays in response to transcription factor (TF) binding. Initial experiments using the nucleosomecaliper construct with GAL4-VP16, a hybrid transcription factor capable of highly-efficient transcription activation, shows that the angular distribution broadens as a result of increasing TF concentrations. Our measurements revealed a dissociation constant in the range of 1-10nM, which agrees with bulk measurements. These results demonstrate the potential of a DNA origami device probing chromatin structure and function in vitro, in particular, the ability to measure structural changes in the range of 10-100nm.
Micropipette aspiration is arguably the most classical technique in mechanical measure-ments and ... more Micropipette aspiration is arguably the most classical technique in mechanical measure-ments and manipulations of single cells. Despite its simplicity, micropipette aspiration has been applied to a variety of experimental systems that span different length scales to study cell mechanics, nanoscale molecular mechanisms in single cells, bleb growth, and nucleus dynamics, to name a few. Enabled by micro/nanotechnology, several novel microfluidic devices have been developed recently with better accuracy, sensitivity, and throughput. Further technical advancements of microfluidics-based micropipette aspira-tion would have broad applications in both fundamental cell mechanics studies and for disease diagnostics. [DOI: 10.1115/1.4029936]
Abstract Micro-fluidic devices have recently received significant attention since many applicatio... more Abstract Micro-fluidic devices have recently received significant attention since many applications in biotechnology involve manipulation of small objects, eg DNA or protein molecules, in liquid media. Microchannel liquid flow requires large pressure to be driven. Hence, surface-driven flows such as electroosmotic flow, taking advantage of surface charge, are becoming the popular choice. Typical negative surface charge results in a plug flow in the direction of the applied electric field. Since both the velocity and the length ...
Metastatic cancer cells migrate through constricted spaces and experience significant compressive... more Metastatic cancer cells migrate through constricted spaces and experience significant compressive stress, but mechanisms enabling migration in confined geometries remain unclear. Cancer cell migration within confined 3-dimensional (3D) microfluidic channels has been shown to be distinct from 2D cell migration. However, whether 3D confined migration can be manipulated by mechanosensory components has not been examined in detail. In this work, we exogenously introduced a mechanosensitive channel of large conductance (MscL) into metastatic breast cancer cells MDA-MB-231. We discovered that inducing expression of a gain-of-function G22S mutant of MscL in MDA-MB-231 cells significantly reduced spontaneous lung metastasis without affecting the growth of orthotopic tumor implants. To further investigate the effects of G22S MscL on cell migration, we designed a microfluidic device with channels of various cross-sections ranging from a 2D planar environment to narrow 3D constrictions. Both MscL G22S and control breast cancer cells migrated progressively slower in more constricted environments. Migration of cells expressing MscL G22S did not differ from control cells, even though MscL was activated in cells in constricted channels of 3 lm width. Interestingly, we found MscL expressing cells to be more frequently "stuck" at the entrance of the 3 lm channels and failed to migrate into the microchannel. Our work demonstrates the possibility of engineering mechanotransduction for controlling confined cell migration.
Isolation of pure populations of mesenchymal stem cells from bone marrow aspirate is a critical n... more Isolation of pure populations of mesenchymal stem cells from bone marrow aspirate is a critical need in regenerative medicine such as orthopedic and cartilage reconstruction with important clinical and therapeutic implications.
Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hyper... more Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hypertrophic cardiomyopathy (HCM). Haploinsufficiency of full-length MYBPC3 and disruption of proteostasis have both been proposed as central to HCM disease pathogenesis. Discriminating the relative contributions of these 2 mechanisms requires fundamental knowledge of how turnover of WT and mutant MYBPC3 proteins is regulated. We expressed several disease-causing mutations in MYBPC3 in primary neonatal rat ventricular cardiomyocytes. In contrast to WT MYBPC3, mutant proteins showed reduced expression and failed to localize to the sarcomere. In an unbiased coimmunoprecipitation/mass spectrometry screen, we identified HSP70-family chaperones as interactors of both WT and mutant MYBPC3. Heat shock cognate 70 kDa (HSC70) was the most abundant chaperone interactor. Knockdown of HSC70 significantly slowed degradation of both WT and mutant MYBPC3, while pharmacologic activation of HSC70 and HSP70 ac...
Directed evolution has long been a key strategy to generate enzymes with desired properties like ... more Directed evolution has long been a key strategy to generate enzymes with desired properties like high selectivity, but experimental barriers and analytical costs of screening enormous mutant libraries have limited such efforts. Here, we describe an ultrahigh-throughput dual-channel microfluidic droplet screening system that can be used to screen up to ~10enzyme variants per day. As an example case, we use the system to engineer the enantioselectivity of an esterase to preferentially produce desired enantiomers of profens, an important class of anti-inflammatory drugs. Using two types of screening working modes over the course of five rounds of directed evolution, we identify (from among 5 million mutants) a variant with 700-fold improved enantioselectivity for the desired (S)-profens. We thus demonstrate that this screening platform can be used to rapidly generate enzymes with desired enzymatic properties like enantiospecificity, chemospecificity, and regiospecificity.
Various micro-engineered tools or platforms have been developed recently for cell mechanics studi... more Various micro-engineered tools or platforms have been developed recently for cell mechanics studies based on acoustic, magnetic, and optical actuations. Compared with other techniques for single cell manipulations, microfluidics has the advantages with simple working principles and device implementations. In this work, we develop a multi-layer microfluidic pipette aspiration device integrated with pneumatically actuated microfluidic control valves. This configuration enables decoupling of cell trapping and aspiration, and hence causes less mechanical perturbation on trapped single cells before aspiration. A high trapping efficiency is achieved by the microfluidic channel design based on fluid resistance model and deterministic microfluidics. Compared to conventional micropipette aspiration, the suction pressure applied on the aspirating cells is highly stable due to the viscous nature of low Reynolds number flow. As a proof-of-concept of this novel microfluidic technology, we built ...
Page 1. Optofluidic Microscopy: Chip-scale imaging cell cytometry Guoan Zheng, Seung Ah Lee, Sean... more Page 1. Optofluidic Microscopy: Chip-scale imaging cell cytometry Guoan Zheng, Seung Ah Lee, Sean Pang, Chao Han, Lapman Lee, Changhuei Yang Electrical Engineering Department, California institute of Technology, Pasadena CA 91125 Abstract We will discuss our recent work on chip-scale microscopy, including fluorescence and laser-scanning imaging techniques. The optofluidic microscope (OFM) represents a significant departure from the conventional microscope design in several significant ways. ...
The completion of my thesis is based on support and help from many individuals. First, I would li... more The completion of my thesis is based on support and help from many individuals. First, I would like to express my gratitude to my PhD advisor Prof. Changhuei Yang for offering me a chance to work on an emerging research field of optofluidics and participate in the development of optofluidic microscopy (OFM), which leads to many successful results. His guidance has led the OFM project from an elegant engineering idea to reality. His enthusiasm and creativity in conducting academic research is forever young, motivating us to pursue excellence in our projects. I thank him for introducing me to biophotonics and allowing me to play a role in contributing to the field. I want to thank Prof. Yu-Chong Tai for being my thesis committee chair. His pioneering work in MEMS has always been my motivation to pursue something ‘big ’ in the world of ‘small’. You will find yourself learning something new every time you interact with him, not only in science but also in life. I want to thank Prof. Chi...
Journal of nanotechnology in engineering and medicine, 2014
Micropipette aspiration is arguably the most classical technique in mechanical measurements and m... more Micropipette aspiration is arguably the most classical technique in mechanical measurements and manipulations of single cells. Despite its simplicity, micropipette aspiration has been applied to a variety of experimental systems that span different length scales to study cell mechanics, nanoscale molecular mechanisms in single cells, bleb growth, and nucleus dynamics, to name a few. Enabled by micro/nanotechnology, several novel microfluidic devices have been developed recently with better accuracy, sensitivity, and throughput. Further technical advancements of microfluidics-based micropipette aspiration would have broad applications in both fundamental cell mechanics studies and for disease diagnostics.
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