Co-author: Arthur D. Lander, Professor in Developmental & Cell Biology, UCI
The development of multicellular organisms involves the differentiation of genetically identical cells into distinct cell type (e.g., liver or heart cells). However, as one can easily imagine, a correctly patterned organism would not result if each cell were to differentiate into any cell type at random. Rather, each cell type must differentiate into the type which is appropriate to its position, and thus biological pattern formation depends on the ability of cells to measure their positions.
Since cells aren’t equipped with rulers, the conveyance and measurement of positional informations constitutes a central problem in developmental biology. Typically, positional information is communicated via intercellular signaling molecules, most of which having been well characterized and being remarkably conserved evolutionarily. These signaling molecules are typically produced in only specific locations within tissues, and it is the concentration gradients which form as these signaling molecules spread from these locations which provide positional information to cells.
In my research, I use genetic experiments on fruit flies and mathematical modeling to study how patterning is coordinated with growth of the organism (in order to ensure that differently sized individuals maintain correct proportions). Specifically, most of my experiments are performed in the fruit fly wing, which is one of the best understood tissues in terms of pattern formation. The intercellular signaling molecules involved in patterning the fly wing are well conserved evolutionarily, and thus are also involved in patterning humans. One striking discovery I made is that the intercellular signaling molecules aren’t so much providing positional information to cells, as much as instructing them how much to grow. Furthermore, the patterns are actually controlling the concentration gradients of the signaling molecules, rather than just responding to them. Although still preliminary, both of these discoveries, if true, would suggest a significant departure from the conventional model of how intercellular signaling orchestrates the formation of biological pattern.
American Cancer Society predicts an estimate of 10,450 new cancer cases and 1350 deaths among children, while 5,330 new cases and 610 deaths among adolescents for 2014. Millions of dollars are being spent in cancer research and treatment every year. So, the knowledge of cancer type of a patient becomes really important in its diagnosis. However, since the concentration of CTC cells (circulating tumor cells) cells in our body is low (1-10 per mL) enrichment is needed. Hence, the goal of my research is to design a microfluidic device to enrich the CTC using Lateral Cavity Acoustic Transducer (LCATs) developed in Prof. Lee’s Lab. LCATs are microfluidic devices which utilize an array of acoustically actuated air/liquid interfaces generated using dead-end side channels. By analyzing the genes from progenitor CTC cells, the knowledge of cancer type can be determined. Besides this, effectiveness of drug can also be tested using CTC cells. Nowadays, cellsearch is the only company whose kit is intended for the immune-magnetic selection, identification, and enumeration of circulating tumor cells (CTCs) of epithelial origin in whole blood. The technology used by cellsearch is a bench-top mechanism where large quantity of costly reagents is required, along with costly equipment which can be eliminated by the employment of microfluidic technologies.
Furthermore, microfluidic technology requires small sample volumes and reduces the reagent consumption as well. Additionally, microfluidics platforms are amenable to integration with upstream and downstream process steps, which is essential for developing a complete Lab on a Chip (LOC) system. Also, to date, most cell sorting microfluidic devices use external pumps which lead to the reduction of cell concentration. A microfluidic device where enrichment can be done without the usage of any external pumps will greatly serve the purposes. Furthermore, the length scales of microfluidic device features are of the same order of magnitude as the cells and particles. The LCAT technology lends itself well its ability in the development of point of care devices as it forms self-contained microfluidic platforms capable of pumping the sample, separating cells based on size, DNA shearing, cell-particle sorting, enrichment etc. My goal is to design a portable device that integrates sample introduction, cell sorting and the downstream biological assays.
Co-author: Do-Hyun Lee, BioMint Lab, Department of Biomedical Engineering, UCI
Single-cell analysis provides precise metabolic and genetic information of individual cells, whereas traditional bulk tests neglect heterogeneity and stochastic effect among cell population, which is essential in determining key cellular activities. Clonal populations of cancer cells get distinct fate outcomes in response to uniform chemotherapy, because of the heterogeneity in regulatory-protein expressions and apoptosis regulation. Therefore, single-cell analysis is a more powerful and effective way for cancer characterization.
Here we fabricate a microfluidic diagnostic chip, integrating cell separation and single-cell trapping arrays with an open interface, enabling external micro-manipulating instruments to enter into individual cells for analysis. In particular, single-cell mRNA extraction is performed by atomic force microscope (AFM). The device is fabricated by standard PDMS soft lithography based on the silicon mold made by negative photo-lithography.
Slanted obstacles and filtration obstacles are used for hydrophoresis size-based, label-free cell separation, with a resolution higher than 90%. Slanted obstacles in the channel drive helical recirculations. Along the transverse flow, cells are focused to the sidewall. Cells larger than the gap of filtration obstacles are blocked and move through the filtration pore, whereas smaller ones pass the gaps and stay in focused position.
Cells are then trapped by hydrodynamic flow in conjunction with grooves arrayed on a serpentine cell-delivery channel (arranged in 5-column format, 20 traps each column). An array of cross-flow channels connects each section of the serpentine channel. Unlike traditional microfluidic chips with closed interface, a 10μm thick PDMS film seals the array top. External devices, e.g. AFM tips and micro-manipulators, can punch through the membrane and enter into individual cells.
The AFM tip is modified into a dielectrophoretic nanotweezer (DENT) to extract mRNA from nucleus. Application of an AC electric field between the inner and outer electrodes of the DENT creates a large electric field gradient, resulting in a dielectrophoretic force to extract mRNA. Selective extraction is achieved by decorating the tip with oligonucleotide probes hybridizing target mRNA. This approach is highly specific, fast, nondestructive, and requires no cell lysis or mRNA purification. The mRNA is then released and tested by q-PCR.
This device addresses the need for clinically compatible single-cell analysis in cancer diagnosis. It will be tested in melanoma cancer characterization. On the chip, melanoma cells extracted from skin biopsies are separated with surrounding cells (fibroblasts, keratinocytes), then tyrosinase mRNA is extracted and tested by q-PCR to determine the cancer stage of a certain cell.
Co-authors: Trisha Westerhof (School of Biological Sciences), Edward Nelson (School of Medicine), and Jered Haun (School of Engineering)
Cancer is the second leading cause of death in the Western world, and thus there is a tremendous need for new approaches and technologies that will help us better detect and treat this deadly disease. Nearly all cancer types form solid tumors. Tumors are highly heterogeneous, consisting not only of cancer cells but stroma, immune cells, and blood vessels. These ubiquitous tumor characteristics have driven intense interest in replacing general chemotherapies with molecularly-targeted agents to achieve personalized therapies for cancer patients. However, realizing this goal will only be possible if molecular measurements can be made are rapid, cost-effective, multiplexed, and at the resolution of single cells. Satisfying all of these needs is extremely challenging for solid tumors because clinical specimens are procured as tissues. Therefore the very first step in approaching single cell molecular analysis is tumor dissociation. Clinical gold standard of tissue dissociation relies on the combination of enzymatic treatment and mechanical disruption. While manual mechanical disruption hinders the speed of clinical diagnostics, enzymatic treatment ultimately destroys certain biomarkers of diagnostic interests. Thus, new diagnostic approaches and technologies are needed for solid tumor tissue specimens to usher in the era of molecular medicine. Microfabrication technologies have advanced the fields of biology and medicine by miniaturizing devices to the scale of cellular samples. In particular, microfluidic systems have enabled precise manipulation of cells and other reagents to achieve systems with high throughput, cost efficiency, and point-of-care operation. Nevertheless, little attention has been given to processing tissues. To advance and automate mechanical dissociation of tumor tissues, we have developed a novel microfluidic device with gradually reduced cross-sections through a series of bifurcating stages. We also introduced constriction and expansion regions on the device to induce flow disturbances that help mix tissues and generate fluidic jets at different length scales to provide shear forces. Using cultured tumor spheroids and recently clinical tumor biopsies, we have demonstrated that our microfluidic dissociation device significantly augmented single cell yields compared to the current clinical gold standard while still maintaining viability. Most importantly, all results were obtained in less than ten minutes of total processing time. In conclusion, we have created a microfluidic device that is capable of dissociating a tumor into single cells at the point-of-care for downstream analysis. We envision our device to serve as one of the core components toward more personalized cancer therapies in the future.
Manasi M. Raje
Co-authors: Siavash Ahrar, Elliot E. Hui
Serial dilution is a fundamental laboratory procedure that produces a logarithmic array of concentrations from an initial sample. Since this procedure is common to a number of laboratory protocols, its automation would be a powerful contribution to lab-on-a-chip systems. We previously reported a microfluidic strategy for serial dilution employing valve-driven circulatory mixing: the serial dilution ladder . While the diluter provides an effective architecture to obtain a series of dilutions from a small amount of sample, it is operated using a vacuum-driven solenoid valve array under computer control. We envision eliminating such unwieldy machinery around the diluter by automating it using on-chip pneumatic logic circuitry. The heart of this system is a four-stage serial diluter, which is a structure with four loops arranged in the form of a ladder. Each loop carries out a 1:1 dilution through circulatory mixing by peristaltic pumping. Peristaltic pumping is established by the coordinated opening and closing of three valves within a loop. We achieved the automation of this pumping pattern by integrating the diluter with an on-chip oscillator . The oscillatory signal provided by the oscillator is routed to the appropriate valves on the active loop via a decoder circuit, a network of pneumatic valves and channels surrounding the dilution ladder. This circuit receives a four-bit input signal which is logically decoded to select the appropriate loop. Once a loop is selected, the oscillatory signal for peristaltic pumping is routed to that loop. Finally, automated selection of the loops can be achieved by using an on-chip four-bit finite state machine to generate the four-bit signal that controls the decoder circuit. We have succeeded in integrating the diluter, oscillator, and decoder circuits, achieving a four-stage serial dilution in approximately one minute. The entire device requires a total of only 4 control inputs. With the integration of the four-bit finite state machine, the device will be fully automated, requiring no external control inputs to function, and powered by a single static vacuum source. This compact and automated serial dilution system is appropriate for point-of-care applications and can be applied to a variety of assays.
1. S. Ahrar, M. Hwang, P. N. Duncan, E. E. Hui, Analyst, 2014, 139, 187-190.
2. P. N. Duncan, T. V. Nguyen and E. E. Hui, Proc. Natl. Acad. Sci. U.S.A., 2013, 110, 18104-18109.
Co-author: Esther Chen (from Dr. Wendy Liu’s lab)
The human immune system is designed to recognize and fight “foreign” objects, pathogens, or domestic diseased cells. Part of the immune system’s defenses includes an inflammatory response in which the area surrounding damaged or infected tissue becomes red and swollen (inflamed). This is in part due to white blood cell activation from the presence of a foreign object or laceration. Inflammation poses a major problem to the successful function of essentially “foreign” implanted medical devices such as vascular stents, heart valves, hip or knee replacements, etc. Conventional implants are permanent and made from surgical grade stainless steel metal which does not provoke an immune response. However, there is a need for non-permanent implant solutions. Biodegradable/bioabsorbable materials are a relatively new movement in the multibillion dollar implantable medical device field. These materials break down into smaller absorbable subunits in the body after it has served its purpose. Despite the promise of non-permanent implants, subunits of degraded bioabsorbable materials can induce a localized inflammatory immune response. This can be seen in a commonly used bioabsorbable material called Poly(lactic-co-glycolic acid) (PLGA), which is currently used as degradable stent and suture material among other things. PLGA degrades into nontoxic lactic and glycolic acid subunits. Although PLGA is a safe material for implantation into the body, accumulation of its acidic subunits in a localized area can cause irritation thereby invoking an inflammatory response. In previous studies, an immunomodulatory protein called CD200 coated on polystyrene surfaces has been found to reduce the activation of white blood cells and their subsequent secretion of inflammatory factors. Our work aims to combine the bioabsorbable property of PLGA and the anti-inflammatory property of CD200. We are using a microfluidic system to create PLGA material that displays CD200. This system is designed to conjugate functionalized CD200 to PLGA to form protein-polymer hybrid particles. We are assessing whether CD200 can be effectively displayed throughout the particle lifetime and effectively inhibit white blood cell activation. In the future, we hope to create vascular stents with this anti-inflammatory CD200-PLGA material which would finally give patients a non-permanent, biodegradable alternative.