Drop-on-Demand Single Cell Isolation and Total RNA Analysis

Peer reviewe

Role of micropillar arrays in cell rolling dynamics

In this study, we present a role of arrayed micropillar structures in cell rolling dynamics. Cell rolling on a ligand coated surface as a means of cell separation was demonstrated using a micropillar-integrated microfluidic channel. This approach allows the separation of cells according to characteristic surface properties, regardless of cell size. In these experiments, different moving trajectories of the cells between a ligand-coated micropost structure and a 1% BSA coated micropost structure were observed using sequential images. Based on the analysis of the angle of travel of cells in the trajectory, the average angles of travel on the ligand-coated microposts were 1.5 degrees and -3.1 degrees on a 1% BSA-coated micropost structure. The overall force equivalent applied to a cell can be analyzed to predict the cell rolling dynamics when a cell is detached. These results show that it will be possible to design chip geometry for delicate operations and to separate target cells. Furthermore, we believe that these control techniques based on a ligand coated micropillar surface can be used for enhancing cell rolling-based separation in a faster and more continuous manner.ope

Enumeration of CD4+ T-Cells Using a Portable Microchip Count Platform in Tanzanian HIV-Infected Patients

Background CD4+ T-lymphocyte count (CD4 count) is a standard method used to monitor HIV-infected patients during anti-retroviral therapy (ART). The World Health Organization (WHO) has pointed out or recommended that a handheld, point-of-care, reliable, and affordable CD4 count platform is urgently needed in resource-scarce settings. Methods HIV-infected patient blood samples were tested at the point-of-care using a portable and label-free microchip CD4 count platform that we have developed. A total of 130 HIV-infected patient samples were collected that included 16 de-identified left over blood samples from Brigham and Women's Hospital (BWH), and 114 left over samples from Muhimbili University of Health and Allied Sciences (MUHAS) enrolled in the HIV and AIDS care and treatment centers in the City of Dar es Salaam, Tanzania. The two data groups from BWH and MUHAS were analyzed and compared to the commonly accepted CD4 count reference method (FACSCalibur system). Results The portable, battery operated and microscope-free microchip platform developed in our laboratory (BWH) showed significant correlation in CD4 counts compared with FACSCalibur system both at BWH (r = 0.94, p<0.01) and MUHAS (r = 0.49, p<0.01), which was supported by the Bland-Altman methods comparison analysis. The device rapidly produced CD4 count within 10 minutes using an in-house developed automated cell counting program. Conclusions We obtained CD4 counts of HIV-infected patients using a portable platform which is an inexpensive (<$1 material cost) and disposable microchip that uses whole blood sample (<10 µl) without any pre-processing. The system operates without the need for antibody-based fluorescent labeling and expensive fluorescent illumination and microscope setup. This portable CD4 count platform displays agreement with the FACSCalibur results and has the potential to expand access to HIV and AIDS monitoring using fingerprick volume of whole blood and helping people who suffer from HIV and AIDS in resource-limited settings.Wallace H. Coulter Foundation (Young Investigation Award in Bioengineering Award)National Institutes of Health (U.S.) (NIH R01AI081534)National Institutes of Health (U.S.) (NIH R21AI087107)National Institutes of Health (U.S.) (NIH grant RR016482)National Institutes of Health (U.S.) (grant AI060354)National Institutes of Health (U.S.) (NIH Fogarty Fellowship

Projection of Cancer Incidence and Mortality From 2020 to 2035 in the Korean Population Aged 20 Years and Older

Objectives: This study aimed to identify the current patterns of cancer incidence and estimate the projected cancer incidence and mortality between 2020 and 2035 in Korea. Methods: Data on cancer incidence cases were extracted from the Korean Statistical Information Service from 2000 to 2017, and data on cancer-related deaths were extracted from the National Cancer Center from 2000 to 2018. Cancer cases and deaths were classified according to the International Classification of Diseases, 10th edition. For the current patterns of cancer incidence, age-standardized incidence rates (ASIRs) and age-standardized mortality rates were investigated using the 2000 mid-year estimated population aged over 20 years and older. A joinpoint regression model was used to determine the 2020 to 2035 trends in cancer. Results: Overall, cancer cases were predicted to increase from 265 299 in 2020 to 474 085 in 2035 (growth rate: 1.8%). The greatest increase in the ASIR was projected for prostate cancer among male (7.84 vs. 189.53 per 100 000 people) and breast cancer among female (34.17 vs. 238.45 per 100 000 people) from 2000 to 2035. Overall cancer deaths were projected to increase from 81 717 in 2020 to 95 845 in 2035 (average annual growth rate: 1.2%). Although most cancer mortality rates were projected to decrease, those of breast, pancreatic, and ovarian cancer among female were projected to increase until 2035. Conclusions: These up-to-date projections of cancer incidence and mortality in the Korean population may be a significant resource for implementing cancer-related regulations or developing cancer treatments

Statistical Modeling of Single Target Cell Encapsulation

High throughput drop-on-demand systems for separation and encapsulation of individual target cells from heterogeneous mixtures of multiple cell types is an emerging method in biotechnology that has broad applications in tissue engineering and regenerative medicine, genomics, and cryobiology. However, cell encapsulation in droplets is a random process that is hard to control. Statistical models can provide an understanding of the underlying processes and estimation of the relevant parameters, and enable reliable and repeatable control over the encapsulation of cells in droplets during the isolation process with high confidence level. We have modeled and experimentally verified a microdroplet-based cell encapsulation process for various combinations of cell loading and target cell concentrations. Here, we explain theoretically and validate experimentally a model to isolate and pattern single target cells from heterogeneous mixtures without using complex peripheral systems.Wallace H. Coulter Foundation (Young Investigator in Bioengineering Award)National Institutes of Health (U.S.) (Grant R01AI081534)National Institutes of Health (U.S.) (Grant R21AI087107

Blood Banking in Living Droplets

Blood banking has a broad public health impact influencing millions of lives daily. It could potentially benefit from emerging biopreservation technologies. However, although vitrification has shown advantages over traditional cryopreservation techniques, it has not been incorporated into transfusion medicine mainly due to throughput challenges. Here, we present a scalable method that can vitrify red blood cells in microdroplets. This approach enables the vitrification of large volumes of blood in a short amount of time, and makes it a viable and scalable biotechnology tool for blood cryopreservation.National Institutes of Health (U.S.) (NIH R21 EB007707)Wallace H. Coulter FoundationUnited States. Army Medical Research and Materiel Command (Acquisition Activity Cooperative Agreement RO1 A1081534)Center for Integration of Medicine and Innovative TechnologyUnited States. Army Medical Research and Materiel Command (Acquisition Activity Cooperative Agreement R21 AI087107)United States. Army. Telemedicine & Advanced Technology Research Cente

Targeting metastatic breast cancer with peptide epitopes derived from autocatalytic loop of Prss14/ST14 membrane serine protease and with monoclonal antibodies

Background In order to develop a new immunotherapeutic agent targeting metastatic breast cancers, we chose to utilize autocatalytic feature of the membrane serine protease Prss14/ST14, a specific prognosis marker for ER negative breast cancer as a target molecule. Methods The study was conducted using three mouse breast cancer models, 4 T1 and E0771 mouse breast cancer cells into their syngeneic hosts, and an MMTV-PyMT transgenic mouse strain was used. Prss14/ST14 knockdown cells were used to test function in tumor growth and metastasis, peptides derived from the autocatalytic loop for activation were tested as preventive metastasis vaccine, and monoclonal and humanized antibodies to the same epitope were tested as new therapeutic candidates. ELISA, immunoprecipitation, Immunofluorescent staining, and flow cytometry were used to examine antigen binding. The functions of antibodies were tested in vitro for cell migration and in vivo for tumor growth and metastasis. Results Prss14/ST14 is critically involved in the metastasis of breast cancer and poor survival rather than primary tumor growth in two mouse models. The epitopes derived from the specific autocatalytic loop region of Prss14/ST14, based on structural modeling acted as efficient preventive metastasis vaccines in mice. A new specific monoclonal antibody mAb3F3 generated against the engineered loop structure could reduce cell migration, eliminate metastasis in PyMT mice, and can detect the Prss14/ST14 protein expressed in various human cancer cells. Humanized antibody huAb3F3 maintained the specificity and reduced the migration of human breast cancer cells in vitro. Conclusion Our study demonstrates that Prss14/ST14 is an important target for modulating metastasis. Our newly developed hybridoma mAbs and humanized antibody can be further developed as new promising candidates for the use in diagnosis and in immunotherapy of human metastatic breast cancer.This work is supported in part by the National Research Foundation (NRF) grant funded by the Korea government (MEST) (No. 2013R1A1A2009892 and No. 2017R1A2B4008109) and Inha Univeristy Research Grant awarded to MGK and (No. 2015R1A2A1A15054021) to SHK

A Planar-Type Micro-Biopsy Tool for a Capsule-Type Endoscope Using a One-Step Nickel Electroplating Process

Millimeter-scale biopsy tools combined with an endoscope instrument have been widely used for minimal invasive surgery and medical diagnosis. Recently, a capsule-type endoscope was developed, which requires micromachining to fabricate micro-scale biopsy tools that have a sharp tip and other complex features, e.g., nanometer-scale end-tip sharpness and a complex scalpel design. However, conventional machining approaches are not cost-effective for mass production and cannot fabricate the micrometer-scale features needed for biopsy tools. Here, we demonstrate an electroplated nickel micro-biopsy tool which features a planar shape and is suitable to be equipped with a capsule-type endoscope. Planar-type micro-biopsy tools are designed, fabricated, and evaluated through in vitro tissue dissection experiments. Various micro-biopsy tools with a long shaft and sharp tip can be easily fabricated using a thick photoresist (SU8) mold via a simple one-step lithography and nickel electroplating process. The characteristics of various micro-biopsy tool design features, including a tip taper angle, different tool geometries, and a cutting scalpel, are evaluated for efficient tissue extraction from mice intestine. These fabricated biopsy tools have shown appropriate strength and sharpness with a sufficient amount of tissue extraction for clinical applications, e.g., cancer tissue biopsy. These micro-scale biopsy tools could be easily integrated with a capsule-type endoscope and conventional forceps

Extending the Shelf-Life of Immunoassay-Based Microfluidic Chips through Freeze-Drying Sublimation Techniques

Point-of-care testing (POCT) platforms utilizing immunoassay-based microfluidic chips offer a robust and specific method for detecting target antibodies, demonstrating a wide range of applications in various medical and research settings. Despite their versatility and specificity, the adoption of these immunoassay chips in POCT has been limited by their short shelf-life in liquid environments, attributed to the degradation of immobilized antibodies. This technical limitation presents a barrier, particularly for resource-limited settings where long-term storage and functionality are critical. To address this challenge, we introduce a novel freeze-dry sublimation process aimed at extending the shelf-life of these microfluidic chips without compromising their functional integrity. This study elaborates on the mechanisms by which freeze-drying preserves the bioactivity of the immobilized antibodies, thereby maintaining the chip&apos;s performance over an extended period. Our findings reveal significant shelf-life extension, making it possible for these POCT platforms to be more widely adopted and practically applied, especially in settings with limited resources. This research paves the way for more accessible, long-lasting, and effective POCT solutions, breaking down previous barriers to adoption and application

Three-Dimensional High-Resolution Digital Inline Hologram Reconstruction with a Volumetric Deconvolution Method

The digital in-line holographic microscope (DIHM) was developed for a 2D imaging technology and has recently been adapted to 3D imaging methods, providing new approaches to obtaining volumetric images with both a high resolution and wide field-of-view (FOV), which allows the physical limitations to be overcome. However, during the sectioning process of 3D image generation, the out-of-focus image of the object becomes a significant impediment to obtaining evident 3D features in the 2D sectioning plane of a thick biological sample. Based on phase retrieved high-resolution holographic imaging and a 3D deconvolution technique, we demonstrate that a high-resolution 3D volumetric image, which significantly reduces wave-front reconstruction and out-of-focus artifacts, can be achieved. The results show a 3D volumetric image that is more finely focused compared to a conventional 3D stacked image from 2D reconstructed images in relation to micron-size polystyrene beads, a whole blood smear, and a kidney tissue sample. We believe that this technology can be applicable for medical-grade images of smeared whole blood or an optically cleared tissue sample for mobile phytological microscopy and laser sectioning