Cell Separations and Sorting

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.analchem.9b05357.NIBIB Grant P41-EB020594COBRE Grant 5P20GM13042

The use of microfluidic technology for cancer applications and liquid biopsy

© 2018 by the authors. There is growing awareness for the need of early diagnostic tools to aid in point-of-care testing in cancer. Tumor biopsy remains the conventional means in which to sample a tumor and often presents with challenges and associated risks. Therefore, alternative sources of tumor biomarkers is needed. Liquid biopsy has gained attention due to its non-invasive sampling of tumor tissue and ability to serially assess disease via a simple blood draw over the course of treatment. Among the leading technologies developing liquid biopsy solutions, microfluidics has recently come to the fore. Microfluidic platforms offer cellular separation and analysis platforms that allow for high throughout, high sensitivity and specificity, low sample volumes and reagent costs and precise liquid controlling capabilities. These characteristics make microfluidic technology a promising tool in separating and analyzing circulating tumor biomarkers for diagnosis, prognosis and monitoring. In this review, the characteristics of three kinds of circulating tumor markers will be described in the context of cancer, circulating tumor cells (CTCs), exosomes, and circulating tumor DNA (ctDNA). The review will focus on how the introduction of microfluidic technologies has improved the separation and analysis of these circulating tumor markers

Microfluidics for studying metastatic patterns of lung cancer

The incidence of lung cancer continues to rise worldwide. Because the aggressive metastasis of lung cancer cells is the major drawback of successful therapies, the crucial challenge of modern nanomedicine is to develop diagnostic tools to map the molecular mechanisms of metastasis in lung cancer patients. In recent years, microfluidic platforms have been given much attention as tools for novel point-of-care diagnostic, an important aspect being the reconstruction of the body organs and tissues mimicking the in vivo conditions in one simple microdevice. Herein, we present the first comprehensive overview of the microfluidic systems used as innovative tools in the studies of lung cancer metastasis including single cancer cell analysis, endothelial transmigration, distant niches migration and finally neoangiogenesis. The application of the microfluidic systems to study the intercellular crosstalk between lung cancer cells and surrounding tumor microenvironment and the connection with multiple molecular signals coming from the external cellular matrix are discussed. We also focus on recent breakthrough technologies regarding lab-on-chip devices that serve as tools for detecting circulating lung cancer cells. The superiority of microfluidic systems over traditional in vitro cell-based assays with regard to modern nanosafety studies and new cancer drug design and discovery is also addressed. Finally, the current progress and future challenges regarding printable and paper-based microfluidic devices for personalized nanomedicine are summarized.publishedVersio

Clinical diagnostic biomarker “circulating tumor cells” in breast cancer - a meta-analysis

ObjectiveUsing meta-analysis, we evaluate circulating tumor cells(CTCs) as a potential diagnostic tool for breast cancer.MethodsA document search was conducted using publicly available databases up to May 2021. Specific inclusion and exclusion criteria were formulated and summarize relevant data through literature types, research types, case populations, samples, etc. Subgroup analysis of documents based on regions, enrichment methods, and detection methods. The included research projects were evaluated using DeeKs’ bias, and evaluation indicators such as specificity (SPE), sensitivity (SEN), diagnosis odds ratio (DOR) were used as evaluation indicators.Results16 studies on the use of circulating tumor cells to diagnose breast cancer were included in our meta-analysis. Overall sensitivity value was 0.50 (95%CI:0.48-0.52), specificity value was 0.93 (95%CI:0.92- 0.95), DOR value was 33.41 (95%CI:12.47-89.51), and AUC value was 0.8129.ConclusionIn meta-regressions and subgroup analysis, potential heterogeneity factors were analyzed, but the source of heterogeneity is still unclear. CTCs, as a novel tumor marker, have a good diagnostic value, but its enrichment and detection methods still need to continue to be developed to improve detection accuracy. Therefore, CTCs can be used as an auxiliary means of early detection, which is helpful to the diagnosis and screening of breast cancer

모세관 현상 기반의 패터닝 기법을 활용한 고효율 삼차원 면역세포 항암효능 평가 플랫폼

학위논문 (박사) -- 서울대학교 대학원 : 공과대학 기계공학부, 2020. 8. 전누리.Organs-on-chips have been developed for recapitulating human organ functions in in vitro as microfabrication techniques meet biology since the early 2000s. Specifically, polydimethylsiloxane (PDMS) based microfluidic devices enabled to mimic organ functions by providing spatially compartmented cell patterning for culturing cells with in vivo like layout. The selective cell patterning enabled 3D cell culture and spatiotemporal analysis which were challenging to conduct with conventional cell culturewares such as petri-dishes, flasks, and well-plates. However, traditional organs-on-chips have limitations in salability, experimental throughput, and absence of standard due to their closed channel designs based on PDMS. Here, we introduce two capillarity guided patterning (CGP) methods by integrating microstructures with conventional cell culturewares. First, we fabricated micropillar arrays on open polystyrene (PS) surfaces and the micropillars can capture liquids swept over the surface. Using the devices, we demonstrated 3D culture applications, single cell capturing and retrieval and multiple cell co-culture. Second, we integrated rail-structures with microplate. Beneath a rail-structure, hydrogel precursors can selectively remain according to meniscus dynamics when the pre-loaded precursors are aspirated. These two CGP methods can be produced with injection molding and provide enhanced experimental throughput. Using the rail-based CGP method, we developed a 3D cytotoxicity assay for cancer immunotherapy based on an injection molded plastic culture (CACI-IMPACT) device to assess killing abilities of cytotoxic lymphocytes in 3D microenvironment through a spatiotemporal analysis of the lymphocytes and cancer cells embedded in 3D extra cellular matrix (ECM). Owing to the aspiration-mediated patterning, hydrogel precursors can be patterned in 12 wells within 30 s. For functional evaluation of the cytotoxic lymphocytes engineered for cancer immunotherapy, HeLa cells encapsulated by collagen matrix were patterned beneath low rails and NK-92 cells were loaded into the channel formed by the collagen matrix. We observed infiltration, migration and killing activity of NK-92 cells against HeLa cells in collagen matrix. Through image-based analysis, we found ECM significantly influences migration and cytotoxicity of lymphocytes. Hence, the CACI-IMPACT platform has the potential to be used for pre-clinical evaluation of ex vivo engineered cytotoxic lymphocytes for cancer immunotherapy against solid tumors, and the CGP methods are expected to accelerate the commercialization of organs-on-chips.장기모사칩은 2000년대 초부터 마이크로 공정 기술이 생물학적 연구에 활용됨에 따라 인간 장기 기능을 모사하기 위해 개발되었다. 구체적으로, polydimethylsiloxane (PDMS) 기반 미세유체 장치는 공간적으로 구분된 세포 패터닝을 가능케 함으로써 생체와 유사한 구조로 세포를 배양할 수 있게 해주었다. 이러한 세포 패터닝은 페트리 디쉬, 플라스크, 혹은 웰플레이트와 같은 기존의 세포 배양 도구에서는 수행하기 어려운 삼차원 세포 배양과 그 안에서의 시공간적 분석을 가능하게 하였다. 하지만, 종래의 장기모사칩은 PDMS에 기반한 닫힌 형태의 채널 설계로 인해 낮은 생산성, 낮은 실험 효율, 낮은 장비 호환성을 갖는다. 따라서, 본 연구는 대중적인 세포 배양 장치들에 마이크로 구조물을 통합한 두가지 모세관 현상 기반의 패터닝 방법을 제시한다. 첫번째 방법은 페트리 디쉬나 polystyrene (PS) 필름과 같이 개방된 PS 표면에 마이크로 기둥 어레이를 제작하여 그 위에서 액체가 쓸려 지나갈 때 기둥 구조물들 사이에 액체를 포획하는 방식이다. 마이크로 기둥 어레이의 배치에 따라 나노리터부터 마이크로리터에 이르는 액체를 빠르게 패터닝할 수 있게 한다. 이러한 기둥 구조를 활용하면 다양한 세포의 배치 및 배양이 가능하여, 본 연구에서는 삼차원 환경에서의 단일세포 배양과 다세포 공배양 플랫폼으로의 활용 가능성을 제시하였다. 두번째 방법은 마이크로 레일 형태의 마이크로구조물을 표준화된 마이크로 플레이트의 웰과 통합하여 고효율 삼차원 배양 플랫폼을 제시한다. 레일 구조의 아래에 주입된 액체가 빨아들여질 때 구조물에 의해 형성된 액체-기체 계면들의 순차적 이동을 활용하여 특정 레일의 아래에만 액체를 남기는 기술을 개발하였다. 이 두가지 모세관 현상 기반 패터닝 방법을 위한 장치들은 사출성형으로 대량생산이 가능하고 우수한 실험 효율을 갖는다. 이 중 레일 구조를 활용한 흡인 기반의 패터닝 방법을 이용하여 면역세포치료제의 성능 평가를 위한 사출 성형된 플라스틱 어레이 배양 장치 (CACI-IMPACT)를 개발하였다. 흡인 기반 패터닝 덕분에 20 μl 파이펫으로 빨아들인 하이드로젤 용액을 30 초 이내에 12개의 웰에 패터닝 할 수 있었다. 면역세포치료제의 기능적 평가를 위해, 콜라겐 젤에 포함된 HeLa 세포를 패터닝하고 NK-92 세포의 콜라겐 매트릭스 내부로의 침투, 매트릭스 내부에서의 이동 및 암세포 살해 활동을 관찰하였다. 이를 통해 세포외기질이 세포 독성 림프구의 이동 및 세포 독성에 상당히 영향을 미친다는 것을 확인할 수 있었다. 따라서, 암세포와 세포 독성 림프구의 고효율 삼차원 공동 배양을 가능하게 하는 본 플랫폼은 고형 종양에 대한 면역 치료를 위해 개발된 세포 독성 림프구의 전임상 평가에 사용될 가능성이 있으며, 본 연구에서 개발 및 사용된 모세관 현상 기반 패터닝 기술들은 장기모사칩의 상용화를 가속화시킬 것으로 기대한다.Chapter 1. Introduction 1 1.1. History of organs-on-chips 1 1.2. Challenges in current organs-on-chips 4 1.3. Models for cancer immunotherapy 7 1.4. Purpose of research 8 Chapter 2. Microstructure-guided multi-scale liquid patterning on open surface 11 2.1. Introduction 11 2.2. Materials and Methods 13 2.2.1. Fabrication of the microstructured PS surface 13 2.2.2. Single cell isolation and retrieval of single colony 16 2.2.3. In vitro vasculogenesis 17 2.2.4. Visualization of the in vitro blood vessel 19 2.3. Results and discussion 18 2.3.1. Liquid patterning process 18 2.3.2. Comparison of microliquid trapping with a micropillar array and microwells 30 2.3.3. Arrangement of micropillars for controlling the volume and shape of patterned liquids 33 2.3.4. Single cell culture & recovery platform 37 2.3.5. Sequential patterning for co-culture in a 3D microenvironment 42 2.4. Conclusions 46 Chapter 3. Aspiration-mediated microliquid patterning using rail-based open microfluidics 47 3.1. Introduction 47 3.2 Materials and Methods 50 3.2.1. Fabrication of open microfluidic devices 50 3.2.2. Cell culture 50 3.2.3. Hydrogel micropatterning 51 3.2.4. Image analysis 52 3.3. Results 53 3.3.1. Microstructures for aspiration-mediated patterning 53 3.3.2. Theoretical analysis of microchannel formation 56 3.3.3. Formation of multiple discrete microchannels 63 3.3.4. An application for screening vasculogenic capacities 70 3.4. Conclusions 75 Chapter 4. High-throughput microfluidic 3D cytotoxicity assay for cancer immunotherapy 77 4.1. Introduction 77 4.2. Materials and Methods 81 4.2.1. Cell culture 81 4.2.2. Fluorescent labeling of live and dead cells 81 4.2.3. 3D cytotoxicity assay using gel patterned device 82 4.2.4. Image analysis 83 4.2.5. 2D cytotoxicity assay 84 4.3. Results 84 4.3.1. Design and fabrication of devices 84 4.3.2. Cytotoxicity assay in 3D ECM environment 89 4.3.3. 3D ECM reduce cytotoxicity 94 4.3.4. Dense ECM impede migration of CLs 98 4.4. Conclusions 104 Chapter 5. Concluding Remarks 110 Bibliography 113 Abstract in Korean 124Docto

Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78–83%), high retention of cell viability (71–74%), high tumour cell enrichment against leukocytes (1.7–2 × 10^3), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4–0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research

Measuring cell deformation by microfluidics

Microfluidics is an increasingly popular method for studying cell deformation, with various applications in fields such as cell biology, biophysics, and medical research. Characterizing cell deformation offers insights into fundamental cell processes, such as migration, division, and signaling. This review summarizes recent advances in microfluidic techniques for measuring cellular deformation, including the different types of microfluidic devices and methods used to induce cell deformation. Recent applications of microfluidics-based approaches for studying cell deformation are highlighted. Compared to traditional methods, microfluidic chips can control the direction and velocity of cell flow by establishing microfluidic channels and microcolumn arrays, enabling the measurement of cell shape changes. Overall, microfluidics-based approaches provide a powerful platform for studying cell deformation. It is expected that future developments will lead to more intelligent and diverse microfluidic chips, further promoting the application of microfluidics-based methods in biomedical research, providing more effective tools for disease diagnosis, drug screening, and treatment

Micro/Nano-Chip Electrokinetics

Micro/nanofluidic chips have found increasing applications in the analysis of chemical and biological samples over the past two decades. Electrokinetics has become the method of choice in these micro/nano-chips for transporting, manipulating and sensing ions, (bio)molecules, fluids and (bio)particles, etc., due to the high maneuverability, scalability, sensitivity, and integrability. The involved phenomena, which cover electroosmosis, electrophoresis, dielectrophoresis, electrohydrodynamics, electrothermal flow, diffusioosmosis, diffusiophoresis, streaming potential, current, etc., arise from either the inherent or the induced surface charge on the solid-liquid interface under DC and/or AC electric fields. To review the state-of-the-art of micro/nanochip electrokinetics, we welcome, in this Special Issue of Micromachines, all original research or review articles on the fundamentals and applications of the variety of electrokinetic phenomena in both microfluidic and nanofluidic devices

Biosensing on the centrifugal microfluidic lab-on-a-Disc platform

Lab-on-a-Disc (LoaD) biosensors are increasingly a promising solution for many biosensing applications. In the search for a perfect match between point-of-care (PoC) microfluidic devices and biosensors, the LoaD platform has the potential to be reliable, sensitive, low-cost, and easy-to-use. The present global pandemic draws attention to the importance of rapid sample-to-answer PoC devices for minimising manual intervention and sample manipulation, thus increasing the safety of the health professional while minimising the chances of sample contamination. A biosensor is defined by its ability to measure an analyte by converting a biological binding event to tangible analytical data. With evolving manufacturing processes for both LoaDs and biosensors, it is becoming more feasible to embed biosensors within the platform and/or to pair the microfluidic cartridges with low-cost detection systems. This review considers the basics of the centrifugal microfluidics and describes recent developments in common biosensing methods and novel technologies for fluidic control and automation. Finally, an overview of current devices on the market is provided. This review will guide scientists who want to initiate research in LoaD PoC devices as well as providing valuable reference material to researchers active in the field