Spheroids-on-a-chip: Recent advances and design considerations in microfluidic platforms for spheroid formation and culture

© 2018 Elsevier B.V. A cell spheroid is a three-dimensional (3D) aggregation of cells. Synthetic, in-vitro spheroids provide similar metabolism, proliferation, and species concentration gradients to those found in-vivo. For instance, cancer cell spheroids have been demonstrated to mimic in-vivo tumor microenvironments, and are thus suitable for in-vitro drug screening. The first part of this paper discusses the latest microfluidic designs for spheroid formation and culture, comparing their strategies and efficacy. The most recent microfluidic techniques for spheroid formation utilize emulsion, microwells, U-shaped microstructures, or digital microfluidics. The engineering aspects underpinning spheroid formation in these microfluidic devices are therefore considered. In the second part of this paper, design considerations for microfluidic spheroid formation chips and microfluidic spheroid culture chips (μSFCs and μSCCs) are evaluated with regard to key parameters affecting spheroid formation, including shear stress, spheroid diameter, culture medium delivery and flow rate. This review is intended to benefit the microfluidics community by contributing to improved design and engineering of microfluidic chips capable of forming and/or culturing three-dimensional cell spheroids

Simultaneous Culturing of Cell Monolayers and Spheroids on a Single Microfluidic Device for Bridging the Gap between 2D and 3D Cell Assays in Drug Research

Two‐dimensional (2D) cell cultures have been the primary screening tools to predict drug impacts in vitro for decades. However, owing to the lack of tissue‐specific architecture of 2D cultures, secondary screening using three‐dimensional (3D) cell culture models is often necessary. A microfluidic approach that facilitates side‐by‐side 2D and 3D cell culturing in a single microchannel and thus combines the benefits of both set‐ups in drug screening; that is, the uniform spatiotemporal distributions of oxygen, nutrients, and metabolic wastes in 2D, and the tissue‐like architecture, cell–cell, and cell–extracellular matrix interactions only achieved in 3D. The microfluidic platform is made from an organically modified ceramic material, which is inherently biocompatible and supports cell adhesion (2D culture) and metal adhesion (for integration of impedance electrodes to monitor cell proliferation). To induce 3D spheroid formation on another area, a single‐step lithography process is used to fabricate concave microwells, which are made cell‐repellant by nanofunctionalization (i.e., plasma porosification and hydrophobic coating). Thanks to the concave shape of the microwells, the spheroids produced on‐chip can also be released, with the help of microfluidic flow, for further off‐chip characterization after culturing. In this study, the methodology is evaluated for drug cytotoxicity assessment on human hepatocytes.Peer reviewe

Protein and Cell Micropatterning and its Integration With Micro/Nanoparticles Assembly

Ph.DDOCTOR OF PHILOSOPH

Miniaturized analytical platforms from nanoparticle components: studies in the construction, characterization, and high-throughput usage of these novel architectures

The future advancement of nearly all basic and applied sciences is linked in some aspect to understanding and/or controlling of events that occur on the nanometer size scale. One such tool that is being investigated to assist in these studies is nanoparticles. This thesis revolves around the theme of self-assembling nanoparticles to either understand the behavior of the nanoparticles themselves or create analytically useful architectures. The beginning of this thesis examines the interplay between various polymeric nanoparticle properties (i.e., surface chemistry) and patterns of monolayers that display differing chemical moieties. This investigation then leads to the development of guidelines for building two-dimensional patterns through solution deposition and self-assembly of nanoparticles on the underlying.;The properties of polymeric nanoparticles are further employed to create miniaturized platforms with a process involving both the layer-by-layer and photolithographic methodologies. This scheme creates structures with control over the magnitude of all three-dimensions. The analytical utility of the three dimensional structures is demonstrated through creation of a massively dense array of sub-femtoliter volume wells. These microwells are shown to be capable of isolating compounds micrometers apart from one another. Moreover, this characteristic, along with other properties of these wells, is exploited to develop a miniaturized, immunodiagnostic platform.;Finally, the theme of self-assembly of nanoparticles for the purpose of immunodiagnostics is advanced through the creation of a unique height based, bar-code read-out concept. The surfaces of gold and silica nanoparticles are designed to aggregate in a pre-determined pattern when in the presence of a specific analyte. An atomic force microscope (AFM) is employed to analyze the bar-code design and positively identify the presence of the analyte. A proof-of-concept experiment with that exploits the specificity of biotin-streptavidin binding is employed to validate the concept

Electrical and Elastic Properties of Individual Single-Layer Nb₄C₃Tₓ MXene Flakes

2D carbides and nitrides (MXenes) are widely recognized for their exceptional promise for numerous applications. However, physical property measurements of their individual monolayers remain very limited despite their importance for revealing the intrinsic physical properties of MXenes. The first mechanical and electrical measurements of individual single‐layer flakes of Nb4C3Tx MXene, which are prepared via an improved synthetic method are reported. Characterization of field‐effect transistor devices based on individual single‐layer Nb4C3Tx flakes shows an electrical conductivity of 1024 ± 165 S cm−1, which is two orders of magnitude higher than the previously reported values for bulk Nb4C3Tx assemblies, and an electron mobility of 0.41 ± 0.27 cm2 V−1 s−1. Atomic force microscopy nanoindentation measurements of monolayer Nb4C3Tx membranes yield an effective Young's modulus of 386 ± 13 GPa, assuming a membrane thickness of 1.26 nm. This is the highest value reported for nanoindentation measurements of solution‐processable 2D materials, revealing the potential of Nb4C3Tx as a primary component for various mechanical applications. Finally, the agreement between the mechanical properties of 2D Nb4C3Tx MXene and cubic NbC suggests that the extensive experimental data on bulk carbides could be useful for identifying new MXenes with improved functional characteristics

Development of a microfluidic device for patterning multiple species by scanning probe lithography

Scanning Probe Lithography (SPL) is a versatile nanofabrication platform that leverages microfluidic “ink” delivery systems with Scanning Probe Microscopy (SPM) for generating surface-patterned chemical functionality on the sub-100 nm length scale. One of the prolific SPL techniques is Dip Pen Nanolithography™ (DPN™). High resolution, multiplexed registration and parallel direct-write capabilities make DPN (and other SPL techniques) a power tool for applications that are envisioned in micro/nano-electronics, molecular electronics, catalysis, cryptography (brand protection), combinatorial synthesis (nano-materials discovery and characterization), biological recognition, genomics, and proteomics. One of the greatest challenges for the successful performance of the DPN process is the delivery of multiple inks to the scanning probe tips for nano-patterning. The purpose of the present work is to fabricate a microfluidic ink delivery device (called “Centiwell”) for DPN (and other SPL) applications. The device described in this study maximizes the number of chemical species (inks) for nanofabrication that can be patterned simultaneously by DPN to conform the industrial standards for fluid handling for biochemical assays (e.g., genomic and proteomic). Alternate applications of Centiwell are also feasible for the various envisioned applications of DPN (and other SPL techniques) that were listed above. The Centiwell consists of a two-dimensional array of 96 microwells that are bulk micromachined on a silicon substrate. A thermoelectric module is attached to the back side of the silicon substrate and is used to cool the silicon substrate to temperatures below the dew point. By reducing the temperature of the substrate to below the dew point, water droplets are condensed in the microwell array. Microbeads of a hygroscopic material (e.g., poly-ethylene glycol) are dispensed into the microwells to prevent evaporation of the condensed water. Furthermore, since poly-ethylene glycol (PEG) is water soluble, it forms a solution inside the microwells which is subsequently used as the ink for the DPN process. The delivery of the ink to the scanning probe tip is performed by dipping the tip (or multiple tips in an array) into the microwells containing the PEG solution. This thesis describes the various development steps for the Centiwell. These steps include the mask design, the bulk micromachining processes explored for the micro-fabrication of the microwell array, the thermal design calculations performed for the selection of the commercially available thermoelectric coolers, the techniques explored for the synthesis of the PEG microbeads, and the assembly of all the components for integration into a functional Centiwell. Finally, the successful implementation of the Centiwell for nanolithography of PEG solutions is also demonstrated

환자맞춤형 치료를 위한 체외 항암제 스크리닝용 바이오칩

학위논문 (박사) -- 서울대학교 대학원 : 공과대학 전기·정보공학부, 2020. 8. 권성훈.정밀의학(Precision Medicine) 혹은 개인맞춤의학(Personalized Medicine)은 개개인의 최적화된 치료방법을 결정하는 것을 목표로 하는 의학의 패러다임이다. 특히, 임상종양학에서는 차세대염기서열분석(NGS), 전사체서열분석, 그리고 질량분석법들을 통한 환자의 분자 프로파일(molecular profile) 방법이 발전해오고 있으며, 이를 바탕으로 환자를 세분화하여 맞춤형 치료를 구현하려고 노력해오고 있다. 하지만, 여전히 현 수준에서 이해되지 못하는 수준의 종양 이질성(tumor heterogeneity)과 오랜 처방기록을 가진 환자군들의 항암제 획득내성(acquired resistance) 등의 원인으로 맞춤형 환자 처방은 쉽지 않은 경우가 많다. 이러한 경우 환자로부터 얻어진 암세포, 조직으로부터 얻어진 일차세포 혹은 체외 배양된 세포, 스페로이드, 장기유사체 등을 이용하여 고속다중약물스크리닝기술을 통한 맞춤형 항암제를 선별해내는 체외 약물진단 기술을 생각해낼 수 있는데, 이는 기존의 유전체 기반의 시도와 병행되어 개개의 환자들에게 더욱 적합한 치료방법을 찾는 것이 가능하게 한다. 하지만 이러한 목적의 고속다중약물스크리닝기술은 높은 활용가능성에도 불구하고, 광범위한 보급과 활용이 되기에는 제약점이 많았다. 기존의 고속다중약물스크리닝기술은 많은 양의 샘플이 소모되고, 값비싼 시약의 소모량도 적지 않았다. 게다가, 수천 가지 이상의 서로 다른 물질들을 탐색하기 위해 반드시 필요한 고가의 자동화된 액체 운반기(liquid handler) 등이 필요하였는데, 이러한 문제로 대형 제약사, 연구소 등을 제외하고는 도입이 쉽지가 않아 기술접근성이 제한되어 있었다. 본 연구에서는 반도체공정에서의 노광기술을 이용하여 개개의 식별할 수 있는 코드를 가지고 있는 코드화된 하이드로젤 기반의 광경화성폴리머 미세입자를 만들어, 이를 원하는 암세포에 약물 스크리닝을 해보고자 하는 다양한 약물라이브러리를 이용 각각의 코드화된 미세입자에 흡수시켜 약물-미세입자 라이브러리를 제작한다. 그후, 값비싼 어레이 제작용 스포터 혹은 디스펜서 장비없이 간단한 자기조립을 통해 대규모의 다양한 약물-하이드로젤 어레이를 제작할 수 있는 기술을 개발하였다. 또한, 소량의 세포들 만으로도 미세우물(microwell) 기반의 세포칩에 도포하는 방식을 개발하였으며, 이를통해 약물-하이드로젤 어레이와 미세우물기반의 세포칩의 결합으로 수백-수천의 다양한 어세이를 적은 수의 샘플만으로도 한번에 수행할 수 있는 고속다중약물스크리닝 기술을 수행할 수 있게 만들었다. 본 연구에서 제시한 소형화된 체외 항암제 스크리닝용 약물플랫폼은 다음과 같은 의의를 가진다. 적은 수의 환자세포 혹은 샘플의 양에 적용할 수 있는, 사용하기 손쉬운 기술로서, 기존의 값비싼 장비, 시약의 사용량을 획기적으로 줄일 수 있는 기술이다. 본 연구에서 제안된 기술을 통해 기존의 장비를 사용할 때 시약의 값이 비싸거나, 장비의 가격이 비싸서, 혹은 다루고자 하는 샘플의 양이 제한적이어서 기존에 접근하기 힘들었던 다양한 학술연구에 적용할 수 있으며, 병원에서의 임상연구 및 실제 환자맞춤형 치료에 사용 될 수 있는 접근성을 획기적으로 높일 수 있다. 특히, 비교적 중,소 규모의 연구환경에서도 다양한 희귀한 환자유래세포 혹은 환자유래오가노이드 등과 접목하여 사용된다면 본 플랫폼의 가능성을 더욱 극대화 할 수 있을 것으로 기대한다.Precision or Personalized Medicine is a medical paradigm aimed to determine optimal therapy for individual patient. In particular, clinical oncology has been using methods of molecular profiling for each patient through next-generation sequencing (NGS), mRNA-sequencing, and mass spectrometry, and has been trying to implement personalized treatment. However, personalized treatment based on molecular profiling to each patient is not always possible due to the high level of heterogeneity of tumor that is still not fully understood at the current level and acquired resistance of anti-cancer drug due to cumulative targeted therapy. In such cases, in vitro drug testing platform using primary cells obtained from patients, or patient-derived cells, spheroids, and organoids can make it possible to find a more appropriate treatment for each individual patient. However, though high-throughput drug screening technology for this purpose is of the utmost importance in saving lives, there were many limitations to its wide use in many hospitals. The existing high-throughput drug combination screening technology consumes a large number of samples and consumes a considerable amount of expensive reagents. In addition, expensive automated liquid handlers, which were essential for exploring thousands of different pipetting, were not easy to introduce except for large-sized pharmaceutical companies and research institutes, which limited access to technology. In this study, I construct a heterogeneous drug-loaded microparticle library by fabricating encoded photocurable polymer particle that has individually identifiable codes to track loaded drug. and I load various drug molecules, which I want to test to target cells, into each coded microparticle. Then, I developed to produce heterogeneous drug-laden microparticle arrays through simple self-assembly without the need for a microarray spotter or dispensing machine for generating microarray. I also have developed cell seeding method of seeding small-volume samples into the microwell-based cell chip. By utilizing the drug-laden microparticle hydrogel array and microwell-based cell chip technology, hundreds to thousands of different assays can be done at once with just a small number of samples and low cost. Through the implemented platform, the anti-cancer drug sequential combination screening was conducted on the triple-negative breast cooler (TNBC) cells, which are generally known to be difficult to treat due to lack of known drug target, and the results of screening were analyzed by establishing a library of drugs in the EGFR inhibitory type and drugs in the genotoxin type. In addition, another study was conducted to find optimal drug combinations using patient-derived cells derived from tumors in patients with non-small cell lung cancer that have obtained acquired resistance. Finally, as the growing need for three-dimensional culture, such as spheroid and organoid for having a similar response to in vivo drug testing, it was also developed that microwell-based cell chip that is capable of 3D culture with low-cost and small-volume of cells. The miniaturized in vitro anticancer drug screening platform presented in this study has the following significance. An easy-to-use technique that can be applied to a small number of patient cells or samples, which can dramatically reduce the use of conventional expensive equipment, reagents. The proposed technology in this study can be applied to a variety of academic studies previously inaccessible to high-throughput screening due to the high cost of reagents, the high price of equipment, or the limited amount of samples in conventional drug screening. and this platform can also dramatically increase access to clinical research in hospitals for personalized treatments. In particular, it is expected that the possibility of this platform will be further maximized if it is used in a relatively small and medium-sized research environment by the combined use of various rare samples such as patient-derived cells or patient-derived organoids.Chapter 1 Introduction １ 1.1 Motivation of this research ２ 1.2 Competing technologies and Previous works ８ 1.3 Main Concept: In vitro drug testing using miniaturized encoded drug-laden hydrogel array technology １５ Chapter 2 Platform Development of Drug Releasing Hydrogel Microarray ２０ 2.1 Encoded Drug-Laden Hydrogel & Library construction ２１ 2.2 Array generation of heterogenous drug-laden microparticles. ３４ 2.3 Cell Culturing on Cell Chip and bioassay ３６ Chapter 3 Sequential Drug Combination Screening Assy on TNBC ４０ 3.1 Background : Sequential Drug Combination as promising therapeutic option ４１ 3.2 Experimental design with sequential drug treatment assay ４３ 3.3 Technical Issue & its engineering solution ４４ 3.4 Assay Result ４９ Chapter 4 Drug Combination Assay on Patient-Derived Cells ５８ 4.1 Background : Simultaneous Combination Treatment using Patient-Derived Cells ５９ 4.2 Improvement of Platform for facilitating translational study ６２ 4.3 Study Design for small-volume drug combinatorial screening with NSCLC patient derived cell ６５ 4.4 Assay Result ６９ Chapter 5 Development of platform for 3D culture model ７２ 5.1 3D culturable platform ７３ 5.2 Development of 3D culture platform based Matrigel scaffold. ７８ 5.3 Advantage over conventional 3D culture-based drug testing platform. ８５ Chapter 6 Conclusion ８７ Bibliography ９０ Abstract in Korean ９７Docto