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

원심력 기반 유체 시스템의 생물의학적 응용에 관한 연구

학위논문 (박사)-- 서울대학교 대학원 : 바이오엔지니어링전공, 2017. 2. 김희찬.This dissertation focuses on the design, fabrication, evaluation, and application of a centrifugal force-based fluidic system based on macro and micro scale engineering disciplines. Unlike other fluid control forces including electrical force, compression force, magnetic force, etc., centrifugal force is capable of manipulating fluids ranging from macro- to micro-scales with high efficiencies regardless of fluid properties. Accordingly, centrifugal force has been extensively used for a great number of biomedical applications. However, the design optimization of such centrifugal force-based fluidic system for practical use is still under investigation due to the inadequate integrating technique, especially for clinical settings, and the strong dependency on geometric designs within spatially varying three different rotational forces (centrifugal, Coriolis, and Euler forces) to precisely regulate the flow of the fluid. Therefore, this dissertation aims to develop a centrifugal force-based fluidic system appropriate for either clinical or biological research environment based on thorough investigations of the fluid flow, the environments created by the rotational forces, and the geometric designs of the system at both the macro- and micro-scale. The macro-scale study involves the evaluation of design strategies for developing a smart all-in-one cardiopulmonary circulatory support device (CCSD) applicable to diverse clinical environments (emergency room (ER), intensive care unit (ICU), operation room (OR), etc.) (Chapter 2, Section 2.1), the evaluation of hemolytic characteristics of centrifugal blood pump (Chapter 2, Section 2.2), and the evaluation of drug sequestration (Chapter 2, Section 2.3) in CCSD component. Smart all-in-one CCSD equipped with a qualified low hemolytic centrifugal blood pump developed in this study resulted in low hemolysis with a free plasma hemoglobin level far less than 50 mg/dL, and an oxygenator membrane made of polyurethane fibers was turned out to be especially susceptible to the analgesic drug loss (41.8%). The micro-scale study involves the numerical evaluation of the Coriolis effects on fluid flow inside a rotating microchannel (Chapter 3, Section 3.1), the feasibility study for the development of a centrifugal microfluidic-based viscometer (Chapter 3, Section 3.2), the evaluation of hypergravity-induced spheroid formation (Chapter 3, Section 3.3), and the cellular adaptation study to hypergravity conditions using human adipose derived stem cell (hASC) and human lung fibroblast (MRC-5) (Chapter 3, 3.4). Application studies performed under fundamental understanding of the microfluidic flows in rotating platform demonstrated new potential uses for centrifugal microfluidic technologies especially for cell research, revealing that hypergravity conditions can be an important environmental cues affecting cellular interactions. Through evaluating various types of centrifugal force-based fluidic system designs for both practical applications and bench-scale experiments, considerable potential of centrifugal force-based fluidic system for introducing new paradigms in the development of medical devices and biomedical research has been demonstrated. The unprecedented integration technique to further miniaturize and improve usability of the centrifugal force-based system might facilitate product innovations, fostering its wide acceptance in the future (Chapter 4).Chapter 1. Introduction 1 1.1 Centrifugal force 1 1.2 Centrifugal force-based biomedical system 2 1.2.1 Cardiopulmonary support system: Macro-scale 3 1.2.2 Centrifugal micro-fluidic biochip: Micro-scale 5 1.3 Research Aims 8 Chapter 2. Macro scale centrifugal-fluidic system for biomedical application 10 2.1 Development of a smart all-in-one cardiopulmonary circulatory support device 10 2.1.1 Introduction 11 2.1.2 Materials and Methods 12 2.1.3 Results and Discussion 14 2.1.4 Conclusion 15 2.2 Evaluation of hemolytic characteristics of centrifugal blood pump 22 2.2.1 Introduction 23 2.2.2 Materials and Methods 26 2.2.3 Results and Discussion 29 2.2.4 Conclusion 33 2.3 Evaluation of drug sequestration in the extracorporeal membrane oxygenation (ECMO) circuit 45 2.3.1 Introduction 45 2.3.2 Materials and Methods 47 2.3.3 Results 50 2.3.4 Discussion 51 2.3.5 Conclusion 54 Chapter 3. Micro scale centrifugal-fluidic system for biomedical application 60 3.1 A numerical study of the Coriolis effect in centrifugal microfluidics with different channel arrangements 60 3.1.1 Introduction 61 3.1.2 Model problem 64 3.1.3 Analytical solution 69 3.1.4 Numerical solution 71 3.1.5 Results 75 3.1.6 Discussion 79 3.1.7 Summary and Conclusion 83 3.2 Centrifugal microfluidic-based viscometer 103 3.2.1 Introduction 103 3.2.2 Materials and Methods 104 3.2.3 Results 105 3.2.4 Discussion 105 3.2.5 Conclusion 106 3.3 Hypergravity-induced multicellular spheroid generation 110 3.3.1 Introduction 111 3.3.2 Materials and Methods 114 3.3.3 Results and Discussion 119 3.2.4 Conclusion 125 3.4 A study on adipose-derived stem cells adaptions to hypergravity environment 144 3.4.1 Introduction 144 3.4.2 Materials and Methods 147 3.4.3 Results 150 3.4.4 Discussion 151 3.4.5 Conclusion 152 Chapter 4. Conclusion and Perspective 161 References 168 Abstract in Korean 193Docto

Velocity-independent thermal conductivity and volumetric heat capacity measurement of binary gas mixtures

In this paper, we present a single hot wire suspended over a V-groove cavity that is used to measure the thermal conductivity ($k$) and volumetric heat capacity ($\rho c_p$) for both pure gases and binary gas mixtures through DC and AC excitation, respectively. The working principle and measurement results are discussed

Microfabrication Technology for Isolated Silicon Sidewall Electrodes and Heaters

This paper presents a novel microfabricationtechnology for highly doped silicon sidewall electrodesparallel to – and isolated from – the microchannel. Thesidewall electrodes can be utilised for both electricaland thermal actuation of sensor systems. Thetechnology is scalable to a wide range of channelgeometries, simplifies the release etch, and allows forfurther integration with other Surface ChannelTechnology-based systems. Furthermore, thefabrication technology is demonstrated through thefabrication of a relative permittivity sensor. The sensormeasures relative permittivity values ranging from 1 to80, within 3% accuracy of full scale, including waterand water-containing mixtures

Development of a microfluidic platform for multicellular tumour spheroid assays

Microfluidics is a valuable technology for a variety of different biomedical applications. In particular, within cancer research, it can be used to improve upon currently used in vitro screening assays by facilitating the use of 3D cell culture models. One of these models is the multicellular tumour spheroid (MCTS), which provides a more accurate reflection of the tumour microenvironment in vivo by reproducing the cell to cell contact, the development of a nutritional gradient and the formation of a heterogeneous population of cells. Therefore, the MCTS provides a more physiologically relevant in vitro model for testing the efficacy of treatments at the preclinical level. Currently, methods for the formation and culture of spheroids have several limitations, including being labour intensive, being low throughput, producing shear stress towards cells and the hanging drop system being unstable to physical shocks. Recently, microfluidics (especially droplet microfluidics) has been employed for the culture and screening of spheroids, providing a high-throughput methodology which only requires small volumes of fluids and small numbers of cells. However, current issues with droplet microfluidics include complicated droplet gelation procedures and short cell culture times.In this thesis, the use of microfluidic technologies as an approach for spheroid formation and culture are investigated with the aim to create a platform for radiotherapeutic and chemotherapeutic treatment of spheroids using cell lines. Initially, the use of emulsion technology at the macro scale was evaluated to determine the best conditions for spheroid culture. Once this was achieved the spheroids were compared to spheroids using a traditional method and radiotherapeutic treatment was conducted. Subsequently, avenues for miniaturising the developed emulsion-based methods were studied to provide a microfluidic technology. Finally, along with identifying the optimal culture conditions using hydrogels, a microfluidic system that integrated both droplet and single phase microfluidics features was developed for the formation and culture of spheroids. Using the latter, proof of principle experiments were conducted to demonstrate the suitability of the platform for both chemotherapeutic and radiotherapeutic assays within the same device.Microfluidics is a valuable technology for a variety of different biomedical applications. In particular, within cancer research, it can be used to improve upon currently used in vitro screening assays by facilitating the use of 3D cell culture models. One of these models is the multicellular tumour spheroid (MCTS), which provides a more accurate reflection of the tumour microenvironment in vivo by reproducing the cell to cell contact, the development of a nutritional gradient and the formation of a heterogeneous population of cells. Therefore, the MCTS provides a more physiologically relevant in vitro model for testing the efficacy of treatments at the preclinical level. Currently, methods for the formation and culture of spheroids have several limitations, including being labour intensive, being low throughput, producing shear stress towards cells and the hanging drop system being unstable to physical shocks. Recently, microfluidics (especially droplet microfluidics) has been employed for the culture and screening of spheroids, providing a high-throughput methodology which only requires small volumes of fluids and small numbers of cells. However, current issues with droplet microfluidics include complicated droplet gelation procedures and short cell culture times.In this thesis, the use of microfluidic technologies as an approach for spheroid formation and culture are investigated with the aim to create a platform for radiotherapeutic and chemotherapeutic treatment of spheroids using cell lines. Initially, the use of emulsion technology at the macro scale was evaluated to determine the best conditions for spheroid culture. Once this was achieved the spheroids were compared to spheroids using a traditional method and radiotherapeutic treatment was conducted. Subsequently, avenues for miniaturising the developed emulsion-based methods were studied to provide a microfluidic technology. Finally, along with identifying the optimal culture conditions using hydrogels, a microfluidic system that integrated both droplet and single phase microfluidics features was developed for the formation and culture of spheroids. Using the latter, proof of principle experiments were conducted to demonstrate the suitability of the platform for both chemotherapeutic and radiotherapeutic assays within the same device

Scalable production of 3D microtissues using novel microfluidic technologies

Tissue engineering approaches are widely studied with the goal to replace or repair human tissues. However, while studies are often promising in a laboratory environment, there remain difficulties in the translation of laboratory-based studies towards clinical applications due to low in vivo efficiency and/or complex impractical procedures.An interesting strategy for improving therapy effectiveness is by evolving from conventional 2D cell culture to more biomimetic 3D cell culture approaches. While therapy efficiency can be greatly improved using 3D cell culture, current 3D microtissue production techniques are often non-scalable batch processes, limiting clinical and industrial translation. A continuous production method is needed in order to improve the microtissue production rate and improve the feasibility of clinical application.Microfluidics offers the possibility to evolve microtissue production towards a continuous process. Using conventional on-chip microfluidics, microtissues can be produced in a controlled and continuous manner by cell encapsulation in hollow microcapsules. However, conventional on-chip microfluidics offers challenges such as complex multistep processes, the use of potentially harmful oils and surfactants and often low throughputs, which are currently hampering widespread clinical and industrial translation of microfluidically produced microtissues. There is therefore a need to evolve microfluidics towards a clean, fast and single step scalable approach to fulfill the clinical requirements for tissue engineering approaches that take advantage of 3D microtissues.This thesis describes multiple microfluidic solutions that focus on overcoming these challenges hampering the widespread clinical and industrial use of microtissues. A reusable, cleanroom-free, multifunctional microfluidic device is developed using standard cutting and abrasion technology, which allows the production of microtissue-laden microcapsules in a single step-manner. This on-chip process is then evolved towards an off-chip jetting approach which allows for the production of microtissue-laden microcapsules in an ultra-high throughput manner (&gt;10 ml/min) without the need of potentially harmful oils and surfactants. This in-air microfluidic approach is also utilized for mass production of microtissues in larger compartmentalized hydrogels, which are used for the production of large clinical-sized tissues. A multitude of microtissues are formed using these described microfluidic technologies such as human mesenchymal stem cell spheroids, chondrocyte spheroids, fibroblast spheroids, cholangiocyte and cholangiocarcinoma organoids, lumen-forming embryoid bodies, contracting cardiospheres, and clinical sized cartilage tissues.To summarize, this thesis introduces multiple microfluidic systems for scalable microcapsule and microtissue production with the aim to remove the hurdles towards clinical and industrial translation of 3D microtissues.<br/

Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

Development of a New Method for Cell Spheroid Formation Through a Hydrogel Dipping Process

Traditional cell culture systems make use of two-dimensional (2D) monolayer studies which are simple, cheap, and have been successful for a various cell types. However, since this does not reflect on the in vivo physiology, studies have branched out to use three-dimensional (3D) cell culture systems. 3D cell culture allows for studies which make use of the cell connectivity, polarity, and tissue architecture. The use of 3D aggregates called spheroids is one of the most common and versatile of these methods. There are various techniques for spheroid formation and chosen technique is often decided based on the decided spheroid use and size. Many of these methods are limited by the quantity, size, and reproducibility of the spheroids. A new method focused on the manufacturability of the process would overcome these issues. By focusing on the manufacturability of the process, spheroids would be able to be produced in larger quantities and consistently sized. The goal of this study is to manufacture and characterize droplet-on-fiber through a dipping process. The withdrawal of the fiber from a liquid solution will result in a coating due to the balance between the viscous drag and the capillary rise. The thickness of the layer depends upon various parameters of the fluid and dipping process. Above a threshold coating thickness, Rayleigh-Plateau Instability will trigger the formation of droplets. Controlling the process parameters will determine the liquid volume in the droplet and its morphology. Such a simple droplet formation technique will be less resource intensive than existing methods and can produce droplets of various sizes and shapes in a short amount of time. Extruded polylactic acid (PLA) fiber is considered as the substrate for droplet adherence while alginate solution is used for the dipping fluid. The focus of this work is on the shape fidelity and reproducibility of the droplet formation by varying the dipping fluid composition. The aspect ratio between droplet diameter and wetting length is defined as a quantifiable shape-fidelity index which is reported in this work. By varying the dipping fluid composition, the relationship between the viscosity of the dipping fluid (alginate) and the PLA fiber can be identified. The observations made throughout this thesis will allow for further development of this dipping process, as well as determine the optimal concentration of alginate to achieve reproducible droplets with the desired morphology for cell spheroid formation

Development and application of microtechnologies in the design and fabrication of cell culture biomimetic systems

“Lab-On-a-chip” systems have proved to be a promising tool in the field of biology. Currently, cell culture is performed massively on Petri dishes, which have traditionally been used in cell culture laboratories and tissue engineering. However, having proved to be a widely used tool until now, the scientific community has largely described the lack of correlation between the results obtained in the laboratory and the clinical results. This lack of connection between what has been studied in the laboratories and what has been observed in the clinic has led to the search for more advanced alternative tools that allow results to be obtained closer to reality. Thus, the use of microtechnologies in the field of biomedical engineering, presents itself as the perfect tool as an alternative to obsolete traditional media. Thanks to the low volumes of liquid it presents for its use, it also makes it an essential technology for the testing of drugs, new compounds and materials. By being able to more accurately reproduce the biomimetic environment of cell cultures and tissues, they make this technique fundamental as an intermediate step between basic in vitro laboratory tests and preclinical animal tests, resulting from this way in the best alternative for the reduction of both the use of animal models, as in times and costs. For a biomimetic system to be as such, it also needs another series of complementary devices for its better functioning. Micro-valves, micro pumps, flow sensors, O2 sensors, pH, CO2 are fundamental for the correct functioning andsophistication of biomimetic systems. This complexity, on the other hand, is often not perceived by the user since the miniaturization of all these components makes “Lab-On-a-Chip” systems smaller every day, despite numerous control components that can be incorporated.This thesis presents some examples of different microfluidic devices designed and manufactured through the use of microtechnologies, with all applications, focused on their use in biomimetic systems.<br /

Application of Microfluidics in Stem Cell Culture

In this chapter, we review the recent developments, including our studies on the microfabricated devices applicable to stem cell culture. We will focus on the application of pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells. In the first section, we provide a background on microfluidic devices, including their fabrication technology, characteristics, and the advantages of their application in stem cell culture. The second section outlines the use of micropatterning technology in stem cell culture. The use of microwell array technology in stem cell culture is explored in the third section. In the fourth section, we discuss the use of the microfluidic perfusion culture system for stem cell culture, and the last section is a summary of the current state of the art and perspectives of microfluidic technologies in stem cell culture