Cellular spheroids are aggregates of cells that are being explored to address fundamental biological questions and as building blocks for engineered tissues. Spheroids possess distinct advantages over cellular monolayers or cell encapsulation in 3D natural and synthetic hydrogels, including direct cell-cell interactions and high cell densities, which better mimic aspects of many tissues. Despite these advantages, spheroid cultures often exhibit uncontrollable growth and may be too simplistic to mimic complex tissue structures. To address this, biomaterials are being leveraged to further expand the use of cellular spheroids for biomedical applications. In this review, we provide an overview of recent studies that utilize engineered biomaterials to guide spheroid formation and function, as well as their fabrication into tissues for use as tissue models and for therapeutic applications. First, we describe biomaterial strategies that allow the high-throughput fabrication of homogeneously-sized spheroids. Next, we summarize how engineered biomaterials are introduced into spheroid cultures either internally as microparticles or externally as hydrogel microenvironments to influence spheroid behavior (e.g., differentiation, fusion). Lastly, we discuss a variety of biofabrication strategies (e.g., 3D bioprinting, melt electrowriting) that have been used to develop macroscale tissue models and implantable constructs through the guided assembly of spheroids. Overall, the goal of this review is to provide a summary of how biomaterials are currently being engineered and leveraged to support spheroids in biomedical applications, as well as to provide a future outlook of the field. STATEMENT OF SIGNIFICANCE: Cellular spheroids are becoming increasingly used as in vitro tissue models or as 'building blocks' for tissue engineering and repair strategies. Engineered biomaterials and their processing through biofabrication approaches are being leveraged to structurally support and guide spheroid processes. This review summarizes current approaches where such biomaterials are being used to guide spheroid formation, function, and fabrication into tissue constructs. As the field is rapidly expanding, we also provide an outlook on future directions and how new engineered biomaterials can be implemented to further the development of biofabricated spheroid-based tissue constructs.

In the 2000s, advances in cellular micropatterning using microfabrication contributed to the development of cell-based biosensors for the functional evaluation of newly synthesized drugs, resulting in a revolutionary evolution in drug screening. To this end, it is essential to utilize cell patterning to control the morphology of adherent cells and to understand contact and paracrine-mediated interactions between heterogeneous cells. This suggests that the regulation of the cellular environment by means of microfabricated synthetic surfaces is not only a valuable endeavor for basic research in biology and histology, but is also highly useful to engineer artificial cell scaffolds for tissue regeneration. This review particularly focuses on surface engineering techniques for the cellular micropatterning of three-dimensional (3D) spheroids. To establish cell microarrays, composed of a cell adhesive region surrounded by a cell non-adherent surface, it is quite important to control a protein-repellent surface in the micro-scale. Thus, this review is focused on the surface chemistries of the biologically inspired micropatterning of two-dimensional non-fouling characters. As cells are formed into spheroids, their survival, functions, and engraftment in the transplanted site are significantly improved compared to single-cell transplantation. To improve the therapeutic effect of cell spheroids even further, various biomaterials (e.g., fibers and hydrogels) have been developed for spheroid engineering. These biomaterials not only can control the overall spheroid formation (e.g., size, shape, aggregation speed, and degree of compaction), but also can regulate cell-to-cell and cell-to-matrix interactions in spheroids. These important approaches to cell engineering result in their applications to tissue regeneration, where the cell-biomaterial composite is injected into diseased area. This approach allows the operating surgeon to implant the cell and polymer combinations with minimum invasiveness. The polymers utilized in hydrogels are structurally similar to components of the extracellular matrix in vivo, and are considered biocompatible. This review will provide an overview of the critical design to make hydrogels when used as cell scaffolds for tissue engineering. In addition, the new strategy of injectable hydrogel will be discussed as future directions.

Bioprinting has emerged as one of the most promising strategies for fabrication of functional organs in the lab as an alternative to transplant organs. While progress in the field has mostly been restricted to a few miniaturized tissues with minimal biological functionality until a few years ago, recent progress has advanced the concept of building three-dimensional multicellular organ complexity remarkably. This review discusses a series of milestones that have paved the way for bioprinting of tissue constructs that have advanced levels of biological and architectural functionality. Critical materials, engineering and biological challenges that are key to addressing the desirable function of engineered organs are presented. These are discussed in light of the many difficulties to replicate the heterotypic organization of multicellular solid organs, the nanoscale precision of the extracellular microenvironment in hierarchical tissues, as well as the advantages and limitations of existing bioprinting methods to adequately overcome these barriers. In summary, the advances of the field toward realistic manufacturing of functional organs have never been so extensive, and this manuscript serves as a road map for some of the recent progress and the challenges ahead.

It is often assumed that carbon nanotubes (CNTs) stimulate neuronal differentiation by transferring electrical signals and enhancing neuronal excitability. Given this, CNT-hydrogel composites are regarded as potential materials able to combine high electrical conductivity with biocompatibility, and therefore promote nerve regeneration. However, whether CNT-hydrogel composites actually influence neuronal differentiation and maturation, and how they do so remain elusive. In this study, CNT-hydrogel composites are prepared by in situ polymerization of poly(ethylene glycol) around a preformed CNT meshwork. It is demonstrated that the composites facilitate long-term survival and differentiation of pheochromocytoma 12 cells. Adult neural stem cells cultured on the composites show an increased neuron-to-astrocyte ratio and higher synaptic connectivity. Moreover, primary hippocampal neurons cultured on composites maintain morphological synaptic features as well as their neuronal network activity evaluated by spontaneous calcium oscillations, which are comparable to neurons cultured under control conditions. These results indicate that the composites are promising materials that could indeed facilitate neuronal differentiation while maintaining neuronal homeostasis.

Three-dimensional (3D) multicellular spheroids are a new generation in vitro cell model, however, their applications are severely limited by difficulties in their generation. Here patterned poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel films were synthesized for their generation. Instead of polymerization of HEMA monomers in the presence of a cross-linker, here the PHEMA films were synthesized by cross-linking furan-functionalized linear PHEMA, PHEMA-furan, and maleimide-functionalized linear PHEMA, PHEMA-mal, via Diels-Alder (DA) reaction between furan and maleimide groups. A thermal treatment temperature of 75 °C was chosen for the cross-linking reaction. The occurrence of DA reaction was confirmed by IR spectra. Using this method, cross-linked PHEMA films with smooth surface were successfully synthesized in situ in the well of cell culture plates. The films were then patterned by simply adding water to swell them. Highly ordered, honeycomb-like wrinkling patterns were successfully obtained by adjusting the furan and maleimide contents in the precursor linear polymers. The patterned hydrogel films were used to generate multicellular spheroids. Guided by the patterns, 3D spheroids with narrow size distribution, tunable size, and high cell viability were successfully obtained. The patterned PHEMA films reported here exhibited a lot of advantages. The patterning method was quite simple and required no template or special equipment. They were synthesized in situ in commercial cell culture plates. Particularly, thanks to the clean nature of the DA reaction, no low molecular weight monomer, cross-linker, initiator, or catalyst, which were potentially cytotoxic, was involved in the film synthesis, and no byproduct was produced and left in the film. The resulting films presented a high biocompatibility, allowing the avoidance of the tedious washing step. The films synthesized here were expected to have high potential for massive production of well-defined multicellular spheroids.

Many existing clinical treatments are limited in their ability to completely restore decreased or lost tissue and organ function, an unenviable situation only further exacerbated by a globally aging population. As a result, the demand for new medical interventions has increased substantially over the past 20 years, with the burgeoning fields of gene therapy, tissue engineering, and regenerative medicine showing promise to offer solutions for full repair or replacement of damaged or aging tissues. Success in these fields, however, inherently relies on biomaterials that are engendered with the ability to provide the necessary biological cues mimicking native extracellular matrixes that support cell fate. Accelerating the development of such "directive" biomaterials requires a shift in current design practices toward those that enable rapid synthesis and characterization of polymeric materials and the coupling of these processes with techniques that enable similarly rapid quantification and optimization of the interactions between these new material systems and target cells and tissues. This manuscript reviews recent advances in combinatorial and high-throughput (HT) technologies applied to polymeric biomaterial synthesis, fabrication, and chemical, physical, and biological screening with targeted end-point applications in the fields of gene therapy, tissue engineering, and regenerative medicine. Limitations of, and future opportunities for, the further application of these research tools and methodologies are also discussed.

The interrogation of single cells has revolutionised biology and medicine by providing crucial unparalleled insights into cell-to-cell heterogeneity. Flow cytometry (including fluorescence-activated cell sorting) is one of the most versatile and high-throughput approaches for single-cell analysis by detecting multiple fluorescence parameters of individual cells in aqueous suspension as they flow past through a focus of excitation lasers. However, this approach relies on the expression of cell surface and intracellular biomarkers, which inevitably lacks spatial and temporal phenotypes and activities of cells, such as secreted proteins, extracellular metabolite production, and proliferation. Droplet microfluidics has recently emerged as a powerful tool for the encapsulation and manipulation of thousands to millions of individual cells within pico-litre microdroplets. Integrating flow cytometry with microdroplet architectures surrounded by aqueous solutions (e.g., water-in-oil-in-water (W/O/W) double emulsion and hydrogel droplets) opens avenues for new cellular assays linking cell phenotypes to genotypes at the single-cell level. In this review, we discuss the capabilities and applications of droplet flow cytometry (DFC). This unique technique uses standard commercially available flow cytometry instruments to characterise or select individual microdroplets containing single cells of interest. We explore current challenges associated with DFC and present our visions for future development.

This study demonstrates the rapid fabrication and utility of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer film for cell patterning. The film was obtained on a cell culture surface by microcasting MPC polymer ethanol solution into a degassed polydimethylsiloxane mold with a desired pattern. After removal of the mold, 293AD cells were cultured on the surface of the polymer film with the patterned microstructures. Patterned cell adhesion restricted by the film was successfully maintained during at least a 168-h cultivation. The microcast MPC polymer film can be prepared rapidly and used for efficient long-term cell confinement.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor, accounting for 50% of all cases. GBM patients have a five-year survival rate of merely 5.6% and a median overall survival of 14.6 months with the "Stupp" regimen, 20.9 months with tumor treatment fields (TTF, OptuneR) in patients who participated in clinical trials, and 11 months for all GBM patients prior to TTF use. Objective: Our group recently developed a brain cancer chip which generates tumor spheroids, and provides large-scale assessments on the response of tumor cells to various concentrations and combinations of drugs. This platform could optimize the use of tumor samples derived from GBM patients to provide valuable insight on the tumor growth and responses to drug therapies. To minimize any sample loss in vitro, we improved our brain cancer chip system by adding an additional laminar flow distribution layer, which reduces sample loss during cell seeding and prevents spheroids from escaping from the microwells. Methods: In this study, we cultured 3D spheroids from GBM cell lines and patient-derived GBM cells in vitro, and investigated the effect of the combination of Temozolomide and nuclear factor-κB inhibitor on tumor growth. Results: Our study revealed that these drugs have synergistic effects in inhibiting spheroid formation when used in combination. Conclusions: These results suggest that the brain cancer chip enables large-scale, inexpensive and sample-effective drug screening to 3D cancer tumors in vitro, and could be applied to related tissue engineering drug screening studies.

Despite the precise controllability of droplet samples in digital microfluidic (DMF) systems, their capability in isolating single cells for long-time culture is still limited: typically, only a few cells can be captured on an electrode. Although fabricating small-sized hydrophilic micropatches on an electrode aids single-cell capture, the actuation voltage for droplet transportation has to be significantly raised, resulting in a shorter lifetime for the DMF chip and a larger risk of damaging the cells. In this work, a DMF system with 3D microstructures engineered on-chip is proposed to form semi-closed micro-wells for efficient single-cell isolation and long-time culture. Our optimum results showed that approximately 20% of the micro-wells over a 30 × 30 array were occupied by isolated single cells. In addition, low-evaporation-temperature oil and surfactant aided the system in achieving a low droplet actuation voltage of 36V, which was 4 times lower than the typical 150 V, minimizing the potential damage to the cells in the droplets and to the DMF chip. To exemplify the technological advances, drug sensitivity tests were run in our DMF system to investigate the cell response of breast cancer cells (MDA-MB-231) and breast normal cells (MCF-10A) to a widely used chemotherapeutic drug, Cisplatin (Cis). The results on-chip were consistent with those screened in conventional 96-well plates. This novel, simple and robust single-cell trapping method has great potential in biological research at the single cell level.

The McDevitt group has sustained efforts to develop a programmable sensing platform that offers advanced, multiplexed/multiclass chem-/bio-detection capabilities. This scalable chip-based platform has been optimized to service real-world biological specimens and validated for analytical performance. Fashioned as a sensor that learns, the platform can host new content for the application at hand. Identification of biomarker-based fingerprints from complex mixtures has a direct linkage to e-nose and e-tongue research. Recently, we have moved to the point of big data acquisition alongside the linkage to machine learning and artificial intelligence. Here, exciting opportunities are afforded by multiparameter sensing that mimics the sense of taste, overcoming the limitations of salty, sweet, sour, bitter, and glutamate sensing and moving into fingerprints of health and wellness. This article summarizes developments related to the electronic taste chip system evolving into a platform that digitizes biology and affords clinical decision support tools. A dynamic body of literature and key review articles that have contributed to the shaping of these activities are also highlighted. This fully integrated sensor promises more rapid transition of biomarker panels into wide-spread clinical practice yielding valuable new insights into health diagnostics, benefiting early disease detection.

The development of microfluidic hydrogels is an attractive method to generate continuous perfusion, induce vascularization, increase solute delivery, and ultimately improve cell viability. However, the transport processes in many in vitro studies still have not been realized completely. To address this problem, we have developed a microchanneled hydrogel with different collagen type I concentrations of 1, 2, and 3 wt% and assessed its physical properties and obtained diffusion coefficient of nutrient within the hydrogel. It is well known that microchannel geometry has critical role in maintaining stable perfusion rate. Therefore, in this study, a computational modeling was applied to simulate the 3D microfluidic hydrogel and study the effect of geometric parameters such as microchannel diameters and their distance on the nutrient diffusion. The simulation results showed that the sample with 3 channels with a diameter of 300 μm has adequate diffusion rates and efficiency (56%). Moreover, this system provides easy control and continuous perfusion rate during 5 days of cell culturing. The simulation results were compared with experimental data, and a good correlation was observed for nutrient profiles and cell viability across the hydrogel.

Conventional cell-sized well arrays have advantages of high occupancy, simple operation, and low cost for capturing single-cells. However, they have insufficient space for including reagents required for cell treatment or analysis, which restricts the wide application of cell-sized well arrays as a single-cell research tool alone. Here, we present a novel dual-well array chip, which integrates capture-wells (20 μm in diameter) with reaction-wells (100 μm in diameter) and describe a flow method for convenient single-cell analysis requiring neither complicated infra-structure nor high expenditure, while enabling highly efficient single cell trapping (75.8%) with only 11.3% multi-cells. Briefly, the cells are first loaded into the dual-wells by gravity and then multi-cells in the reaction-wells are washed out by phosphate buffer saline. Next, biochemical reagents are loaded into reaction-wells using the scraping method and the chip is packed as a sandwich structure. We thereby successfully measured intracellular β-galactosidase activity of K562 cells at the single-cell level. We also used computational simulations to illustrate the working principle of dual-well structure and found out a relationship between the wall shear stress distribution and the aspect ratio of the dual-well array chip which provides theoretical guidance for designing multi-wells chip for convenient single-cell analysis. Our work produced the first dual-well chip that can simultaneously provide a high occupancy rate for single cells and sufficient space for reagents, as well as being low in cost and simple to operate. We believe that the feasibility and convenience of our method will enhance its use as a practical single-cell research tool.

We have developed a sequential stereolithographic co-printing process using two different resins for fabricating porous barriers in microfluidic devices. We 3D-printed microfluidic channels with a resin made of poly(ethylene glycol) diacrylate (MW = 258) (PEG-DA-258), a UV photoinitiator, and a UV sensitizer. The porous barriers were created within the microchannels in a different resin made of either PEG-DA (MW = 575) (PEG-DA-575) or 40% (w/w in water) PEG-DA (MW = 700) (40% PEG-DA-700). We showed selective hydrogen ion diffusion across a 3D-printed PEG-DA-575 porous barrier in a cross-channel diffusion chip by observing color changes in phenol red, a pH indicator. We also demonstrated the diffusion of fluorescein across a 3D-printed 40% PEG-DA-700 porous barrier in a symmetric-channel diffusion chip by measuring fluorescence intensity changes across the porous barrier. Creating microfluidic chips with integrated porous barriers using a semi-automated 3D printing process shortens the design and processing time, avoids assembly and bonding complications, and reduces manufacturing costs compared to micromolding processes. We believe that our digital manufacturing method for fabricating selective porous barriers provides an inexpensive, simple, convenient and reproducible route to molecule delivery in the fields of molecular filtration and cell-based microdevices.

We herein report the preparation of a surface that behaves in a hydrophobic manner but does not undergo protein adsorption in an aqueous/organic two-phase system. We found that polyethylene-glycol (PEG)-modified poly(dimethylsiloxane) (PDMS) exhibits hydrophobic properties when the surface is immersed in an organic solution, while the PEG moiety prevents protein adsorption on the PDMS surface in an aqueous solution at high protein concentrations due to the dynamic behaviour of the PEG moiety. As such, we demonstrated the in-well droplet formation of an aqueous solution containing a high protein concentration. In addition, to demonstrate the feasibility of this method in single cell analyses, a droplet array of a liquid medium containing 10% fetal bovine serum and HeLa cells was formed. The preparation of a droplet array using our PDMS-PEG surface to promote in-well droplet formation avoided the use of flow control equipment and complicated microstructures. We therefore expect that the dynamic wettability of our reported surface will be applicable in single cell and biochemical analyses, such as protein characterisation using crystallography or immunoassays.

Controllable organization and transport of cell chain in a fluid, which is of great importance in biological and medical fields, have attracted increasing attentions in recent years. Here we demonstrate an optofluidic strategy, by implanting the microfluidic technique with a large-tapered-angle fiber probe (LTAP), to organize and transport a cell chain in a noncontact and noninvasive manner. After a laser beam at 980-nm wavelength launched into LTAP, the E. coli cells were continuously trapped and then arranged into a cell chain one after another. The chain can be transported by adjusting the magnitudes of optical force and flow drag force. The proposed technique can also be applied for the eukaryotic cells (e. g., yeast cell) and human red blood cells (RBCs). Experiment results were interpreted by the numerical simulation, and the stiffness of cell chain was also discussed.

Array-format cell-culture carriers providing tunable matrix cues are instrumental in current cell biology and bioengineering. A new solvent-assisted demolding approach for the fabrication of microcavity arrays with very small feature sizes down to single-cell level (3 µm) of very soft biohybrid glycosaminoglycan-poly(ethylene glycol) hydrogels (down to a shear modulus of 1 kPa) is reported. It is further shown that independent additional options of localized conjugation of adhesion ligand peptides, presentation of growth factors through complexation to gel-based glycosaminoglycans, and secondary gel deposition for 3D cell embedding enable a versatile customization of the hydrogel microcavity arrays for cell culture studies. As a proof of concept, cell-instructive hydrogel compartment arrays are used to analyze the response of human hematopoietic stem and progenitor cells to defined biomolecular and spatial cues.

Additive manufacturing, or 3D-printing techniques have recently begun to enable simpler, faster, and cheaper production of millifluidic devices at resolutions approaching 100-200 μm. At this resolution, cell culture devices can be constructed that more accurately replicate natural environments compared with conventional culturing techniques. A number of microfluidics researchers have begun incorporating additive manufacturing into their work, using 3D-printed devices in a wide array of chemical, fluidic, and even some biological applications. Here, we describe a 3D-printed cell culture platform and demonstrate its use in culturing Pseudomonas putida KT2440 bacteria for 44 h under a differential substrate gradient. Polyethylene glycol diacrylate (PEGDA) hydrogel barriers are patterned in situ within a 3D-printed channel. Transport of the toluidine blue tracer dye through the hydrogel barriers is characterized. Nutrients and oxygen were delivered to cells in the culture region by diffusion through the PEGDA hydrogel barriers from adjacent media or saline perfusion channels. Expression of green fluorescent protein by P. putida KT2440 enabled real time visualization of cell density within the 3D-printed channel, and demonstrated cells were actively expressing protein over the course of the experiment. Cells were observed clustering near hydrogel barrier boundaries where fresh substrate and oxygen were being delivered via diffusive transport, but cells were unable to penetrate the barrier. The device described here provides a versatile and easy to implement platform for cell culture in readily controlled gradient microenvironments. By adjusting device geometry and hydrogel properties, this platform could be further customized for a wide variety of biological applications.

Dielectric spectroscopy (DS) is a noninvasive, label-free, fast, and promising technique for measuring dielectric properties of biological cells in real time. We demonstrate a microchip that consists of electro-activated microwell arrays for positive dielectrophoresis assisted cell capture, DS measurements, and negative dielectrophoresis driven cell unloading; thus, providing a high-throughput cell analysis platform. To the best of our knowledge, this is the first microfluidic chip that combines electro-activated microwells and DS to analyze biological cells. Device performance is tested using Saccharomyces cerevisiae (yeast) cells. DEP response of yeast cells is determined by measuring their Clausius-Mossotti factor using biophysical models in parallel plate microelectrode geometry. This information is used to determine the excitation frequency to load and unload wells. Effect of yeast cells on the measured impedance spectrum was examined both experimentally and numerically. Good match between the numerical and experimental results establishes the potential use of the microchip device for extracting subcellular properties of biological cells in a rapid and nonexpensive manner.

Heterogeneity among individual molecules and cells has posed significant challenges to traditional bulk assays, due to the assumption of average behavior, which would lose important biological information in heterogeneity and result in a misleading interpretation. Single molecule/cell analysis has become an important and emerging field in biological and biomedical research for insights into heterogeneity between large populations at high resolution. Compared with the ensemble bulk method, single molecule/cell analysis explores the information on time trajectories, conformational states, and interactions of individual molecules/cells, all key factors in the study of chemical and biological reaction pathways. Various powerful techniques have been developed for single molecule/cell analysis, including flow cytometry, atomic force microscopy, optical and magnetic tweezers, single-molecule fluorescence spectroscopy, and so forth. However, some of them have the low-throughput issue that has to analyze single molecules/cells one by one. Flow cytometry is a widely used high-throughput technique for single cell analysis but lacks the ability for intercellular interaction study and local environment control. Droplet microfluidics becomes attractive for single molecule/cell manipulation because single molecules/cells can be individually encased in monodisperse microdroplets, allowing high-throughput analysis and manipulation with precise control of the local environment. Moreover, hydrogels, cross-linked polymer networks that swell in the presence of water, have been introduced into droplet microfluidic systems as hydrogel droplet microfluidics. By replacing an aqueous phase with a monomer or polymer solution, hydrogel droplets can be generated on microfluidic chips for encapsulation of single molecules/cells according to the Poisson distribution. The sol-gel transition property endows the hydrogel droplets with new functionalities and diversified applications in single molecule/cell analysis. The hydrogel can act as a 3D cell culture matrix to mimic the extracellular environment for long-term single cell culture, which allows further heterogeneity study in proliferation, drug screening, and metastasis at the single-cell level. The sol-gel transition allows reactions in solution to be performed rapidly and efficiently with product storage in the gel for flexible downstream manipulation and analysis. More importantly, controllable sol-gel regulation provides a new way to maintain phenotype-genotype linkages in the hydrogel matrix for high throughput molecular evolution. In this Account, we will review the hydrogel droplet generation on microfluidics, single molecule/cell encapsulation in hydrogel droplets, as well as the progress made by our group and others in the application of hydrogel droplet microfluidics for single molecule/cell analysis, including single cell culture, single molecule/cell detection, single cell sequencing, and molecular evolution.

There is an immense need for tissue engineered blood vessels. However, current tissue engineering approaches still lack the ability to build native blood vessel-like perfusable structures with multi-layered vascular walls. This paper demonstrated a new method to fabricate tri-layer biomimetic blood vessel-like structures on a microfluidic platform using photocrosslinkable gelatin hydrogel. The presented method enables fabrication of physiological blood vessel-like structures with mono-, bi- or tri-layer vascular walls. The diameter of the vessels, the total thickness of the vessel wall and the thickness of each individual layer of the wall were independently controlled. The developed fabrication process is a simple and rapid method, allowing the physical fabrication of the vascular structure in minutes, and the formation of a vascular endothelial cell layer inside the vessels in 3-5 days. The fabricated vascular constructs can potentially be used in numerous applications including drug screening, development of in vitro models for cardiovascular diseases and/or cancer metastasis, and study of vascular biology and mechanobiology.

Multiarm hydrogel microparticles with varying geometry are fabricated to specifically capture cells expressing epithelial cell adhesion molecule. Results show that particle shape influences cell-capture efficiency due to differences in surface area, hydrodynamic effects, and steric constraints. These findings can lead to improved particle design for cell separation and diagnostic applications.

With the increasing amount of research work in surface studies, a more effective method of producing patterned microstructures is highly desired due to the geometric limitations and complex fabricating process of current techniques. This paper presents an efficient and cost-effective method to generate customizable micro-wavy pattern using direct image lithography. This method utilizes a grayscale Gaussian distribution effect to model inaccuracies inherent in the polymerization process, which are normally regarded as trivial matters or errors. The measured surface profiles and the mathematical prediction show a good agreement, demonstrating the ability of this method to generate wavy patterns with precisely controlled features. An accurate pattern can be generated with customizable parameters (wavelength, amplitude, wave shape, pattern profile, and overall dimension). This mask-free photolithography approach provides a rapid fabrication method that is capable of generating complex and non-uniform 3D wavy patterns with the wavelength ranging from 12 μm to 2100 μm and an amplitude-to-wavelength ratio as large as 300%. Microfluidic devices with pure wavy and wavy-herringbone patterns suitable for capture of circulating tumor cells are made as a demonstrative application. A completely customized microfluidic device with wavy patterns can be created within a few hours without access to clean room or commercial photolithography equipment.

During the last decades, the engineering of well-defined 3D tissues has attracted great attention because it provides in vivo mimicking environment and can be a building block for the engineering of bioartificial organs. In this Review, diverse engineering methods of 3D tissues using microscale devices are introduced. Recent progress of microtechnologies has enabled the development of microplatforms for bottom-up assembly of diverse shaped 3D tissues consisting of various cells. Micro hanging-drop plates, microfluidic chips, and arrayed microwells are the typical examples. The encapsulation of cells in hydrogel microspheres and microfibers allows the engineering of 3D microtissues with diverse shapes. Applications of 3D microtissues in biomedical fields are described, and the future direction of microplatform-based engineering of 3D micro-tissues is discussed.

In this chapter the state of the art of live cell microarrays for high-throughput biological assays are reviewed. The fabrication of novel microarrays with respect to material science and cell patterning methods is included. A main focus of the chapter is on various aspects of the application of cell microarrays by providing selected examples in research fields such as biomaterials, stem cell biology and neuroscience. Additionally, the importance of microfluidic technologies for high-throughput on-chip live-cell microarrays is highlighted for single-cell and multi-cell assays as well as for 3D tissue constructs.

Despite significant investments in cancer research and drug discovery/development, the rate of new cancer drug approval is ≤5% and most cases of metastatic cancer remain incurable. Ninety-five percent of new cancer drugs fail in clinical development because of a lack of therapeutic efficacy and/or unacceptable toxicity. One of the major factors responsible for the low success rate of anticancer drug development is the failure of preclinical models to adequately recapitulate the complexity and heterogeneity of human cancer. For throughput and capacity reasons, high-throughput screening growth inhibition assays almost exclusively use two-dimensional (2D) monolayers of tumor cell lines cultured on tissue culture-treated plastic/glass surfaces in serum-containing medium. However, these 2D tumor cell line cultures fail to recapitulate the three-dimensional (3D) context of cells in solid tumors even though the tumor microenvironment has been shown to have a profound effect on anticancer drug responses. Tumor spheroids remain the best characterized and most widely used 3D models; however, spheroid sizes tend to be nonuniform, making them unsuitable for high-throughput drug testing. To circumvent this challenge, we have developed defined size microwell arrays using nonadhesive hydrogels that are applicable to a wide variety of cancer cell lines to fabricate size-controlled 3D microtumors. We demonstrate that the hydrogel microwell array platform can be applied successfully to generate hundreds of uniform microtumors within 3-6 days from many cervical and breast, as well as head and neck squamous cell carcinoma (HNSCC) cells. Moreover, controlling size of the microwells in the hydrogel array allows precise control over the size of the microtumors. Finally, we demonstrate the application of this platform technology to probe activation as well as inhibition of epidermal growth factor receptor (EGFR) signaling in 3D HNSCC microtumors in response to EGF and cetuximab treatments, respectively. We believe that the ability to generate large numbers of HNSCC microtumors of uniform size and 3D morphology using hydrogel arrays will provide more physiological in vitro 3D tumor models to investigate how tumor size influences signaling pathway activation and cancer drug efficacy.

Single cell investigations have enabled unexpected discoveries, such as the existence of biological noise and phenotypic switching in infection, metabolism and treatment. Herein, we review methods that enable such single cell investigations specific to metabolism and bioenergetics. Firstly, we discuss how to isolate and immobilize individuals from a cell suspension, including both permanent and reversible approaches. We also highlight specific advances in microbiology for its implications in metabolic engineering. Methods for probing single cell physiology and metabolism are subsequently reviewed. The primary focus therein is on dynamic and high-content profiling strategies based on label-free and fluorescence microspectroscopy and microscopy. Non-dynamic approaches, such as mass spectrometry and nuclear magnetic resonance, are also briefly discussed.

The kinetics of stem and progenitor cell differentiation at the single-cell level provides essential clues to the complexity of the underlying decision-making circuits. In many hematopoietic progenitor cells, differentiation is accompanied by the expression of lineage-specific markers and by a transition from a non-adherent to an adherent state. Here, using the granulocyte-macrophage progenitor (GMP) as a model, we introduce a label-free approach that allows one to follow the course of this transition in hundreds of single cells in parallel. We trap single cells in patterned arrays of micro-wells and use phase-contrast time-lapse movies to distinguish non-adherent from adherent cells by an analysis of Brownian motion. This approach allowed us to observe the kinetics of induced differentiation of primary bone-marrow-derived GMPs into macrophages. The time lapse started 2 hours after addition of the cytokine M-CSF, and nearly 80% of the population had accomplished the transition within the first 20 h. The analysis of Brownian motion proved to be a sensitive and robust tool for monitoring the transition, and thus provides a high-throughput method for the study of cell differentiation at the single-cell level.

Micro-Electro-Mechanical-Systems (MEMS) are desirable for use within medical diagnostics because of their capacity to manipulate and analyze biological materials at the microscale. Biosensors can be incorporated into portable lab-on-a-chip devices to quickly and reliably perform diagnostics procedure on laboratory and clinical samples. In this paper, electrical impedance-based measurements were used to distinguish between benign and cancerous breast tissues using microchips in a real-time and label-free manner. Two different microchips having inter-digited electrodes (10 µm width with 10 µm spacing and 10 µm width with 30 µm spacing) were used for measuring the impedance of breast tissues. The system employs Agilent E4980A precision impedance analyzer. The impedance magnitude and phase were collected over a frequency range of 100 Hz to 2 MHz. The benign group and cancer group showed clearly distinguishable impedance properties. At 200 kHz, the difference in impedance of benign and cancerous breast tissue was significantly higher (3110 Ω) in the case of microchips having 10 µm spacing compared to microchip having 30 µm spacing (568 Ω).

A simple method to generate cell microarrays with high-percentage well occupancy and well-defined cell confinement is presented. This method uses a synergistic combination of vacuum degassing and coverslip sweeping. The vacuum degassing step dislodges air bubbles from the microwells, which in turn enables the cells to enter the microwells, while the physical sweeping step using a glass coverslip removes the excess cells outside the microwells. This low-cost preparation method provides a simple solution to generating cell microarrays that can be performed in basic research laboratories and point-of-care settings for routine cell-based screening assays.

The combination of microfabrication-based technologies with cell biology has laid the foundation for the development of advanced in vitro diagnostic systems capable of analyzing cell cultures under physiologically relevant conditions. In the present review, we address recent lab-on-a-chip developments for stem cell analysis. We highlight in particular the tangible advantages of microfluidic devices to overcome most of the challenges associated with stem cell identification, expansion and differentiation, with the greatest advantage being that lab-on-a-chip technology allows for the precise regulation of culturing conditions, while simultaneously monitoring relevant parameters using embedded sensory systems. State-of-the-art lab-on-a-chip platforms for in vitro assessment of stem cell cultures are presented and their potential future applications discussed.

We present a microfluidic device designed for maintenance and culture of non-adherent mammalian cells, which enables both recirculation and refreshing of medium, as well as easy harvesting of cells from the device. We demonstrate fabrication of a novel microfluidic device utilizing Braille perfusion for peristaltic fluid flow to enable switching between recirculation and refresh flow modes. Utilizing fluid flow simulations and the human promyelocytic leukemia cell line, HL-60, non-adherent cells, we demonstrate the utility of this RECIR-REFRESH device. With computer simulations, we profiled fluid flow and concentration gradients of autocrine factors and found that the geometry of the cell culture well plays a key role in cell entrapping and retaining autocrine and soluble factors. We subjected HL-60 cells, in the device, to a treatment regimen of 1.25% dimethylsulfoxide, every other day, to provoke differentiation and measured subsequent expression of CD11b on day 2 and day 4 and tumor necrosis factor-alpha (TNF-α) on day 4. Our findings display perfusion sensitive CD11b expression, but not TNF-α build-up, by day 4 of culture, with a 1:1 ratio of recirculation to refresh flow yielding the greatest increase in CD11b levels. RECIR-REFRESH facilitates programmable levels of cell differentiation in a HL-60 non-adherent cell population and can be expanded to other types of non-adherent cells such as hematopoietic stem cells.