Facile One Step Formation and Screening of Tumor Spheroids Using Droplet-Microarray Platform.

Tumor spheroids or microtumors are important 3D in vitro tumor models that closely resemble a tumor's in vivo "microenvironment" compared to 2D cell culture. Microtumors are widely applied in the fields of fundamental cancer research, drug discovery, and precision medicine. In precision medicine tumor spheroids derived from patient tumor cells represent a promising system for drug sensitivity and resistance testing. Established and commonly used platforms for routine screenings of cell spheroids, based on microtiter plates of 96- and 384-well formats, require relatively large numbers of cells and compounds, and often lead to the formation of multiple spheroids per well. In this study, an application of the Droplet Microarray platform, based on hydrophilic-superhydrophobic patterning, in combination with the method of hanging droplet, is demonstrated for the formation of highly miniaturized single-spheroid-microarrays. Formation of spheroids from several commonly used cancer cell lines in 100 nL droplets starting with as few as 150 cells per spheroid within 24-48 h is demonstrated. Established methodology carries a potential to be adopted for routine workflows of high-throughput compound screening in 3D cancer spheroids or microtumors, which is crucial for the fields of fundamental cancer research, drug discovery, and precision medicine.

Free-standing three-dimensional hollow bacterial cellulose structures with controlled geometry via patterned superhydrophobic-hydrophilic surfaces.

Bacteria can produce cellulose, one of the most abundant biopolymer on earth, and emerge as an interesting candidate to fabricate advanced materials. Cellulose produced by Komagataeibacter Xylinus, a bacterial strain, is a pure water insoluble biopolymer, without hemicellulose or lignin. Bacterial cellulose (BC) exhibits a nanofibrous porous network microstructure with high strength, low density and high biocompatibility and it has been proposed as cell scaffold and wound healing material. The formation of three dimensional (3D) cellulose self-standing structures is not simple. It either involves complex multi-step synthetic procedures or uses chemical methods to dissolve cellulose and remold it. Here we present an in situ single-step method to produce self-standing 3D-BC structures with controllable wall thickness, size and geometry in a reproducible manner. Parameters such as hydrophobicity of the surfaces, volume of the inoculum and time of culture define the resulting 3D-BC structures. Hollow spheres and convex domes can be easily obtained by changing the surface wettability. The potential of these structures as a 3D cell scaffold is exemplified supporting the growth of mouse embryonic stem cells within a hollow spherical BC structure, indicating its biocompatibility and future prospective.

Assembly of Multi-Spheroid Cellular Architectures by Programmable Droplet Merging.

Artificial multicellular systems are gaining importance in the field of tissue engineering and regenerative medicine. Reconstruction of complex tissue architectures in vitro is nevertheless challenging, and methods permitting controllable and high-throughput fabrication of complex multicellular architectures are needed. Here, a facile and high-throughput method is developed based on a tunable droplet-fusion technique, allowing programmed assembly of multiple cell spheroids into complex multicellular architectures. The droplet-fusion technique allows for construction of various multicellular architectures (double-spheroids, multi-spheroids, hetero-spheroids) in a miniaturized high-density array format. As an example of application, the propagation of Wnt signaling is investigated within hetero-spheroids formed from two fused Wnt-releasing and Wnt-reporter cell spheroids. The developed method provides an approach for miniaturized, high-throughput construction of complex 3D multicellular architectures and can be applied for studying various biological processes including cell signaling, cancer invasion, embryogenesis, and neural development.

High-Throughput Screening of Cell Transfection Enhancers Using Miniaturized Droplet Microarrays.

DNA delivery is a powerful research tool for biological research and clinical therapies. However, many nonviral transfection reagents have relatively low transfection efficiency. It is hypothesized that by treating cells with small molecules, the transfection efficiency can be improved. However, in order to identify such transfection-enhancing molecules, thousands of molecules must be tested. Current high-throughput screening (HTS) technologies based on microtiter plates are not suitable for such screenings due to the prohibitively high costs of reagents and operation. Here, the use of the droplet microarray (DMA) platform to screen 774 FDA-approved drugs with CHO-K1, Jurkat and HEK293T cells is reported. The volume of individual aqueous compartments is 20 nL, requiring 0.84 mL of cell suspension and 200 pmoles of each drug (total 0.02 moles) to perform the screening. Thus, the requirement for cells and reagents is 2500 times less than that for the same experiment performed in 384-well plates. The results reveal the potential of the DMA platform as a more cost-effective and less labor-intensive approach to HTS. Furthermore, an increase (approximately two- to fivefold) in transfection efficiency is achieved by treating cells with some molecules. This study clearly demonstrates the potential of the DMA platform for miniaturization of biochemical and cellular HTS.

In vitro investigation of an intracranial flow diverter with a fibrin-based, hemostasis mimicking, nanocoating.

Flow diversion aims at treatment of intracranial aneurysms via vessel remodeling mechanisms, avoiding the implantation of foreign materials into the aneurysm sack. However, complex implantation procedure, high metal surface and hemodynamic disturbance still pose a risk for thromboembolic complications in the clinical praxis. A novel fibrin and heparin based nano coating considered as a hemocompatible scaffold for neointimal formation was investigated regarding thrombogenicity and endothelialization. The fibrin-heparin coating was compared to a bare metal as well as fibrin- or heparin-coated flow diverters. The implants were tested separately in regard to inflammation and coagulation markers in two different in vitro hemocompatibility models conducted with human whole blood (n = 5). Endothelialization was investigated through a novel dynamic in vitro cell seeding model containing primary human cells with subsequent viability assay. It was demonstrated that platelet loss and platelet activation triggered by presence of a bare metal stent could be significantly reduced by applying the fibrin-heparin, fibrin and heparin coating. Viability of endothelial cells after proliferation was similar in fibrin-heparin compared to bare metal implants, with a slight, non-significant improvement observed in the fibrin-heparin group. The results suggest that the presented nanocoating has the potential to reduce thromboembolic complications in a clinical setting. Though the new model allowed for endothelial cell proliferation under flow conditions, a higher number of samples is required to assess a possible effect of the coating.

Bacterial Cellulose Promotes Long-Term Stemness of mESC.

Stem cells possess unique properties, such as the ability to self-renew and the potential to differentiate into an organism's various cell types. These make them highly valuable in regenerative medicine and tissue engineering. Their properties are precisely regulated in vivo through complex mechanisms that include multiple cues arising from the cell interaction with the surrounding extracellular matrix, neighboring cells, and soluble factors. Although much research effort has focused on developing systems and materials that mimic this complex microenvironment, the controlled regulation of differentiation and maintenance of stemness in vitro remains elusive. In this work, we demonstrate, for the first time, that the nanofibrous bacterial cellulose (BC) membrane derived from Komagataeibacter xylinus can inhibit the differentiation of mouse embryonic stem cells (mESC) under long-term conditions (17 days), improving their mouse embryonic fibroblast (MEF)-free cultivation in comparison to the MEF-supported conventional culture. The maintained cells' pluripotency was confirmed by the mESCs' ability to differentiate into the three germ layers (endo-, meso-, and ectoderm) after having been cultured on the BC membrane for 6 days. In addition, the culturing of mESCs on flexible, free-standing BC membranes enables the quick and facile manipulation and transfer of stem cells between culture dishes, both of which significantly facilitate the use of stem cells in routine culture and various applications. To investigate the influence of the structural and topographical properties of the cellulose on stem cell differentiation, we used the cellulose membranes differing in membrane thickness, porosity, and surface roughness. This work identifies bacterial cellulose as a novel convenient and flexible membrane material enabling long-term maintenance of mESCs' stemness and significantly facilitating the handling and culturing of stem cells.

Droplet microarray: miniaturized platform for rapid formation and high-throughput screening of embryoid bodies.

Stem cells are influenced by various factors present in their in vivo microenvironment, such as interactions with neighboring cells, the extracellular matrix or soluble molecules. This demonstrates the high complexity of the in vivo microenvironment. Hence, many advances have been made in developing 3D screening models mimicking this complexity and the in vivo-like state in order to ensure more biomedically relevant investigations in drug discovery. In the field of stem cell research embryoid bodies are often used as relevant 3D systems. Embryoid bodies are embryonic stem cell aggregates that recapitulate the early embryonic development and that can differentiate into derivatives of the three germ layers. Embryoid bodies enable the investigation of processes underlying embryonic development, tissue generation and identification of drugs with developmental toxicity. The ability to perform high-throughput screenings using embryoid bodies could be extremely important to accelerate the progress in the field of stem cell research and embryonic development. To date, there are no simple methods to create high-density microarrays of embryoid bodies that further enable their high-throughput screening important for biomedical research. Here we demonstrate a new method that enables formation and high-throughput screening of embryoid bodies in arrays of defined, separated microdroplets. Using the superhydrophobic-hydrophilic micropattern of the droplet microarray, we demonstrate rapid and facile one-step formation of a dense array of multiple droplets containing homogeneous, single embryoid bodies. Thorough characterization of the influence of the initial cell number on embryoid body size, roundness and distribution was performed. We applied the embryoid body microarray to screen 774 FDA-approved compounds, identifying compounds with developmental toxicity such as mycophenolate mofetil or embryonic lethality such as eptifibatide. This work demonstrates the potential of the droplet microarray for the rapid formation of high-density microarrays of single embryoid bodies and their high-throughput drug screenings.

Miniaturized platform for high-throughput screening of stem cells.

Over the past decades stem cells have gained great interest in clinical research, tissue engineering and regenerative medicine, due to their ability of self-renewal and potential to differentiate into the various cell types of the organism. The long-term maintenance of these unique properties and the control of stem cell differentiation in vitro, however, remains challenging, thus limiting their applicability in these fields. High-throughput screening (HTS) of stem cells is widely used by the researchers in order to gain more insight in the underlying mechanisms of stem cell fate as well as identifying compounds and factors maintaining stemness. However, limited availability and expandability of stem cells restricts the use of microtiter plates for HTS of stem cells emitting the urge for miniaturized platforms. This review highlights recent advances in the development of miniaturized platforms for HTS of stem cells and presents novel designs of miniaturized HTS systems.

Droplet Microarray Based on Patterned Superhydrophobic Surfaces Prevents Stem Cell Differentiation and Enables High-Throughput Stem Cell Screening.

Over the past decades, stem cells have attracted growing interest in fundamental biological and biomedical research as well as in regenerative medicine, due to their unique ability to self-renew and differentiate into various cell types. Long-term maintenance of the self-renewal ability and inhibition of spontaneous differentiation, however, still remain challenging and are not fully understood. Uncontrolled spontaneous differentiation of stem cells makes high-throughput screening of stem cells also difficult. This further hinders investigation of the underlying mechanisms of stem cell differentiation and the factors that might affect it. In this work, a dual functionality of nanoporous superhydrophobic-hydrophilic micropatterns is demonstrated in their ability to inhibit differentiation of mouse embryonic stem cells (mESCs) and at the same time enable formation of arrays of microdroplets (droplet microarray) via the effect of discontinuous dewetting. Such combination makes high-throughput screening of undifferentiated mouse embryonic stem cells possible. The droplet microarray is used to investigate the development, differentiation, and maintenance of stemness of mESC, revealing the dependence of stem cell behavior on droplet volume in nano- and microliter scale. The inhibition of spontaneous differentiation of mESCs cultured on the droplet microarray for up to 72 h is observed. In addition, up to fourfold increased cell growth rate of mESCs cultured on our platform has been observed. The difference in the behavior of mESCs is attributed to the porosity and roughness of the polymer surface. This work demonstrates that the droplet microarray possesses the potential for the screening of mESCs under conditions of prolonged inhibition of stem cells' spontaneous differentiation. Such a platform can be useful for applications in the field of stem cell research, pharmacological testing of drug efficacy and toxicity, biomedical research as well as in the field of regenerative medicine and tissue engineering.

Droplet Microarray Based on Superhydrophobic-Superhydrophilic Patterns for Single Cell Analysis.

Single-cell analysis provides fundamental information on individual cell response to different environmental cues and is a growing interest in cancer and stem cell research. However, current existing methods are still facing challenges in performing such analysis in a high-throughput manner whilst being cost-effective. Here we established the Droplet Microarray (DMA) as a miniaturized screening platform for high-throughput single-cell analysis. Using the method of limited dilution and varying cell density and seeding time, we optimized the distribution of single cells on the DMA. We established culturing conditions for single cells in individual droplets on DMA obtaining the survival of nearly 100% of single cells and doubling time of single cells comparable with that of cells cultured in bulk cell population using conventional methods. Our results demonstrate that the DMA is a suitable platform for single-cell analysis, which carries a number of advantages compared with existing technologies allowing for treatment, staining and spot-to-spot analysis of single cells over time using conventional analysis methods such as microscopy.