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.

Reproduction of native tissues in vitro is important as a tool, as it both enables investigation of fundamental biological processes, and drug and toxicity screenings. In order to closely mimic complex tissues in vitro, artificial multicellular systems are created from different cell types in spatially ordered structures or welldefined geometries in a 3D microenvironment. [1-3] These systems can be built from building blocks [4-7] such as cell sheets, [8] cell-laden microgels, [5] cell spheroids, [9] and organoids. [10,11] Precise control of cellular composition and spatial distribution of building blocks within artificial multicellular systems allows for reconstitution of native tissues in their healthy and disease state in vitro. [12] There are a number of methodologies developed for fabrication of complex 3D cell systems in vitro. [3-7,13,14] Directed assembly allows manual positioning or stacking building blocks to form 3D architectures. [15,16] Birey et al. applied this method for fusion of two forebrain organoids in order to mimic the human brain development and demonstrate inter-neuronal migration. [15] The method of directed assembly is, however, manual and not compatible with high throughput. Remote assembly, such as, acoustic node, [14,17] magnetic cell levitation, [13,18] optical tweezers, [19] or laser-guided direct writing, [20] can achieve assembly of cells or spheroids against gravity or viscous forces. Chen et al. demonstrated the assembly of hepatic organoids by an acoustic node technique, and the technique was able to achieve formation of bile canaliculi networks resembling native hepatic tissue. [17] Souza et al. used magnetic cell levitation to manipulate a glioblastoma cell spheroid, and a human astrocyte spheroid in order to create a cell invasion model. [18] These methods depend on sophisticated equipment, paramagnetic media, or introduce a risk of laser-induced cell damage. Another common strategy used to fabricate multicellular architecture is assembly of cell-laden hydrogels or microgels, [5,21,22] or cell seeding on scaffolds. [7,23] However, these biomaterial-based methods failed to provide high cell packing density. The use of artificial scaffolds or gel matrices additionally lead to disadvantages for constructing 3D tissue models due to their influence on cell-cell interactions, autocrine, and paracrine signaling. 3D printing [3] is a promising method for designing and achieving multicellular architectures, but it is relatively slow, not always compatible with high throughput and relies on printable bio-inks for maintaining 3D structure and cell viability. 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 prog...

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.

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.

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.

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.

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.

Hydrogels are important functional materials useful for 3D cell culture, tissue engineering, 3D printing, drug delivery, sensors, or soft robotics. The ability to shape hydrogels into defined 3D structures, patterns, or particles is crucial for biomedical applications. Here, the rapid photodegradability of commonly used polymethacrylate hydrogels is demonstrated without the need to incorporate additional photolabile functionalities. Hydrogel degradation depths are quantified with respect to the irradiation time, light intensity, and chemical composition. It can be shown that these parameters can be utilized to control the photodegradation behavior of polymethacrylate hydrogels. The photodegradation kinetics, the change in mechanical properties of polymethacrylate hydrogels upon UV irradiation, as well as the photodegradation products are investigated. This approach is then exploited for microstructuring and patterning of hydrogels including hydrogel gradients as well as for the formation of hydrogel particles and hydrogel arrays of welldefined shapes. Cell repellent but biocompatible hydrogel microwells are fabricated using this method and used to form arrays of cell spheroids. As this method is based on readily available and commonly used methacrylates and can be conducted using cheap UV light sources, it has vast potential to be applied by laboratories with various backgrounds and for diverse applications.various applications in biotechnological and biomedical fields, including actuators, [2] sensors, [3] artificial muscles, [4] drug delivery, [1a] and tissue engineering. [5] These and many other applications require hydrogels with well-defined chemical and physical properties, shape, topography, mechanical properties, porosity and biocompatibility. [6] Several types of photodegradable hydrogels have been introduced in literature to prepare microstructured soft materials or to define chemical and physical properties with spatiotemporal control. [7] However, to achieve hydrogel photodegradability, artificial photoresponsive units or crosslinkers had to be synthesized and incorporated into the hydrogel network, which would then serve as breaking points under UV light. Such photodegradable moieties include o-nitrobenzyl ester, [8] coumarins, [9] disulfides, [10] Ru II polypyridyl complexes, [11] etc. The limited number of available photolabile groups and the need for their synthesis and incorporation into the hydrogel network significantly limit the number and scope of possible applications of photodegradable hydrogels for a broader audience. Here we demonstrate, for the first time, that one of the most commonly used types of hydrogels, i.e., hydrogels based on hydrophilic polymethacrylates, can be efficiently degraded using UV light without requiring any external photolabile moieties ( Figure 1A). The different