Monochorionic Twinning in Bioengineered Human Embryo Models.

Monochorionic twinning of human embryos increases the risk of complications during pregnancy. The rarity of such twinning events, combined with ethical constraints in human embryo research, makes investigating the mechanisms behind twinning practically infeasible. As a result, there is a significant knowledge gap regarding the origins and early phenotypic presentation of monochorionic twin embryos. In this study, a microthermoformed-based microwell screening platform is used to identify conditions that efficiently induce monochorionic twins in human stem cell-based blastocyst models, termed "twin blastoids". These twin blastoids contain a cystic GATA3+ trophectoderm-like epithelium encasing two distinct inner cell masses (ICMs). Morphological and morphokinetic analyses reveal that twinning occurs during the cavitation phase via splitting of the OCT4+ pluripotent core. Notably, each ICM in twin blastoids contains its own NR2F2+ polar trophectoderm-like region, ready for implantation. This is functionally tested in a microfluidic chip-based implantation assay with epithelial endometrium cells. Under defined flow regimes, twin blastoids show increased adhesion capacity compared to singleton blastoids, suggestive of increased implantation potential. In conclusion, the development of technology enabling large-scale formation of twin blastoids, coupled with high-sensitivity readout capabilities, presents an unprecedented opportunity for systematically exploring monochorionic twin formation and its impact on embryonic development.

Multipotent adult progenitor cells prevent functional impairment and improve development in inflammation driven detriment of preterm ovine lungs.

Background: Perinatal inflammation increases the risk for bronchopulmonary dysplasia in preterm neonates, but the underlying pathophysiological mechanisms remain largely unknown. Given their anti-inflammatory and regenerative capacity, multipotent adult progenitor cells (MAPC) are a promising cell-based therapy to prevent and/or treat the negative pulmonary consequences of perinatal inflammation in the preterm neonate. Therefore, the pathophysiology underlying adverse preterm lung outcomes following perinatal inflammation and pulmonary benefits of MAPC treatment at the interface of prenatal inflammatory and postnatal ventilation exposures were elucidated. Methods: Instrumented ovine fetuses were exposed to intra-amniotic lipopolysaccharide (LPS 5 mg) at 125 days gestation to induce adverse systemic and peripheral organ outcomes. MAPC (10 × 106 cells) or saline were administered intravenously two days post LPS exposure. Fetuses were delivered preterm five days post MAPC treatment and either killed humanely immediately or mechanically ventilated for 72 h. Results: Antenatal LPS exposure resulted in inflammation and decreased alveolar maturation in the preterm lung. Additionally, LPS-exposed ventilated lambs showed continued pulmonary inflammation and cell junction loss accompanied by pulmonary edema, ultimately resulting in higher oxygen demand. MAPC therapy modulated lung inflammation, prevented loss of epithelial and endothelial barriers and improved lung maturation in utero. These MAPC-driven improvements remained evident postnatally, and prevented concomitant pulmonary edema and functional loss. Conclusion: In conclusion, prenatal inflammation sensitizes the underdeveloped preterm lung to subsequent postnatal inflammation, resulting in injury, disturbed development and functional impairment. MAPC therapy partially prevents these changes and is therefore a promising approach for preterm infants to prevent adverse pulmonary outcomes.

Microfluidically Aligned Collagen to Maintain the Phenotype of Tenocytes In Vitro.

Tendon is a highly organized tissue that transmits forces between muscle and bone. The architecture of the extracellular matrix of tendon, predominantly from collagen type I, is important for maintaining tenocyte phenotype and function. Therefore, in repair and regeneration of damaged and diseased tendon tissue, it is crucial to restore the aligned arrangement of the collagen type I fibers of the original matrix. To this end, a novel, user-friendly microfluidic piggyback platform is developed allowing the controlled patterned formation and alignment of collagen fibers simply on the bottom of culture dishes. Rat tenocytes cultured on the micropatterns of aligned fibrous collagen exhibit a more elongated morphology. The cells also show an increased expression of tenogenic markers at the gene and protein level compared to tenocytes cultured on tissue culture plastic or non-fibrillar collagen coatings. Moreover, using imprinted polystyrene replicas of aligned collagen fibers, this work shows that the fibrillar structure of collagen per se affects the tenocyte morphology, whereas the biochemical nature of collagen plays a prominent role in the expression of tenogenic markers. Beyond the controlled provision of aligned collagen, the microfluidic platform can aid in developing more physiologically relevant in vitro models of tendon and its regeneration.

Automated, High-Throughput Phenotypic Screening and Analysis Platform to Study Pre- and Post-Implantation Morphogenesis in Stem Cell-Derived Embryo-Like Structures.

Combining high-throughput generation and high-content imaging of embryo models will enable large-scale screening assays in the fields of (embryo) toxicity, drug development, embryogenesis, and reproductive medicine. This study shows the continuous culture and in situ (i.e., in microwell) imaging-based readout of a 3D stem cell-based model of peri-implantation epiblast (Epi)/extraembryonic endoderm (XEn) development with an expanded pro-amniotic cavity (PAC) (E3.5 E5.5), namely XEn/EPiCs. Automated image analysis and supervised machine learning permit the identification of embryonic morphogenesis, tissue compartmentalization, cell differentiation, and consecutive classification. Screens with signaling pathway modulators at different time windows provide spatiotemporal information on their phenotypic effect on developmental processes leading to the formation of XEn/EPiCs. Exposure of the biological model in the microwell platform to pathway modulators at two time windows, namely 0-72 h and 48-120 h, show that Wnt and Fgf/MAPK pathway modulators affect Epi differentiation and its polarization, while modulation of BMP and Tgfß/Nodal pathway affects XEn specification and epithelialization. Further, their collective role is identified in the timing of the formation and expansion of PAC. The newly developed, scalable culture and analysis platform, thereby, provides a unique opportunity to quantitatively and systematically study effects of pathway modulators on early embryonic development.

Direct deep UV lithography to micropattern PMMA for stem cell culture.

Microengineering is increasingly being used for controlling the microenvironment of stem cells. Here, a novel method for fabricating structures with subcellular dimensions in commonly available thermoplastic poly(methyl methacrylate) (PMMA) is shown. Microstructures are produced in PMMA substrates using Deep Ultraviolet lithography, and the effect of different developers is described. Microgrooves fabricated in PMMA are used for the neuronal differentiation of mouse embryonic stem cells (mESCs) directly on the polymer. The fabrication of 3D, curvilinear patterned surfaces is also highlighted. A 3D multilayered microfluidic chip is fabricated using this method, which includes a porous polycarbonate (PC) membrane as cell culture substrate. Besides directly manufacturing PMMA-based microfluidic devices, an application of the novel approach is shown where a reusable PMMA master is created for replicating microstructures with polydimethylsiloxane (PDMS). As an application example, microchannels fabricated in PDMS are used to selectively expose mESCs to soluble factors in a localized manner. The described microfabrication process offers a remarkably simple method to fabricate for example multifunctional topographical or microfluidic culture substrates outside cleanrooms, thereby using inexpensive and widely accessible equipment. The versatility of the underlying process could find various applications also in optical systems and surface modification of biomedical implants.

Complementary Supramolecular Functionalization Enhances Antifouling Surfaces: A Ureidopyrimidinone-Functionalized Phosphorylcholine Polymer.

Fibrosis of implants remains a significant challenge in the use of biomedical devices and tissue engineering materials. Antifouling coatings, including synthetic zwitterionic coatings, have been developed to prevent fouling and cell adhesion to several implantable biomaterials. While many of these coatings need covalent attachment, a conceptually simpler approach is to use a spontaneous self-assembly event to anchor the coating to a surface. This could simplify material processing through highly specific molecular recognition. Herein, we investigate the ability to utilize directional supramolecular interactions to anchor an antifouling coating to a polymer surface containing a complementary supramolecular unit. A library of controlled copolymerization of ureidopyrimidinone methacrylate (UPyMA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) was prepared and their UPy composition was assessed. The MPC-UPy copolymers were characterized by 1H NMR, Fourier transform infrared (FTIR), and gel permeation chromatography (GPC) and found to exhibit similar mol % of UPy as compared to feed ratios and low dispersities. The copolymers were then coated on an UPy elastomer and the surfaces were assessed for hydrophilicity, protein absorption, and cell adhesion. By challenging the coatings, we found that the antifouling properties of the MPC-UPy copolymers with more UPy mol % lasted longer than the MPC homopolymer or low UPy mol % copolymers. As a result, the bioantifouling nature could be tuned to exhibit spatio-temporal control, namely, the longevity of a coating increased with UPy composition. In addition, these coatings showed nontoxicity and biocompatibility, indicating their potential use in biomaterials as antifouling coatings. Surface modification employing supramolecular interactions provided an approach that merges the simplicity and scalability of nonspecific coating methodology with the specific anchoring capacity found when using conventional covalent grafting with longevity that could be engineered by the supramolecular composition itself.

In Situ Covalent Reinforcement of a Benzene-1,3,5-Tricarboxamide Supramolecular Polymer Enables Biomimetic, Tough, and Fibrous Hydrogels and Bioinks.

Synthetic hydrogels often lack the load-bearing capacity and mechanical properties of native biopolymers found in tissue, such as cartilage. In natural tissues, toughness is often imparted via the combination of fibrous noncovalent self-assembly with key covalent bond formation. This controlled combination of supramolecular and covalent interactions remains difficult to engineer, yet can provide a clear strategy for advanced biomaterials. Here, a synthetic supramolecular/covalent strategy is investigated for creating a tough hydrogel that embodies the hierarchical fibrous architecture of the extracellular matrix (ECM). A benzene-1,3,5-tricarboxamide (BTA) hydrogelator is developed with synthetically addressable norbornene handles that self-assembles to form a and viscoelastic hydrogel. Inspired by collagen's covalent cross-linking of fibrils, the mechanical properties are reinforced by covalent intra- and interfiber cross-links. At over 90% water, the hydrogels withstand up to 550% tensile strain, 90% compressive strain, and dissipated energy with recoverable hysteresis. The hydrogels are shear-thinning, can be 3D bioprinted with good shape fidelity, and can be toughened via covalent cross-linking. These materials enable the bioprinting of human mesenchymal stromal cell (hMSC) spheroids and subsequent differentiation into chondrogenic tissue. Collectively, these findings highlight the power of covalent reinforcement of supramolecular fibers, offering a strategy for the bottom-up design of dynamic, yet tough, hydrogels and bioinks.

Polymer film-based microwell array platform for long-term culture and research of human bronchial organoids.

The culture of lung organoids relies on drops of basement membrane matrices. This comes with limitations, for example, concerning the microscopic monitoring and imaging of the organoids in the drops. Also, the culture technique is not easily compatible with micromanipulations of the organoids. In this study, we investigated the feasibility of the culture of human bronchial organoids in defined x-, y- and z-positions in a polymer film-based microwell array platform. The circular microwells have thin round/U-bottoms. For this, single cells are first precultured in drops of basement membrane extract (BME). After they form cell clusters or premature organoids, the preformed structures are then transferred into the microwells in a solution of 50% BME in medium. There, the structures can be cultured toward differentiated and mature organoids for several weeks. The organoids were characterized by bright-field microscopy for size growth and luminal fusion over time, by scanning electron microscopy for overall morphology, by transmission electron microscopy for the existence of microvilli and cilia, by video microscopy for beating cilia and swirling fluid, by live-cell imaging, by fluorescence microscopy for the expression of cell-specific markers and for proliferating and apoptotic cells, and by ATP measurement for extended cell viability. Finally, we demonstrated the eased micromanipulation of the organoids in the microwells by the example of their microinjection.

Molecular Tuning of a Benzene-1,3,5-Tricarboxamide Supramolecular Fibrous Hydrogel Enables Control over Viscoelasticity and Creates Tunable ECM-Mimetic Hydrogels and Bioinks.

Traditional synthetic covalent hydrogels lack the native tissue dynamics and hierarchical fibrous structure found in the extracellular matrix (ECM). These dynamics and fibrous nanostructures are imperative in obtaining the correct cell/material interactions. Consequently, the challenge to engineer functional dynamics in a fibrous hydrogel and recapitulate native ECM properties remains a bottle-neck to biomimetic hydrogel environments. Here, the molecular tuning of a supramolecular benzene-1,3,5-tricarboxamide (BTA) hydrogelator via simple modulation of hydrophobic substituents is reported. This tuning results in fibrous hydrogels with accessible viscoelasticity over 5 orders of magnitude, while maintaining a constant equilibrium storage modulus. BTA hydrogelators are created with systematic variations in the number of hydrophobic carbon atoms, and this is observed to control the viscoelasticity and stress-relaxation timescales in a logarithmic fashion. Some of these BTA hydrogels are shear-thinning, self-healing, extrudable, and injectable, and can be 3D printed into multiple layers. These hydrogels show high cell viability for chondrocytes and human mesenchymal stem cells, establishing their use in tissue engineering applications. This simple molecular tuning by changing hydrophobicity (with just a few carbon atoms) provides precise control over the viscoelasticity and 3D printability in fibrillar hydrogels and can be ported onto other 1D self-assembling structures. The molecular control and design of hydrogel network dynamics can push the field of supramolecular chemistry toward the design of new ECM-mimicking hydrogelators for numerous cell-culture and tissue-engineering applications and give access toward highly biomimetic bioinks for bioprinting.

A pendulum of induction between the epiblast and extra-embryonic endoderm supports post-implantation progression.

Embryogenesis is supported by dynamic loops of cellular interactions. Here, we create a partial mouse embryo model to elucidate the principles of epiblast (Epi) and extra-embryonic endoderm co-development (XEn). We trigger naive mouse embryonic stem cells to form a blastocyst-stage niche of Epi-like cells and XEn-like cells (3D, hydrogel free and serum free). Once established, these two lineages autonomously progress in minimal medium to form an inner pro-amniotic-like cavity surrounded by polarized Epi-like cells covered with visceral endoderm (VE)-like cells. The progression occurs through reciprocal inductions by which the Epi supports the primitive endoderm (PrE) to produce a basal lamina that subsequently regulates Epi polarization and/or cavitation, which, in return, channels the transcriptomic progression to VE. This VE then contributes to Epi bifurcation into anterior- and posterior-like states. Similarly, boosting the formation of PrE-like cells within blastoids supports developmental progression. We argue that self-organization can arise from lineage bifurcation followed by a pendulum of induction that propagates over time.

Modular mixing of benzene-1,3,5-tricarboxamide supramolecular hydrogelators allows tunable biomimetic hydrogels for control of cell aggregation in 3D.

Few synthetic hydrogels can mimic both the viscoelasticity and supramolecular fibrous structure found in the naturally occurring extracellular matrix (ECM). Furthermore, the ability to control the viscoelasticity of fibrous supramolecular hydrogel networks to influence cell culture remains a challenge. Here, we show that modular mixing of supramolecular architectures with slow and fast exchange dynamics can provide a suitable environment for multiple cell types and influence cellular aggregation. We employed modular mixing of two synthetic benzene-1,3,5-tricarboxamide (BTA) architectures: a small molecule water-soluble BTA with slow exchange dynamics and a telechelic polymeric BTA-PEG-BTA with fast exchange dynamics. Copolymerisation of these two supramolecular architectures was observed, and all tested formulations formed stable hydrogels in water and cell culture media. We found that rational tuning of mechanical and viscoelastic properties is possible by mixing BTA with BTA-PEG-BTA. These hydrogels showed high viability for both chondrocyte (ATDC5) and human dermal fibroblast (HDF) encapsulation (>80%) and supported neuronal outgrowth (PC12 and dorsal root ganglion, DRG). Furthermore, ATDC5s and human mesenchymal stem cells (hMSCs) were able to form spheroids within these viscoelastic hydrogels, with control over cell aggregation modulated by the dynamic properties of the material. Overall, this study shows that modular mixing of supramolecular architectures enables tunable fibrous hydrogels, creating a biomimetic environment for cell encapsulation. These materials are suitable for the formation and culture of spheroids in 3D, critical for upscaling tissue engineering approaches towards cell densities relevant for physiological tissues.

Long-Term Controlled Growth Factor Release Using Layer-by-Layer Assembly for the Development of In Vivo Tissue-Engineered Blood Vessels.

The development of a well-designed tissue-engineered blood vessel (TEBV) still remains a challenge. In recent years, approaches in which the host response to implanted biomaterials is used to generate vascular constructs within the patient's body have gained increasing interest. The delivery of growth factors to these in situ-engineered vascular grafts might enhance myofibroblast recruitment and the secretion of essential extracellular matrix proteins, thereby optimizing their functional properties. Layer-by-layer (LbL) coating has emerged as an innovative technology for the controlled delivery of growth factors in tissue engineering applications. In this study, we combined the use of surface-etched polymeric rods with LbL coatings to control the delivery of TGF-ß1, PDGF-BB, and IGF-1 and steer the foreign body response toward the formation of a functional vascular graft. Results showed that the regenerated tissue is composed of elastin, glycosaminoglycans, and circumferentially oriented collagen fibers, without calcification or systemic spill of the released growth factors. Functional controlled delivery was observed, whereas myofibroblast-rich tissue capsules were formed with enhanced collagen and elastin syntheses using TGF-ß1 and TGF-ß1/PDGF-BB releasing rods, when compared to control rods that were solely surface-engineered by chloroform etching. By combining our optimized LbL method and surface-engineered rods in an in vivo bioreactor approach, we could regulate the fate and ECM composition of in situ-engineered vascular grafts to create a successful in vivo vascular tissue-engineered replacement.

3D Lung-on-Chip Model Based on Biomimetically Microcurved Culture Membranes.

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future.

From Mice to Men: Generation of Human Blastocyst-Like Structures In Vitro.

Advances in the field of stem cell-based models have in recent years lead to the development of blastocyst-like structures termed blastoids. Blastoids can be used to study key events in mammalian pre-implantation development, as they mimic the blastocyst morphologically and transcriptionally, can progress to the post-implantation stage and can be generated in large numbers. Blastoids were originally developed using mouse pluripotent stem cells, and since several groups have successfully generated blastocyst models of the human system. Here we provide a comparison of the mouse and human protocols with the aim of deriving the core requirements for blastoid formation, discuss the models' current ability to mimic blastocysts and give an outlook on potential future applications.

Mesoporous Silica-Coated Gold Nanoparticles for Multimodal Imaging and Reactive Oxygen Species Sensing of Stem Cells.

Stem cell (SC)-based therapies hold the potential to revolutionize therapeutics by enhancing the body's natural repair processes. Currently, there are only three SC therapies with marketing authorization within the European Union. To optimize outcomes, it is important to understand the biodistribution and behavior of transplanted SCs in vivo. A variety of imaging agents have been developed to trace SCs; however, they mostly lack the ability to simultaneously monitor the SC function and biodistribution at high resolutions. Here, we report the synthesis and application of a nanoparticle (NP) construct consisting of a gold NP core coated with rhodamine B isothiocyanate (RITC)-doped mesoporous silica (AuMS). The MS layer further contained a thiol-modified internal surface and an amine-modified external surface for dye conjugation. Highly fluorescent AuMS of three different sizes were successfully synthesized. The NPs were non-toxic and efficiently taken up by limbal epithelial SCs (LESCs). We further showed that we can functionalize AuMS with a reactive oxygen species (ROS)-sensitive fluorescent dye using two methods, loading the probe into the mesopores, with or without additional capping by a lipid bilayer, and by covalent attachment to surface and/or mesoporous-functionalized thiol groups. All four formulations displayed a ROS concentration-dependent increase in fluorescence. Further, in an ex vivo SC transplantation model, a combination of optical coherence tomography and fluorescence microscopy was used to synergistically identify AuMS-labeled LESC distribution at micrometer resolution. Our AuMS constructs allow for multimodal imaging and simultaneous ROS sensing of SCs and represent a promising tool for in vivo SC tracing.

The response of three-dimensional pancreatic alpha and beta cell co-cultures to oxidative stress.

The pancreatic islets of Langerhans have low endogenous antioxidant levels and are thus especially sensitive to oxidative stress, which is known to influence cell survival and behaviour. As bioengineered islets are gaining interest for therapeutic purposes, it is important to understand how their composition can be optimized to diminish oxidative stress. We investigated how the ratio of the two main islet cell types (alpha and beta cells) and their culture in three-dimensional aggregates could protect against oxidative stress. Monolayer and aggregate cultures were established by seeding the alphaTC1 (alpha) and INS1E (beta) cell lines in varying ratios, and hydrogen peroxide was applied to induce oxidative stress. Viability, oxidative stress, and the level of the antioxidant glutathione were measured. Both aggregation and an increasing prevalence of INS1E cells in the co-cultures conferred greater resistance to cell death induced by oxidative stress. Increasing the prevalence of INS1E cells also decreased the number of alphaTC1 cells experiencing oxidative stress in the monolayer culture. In 3D aggregates, culturing the alphaTC1 and INS1E cells in a ratio of 50:50 prevented oxidative stress in both cell types. Together, the results of this study lead to new insight into how modulating the composition and dimensionality of a co-culture can influence the oxidative stress levels experienced by the cells.

Polystyrene Pocket Lithography: Sculpting Plastic with Light.

Tissue-culture-ware polystyrene is the gold standard for in vitro cell culture. While microengineering techniques can create advanced cell microenvironments in polystyrene, they require specialized equipment and reagents, which hinder their accessibility for most biological researchers. An economical and easily accessible method is developed and validated for fabricating microstructures directly in polystyrene with sizes approaching subcellular dimensions while requiring minimal processing time. The process involves deep ultraviolet irradiation through a shadow mask or ink pattern using inexpensive, handheld devices followed by selective chemical development with common reagents to generate micropatterns with depths/heights between 5 and 10 µm, which can be used to guide cell behavior. The remarkable straightforwardness of the process enables this class of microengineering techniques to be broadly accessible to diverse research communities.

Desymmetrization via Activated Esters Enables Rapid Synthesis of Multifunctional Benzene-1,3,5-tricarboxamides and Creation of Supramolecular Hydrogelators.

Supramolecular materials based on the self-assembly of benzene-1,3,5-tricarboxamide (BTA) offer an approach to mimic fibrous self-assembled proteins found in numerous natural systems. Yet, synthetic methods to rapidly build complexity, scalability, and multifunctionality into BTA-based materials are needed. The diversity of BTA structures is often hampered by the limited flexibility of existing desymmetrization routes and the purification of multifunctional BTAs. To alleviate this bottleneck, we have developed a desymmetrization method based on activated ester coupling of a symmetric synthon. We created a small library of activated ester synthons and found that a pentafluorophenol benzene triester (BTE) enabled effective desymmetrization and creation of multifunctional BTAs in good yield with high reaction fidelity. This new methodology enabled the rapid synthesis of a small library of BTA monomers with hydrophobic and/or orthogonal reactive handles and could be extended to create polymeric BTA hydrogelators. These BTA hydrogelators self-assembled in water to create fiber and fibrous sheet-like structures as observed by cryo-TEM, and the identity of the BTA conjugated can tune the mechanical properties of the hydrogel. These hydrogelators display high cytocompatibility for chondrocytes, indicating potential for the use of these systems in 3D cell culture and tissue engineering applications. This newly developed synthetic strategy facilitates the simple and rapid creation of chemically diverse BTA supramolecular polymers, and the newly developed and scalable hydrogels can unlock exploration of BTA based materials in a wider variety of tissue engineering applications.

Methodological approaches in aggregate formation and microscopic analysis to assess pseudoislet morphology and cellular interactions.

Microscopy has revolutionised our view on biology and has been vital for many discoveries since its invention around 200 years ago. Recent developments in cell biology have led to a strong interest in generating spheroids and organoids that better represent tissue. However, the current challenge faced by many researchers is the culture and analysis of these three-dimensional (3D) cell cultures. With the technological improvements in reconstructing volumetric datasets by optical sections, it is possible to quantify cells, their spatial arrangement, and the protein distribution without destroying the physical organization. We assessed three different microwell culture plates and four analysis tools for 3D imaging data for their applicability for the analysis of 3D cultures. A key advantage of microwell plates is their potential to perform high-throughput experiments in which cell cultures are generated and analysed in one single system. However, it was shown that this potential could be impacted by the material composition and microwell structure. For example, antibody staining was not possible in a hydrogel microwell, and truncated pyramid-structured microwells had increased background fluorescence due to their structure. Regarding analysis tools, four different software, namely CellProfiler, Fiji/ImageJ, Nikon GA3 and Imaris, were compared for their accuracy and applicability in analysing datasets from 3D cultures. The results showed that the open-access software, CellProfiler and Fiji, could quantify nuclei and cells, yet with varying results compared to manual counting, and may require post-processing optimisation. On the other hand, the GA3 and Imaris software packages showed excellent versatility in usage and accuracy in the quantification of nuclei and cells, and could classify cell localisation. Together these results provide critical considerations for microscopic imaging and analysis of 3D cell cultures.

PEOT/PBT Polymeric Pastes to Fabricate Additive Manufactured Scaffolds for Tissue Engineering.

The advantages of additive manufactured scaffolds, as custom-shaped structures with a completely interconnected and accessible pore network from the micro- to the macroscale, are nowadays well established in tissue engineering. Pore volume and architecture can be designed in a controlled fashion, resulting in a modulation of scaffold's mechanical properties and in an optimal nutrient perfusion determinant for cell survival. However, the success of an engineered tissue architecture is often linked to its surface properties as well. The aim of this study was to create a family of polymeric pastes comprised of poly(ethylene oxide therephthalate)/poly(butylene terephthalate) (PEOT/PBT) microspheres and of a second biocompatible polymeric phase acting as a binder. By combining microspheres with additive manufacturing technologies, we produced 3D scaffolds possessing a tailorable surface roughness, which resulted in improved cell adhesion and increased metabolic activity. Furthermore, these scaffolds may offer the potential to act as drug delivery systems to steer tissue regeneration.