Reproducing the hierarchical organization and dynamic interaction networks of native extracellular matrices, where supramolecular and covalent interactions are combined to provide both dynamic behavior and mechanical stability, remains a major challenge in the design of synthetic hydrogels. Here, we report the synthesis of an aldehyde-functionalized benzene-1,3,5-tricarboxamide hydrogelator designed to combine supramolecular selfassembly with two covalent reinforcement modes. The hydrogelator first self-assembled in aqueous media into fibrous structures forming hydrogels, which underwent an irreversible increase in stiffness upon heating. We have attributed this behavior to proximity-assisted aldehyde self-condensation crosslinking within the preorganized supramolecular network, supported by rheological measurements, control experiments, and the resistance of the heated hydrogels to dissolution. This stabilization preserved the shear-thinning behavior, viscoelastic relaxation, elasticity, and self-healing capacity of the material. The aldehyde groups were able to be exploited to introduce dynamic covalent crosslinks using bifunctional hydrazide or hydroxylamine crosslinkers, which strongly reinforced the hydrogels while also maintaining processability and dynamic behavior. Interestingly, all hydrogels converged toward similar stiffnesses after heating, suggesting that the final reinforcement may be constrained by the underlying supramolecular architecture. Preliminary culture experiments with human mesenchymal stromal cells (seeded on top of the hydrogels) demonstrated preliminary cytocompatibility of the aldehyde-functionalized hydrogels. Overall, this work highlights both the challenges and the potential of imine-type reinforcement in supramolecular hydrogels, which represent promising and versatile platforms for dynamic, self-healing, and mechanically tunable ECM-mimicking materials.

Objectives The limited regenerative capacity of cartilage tissue and the high morbidity associated with injuries and diseases have driven the search for innovative regenerative medicine strategies. The objective of the study was to compare the chondrogenic differentiation of human MSCs in conventional pellet cultures to that of spheroids generated using an innovative microwell system. Design Human bone marrow mesenchymal stem cells (hBMSCs) were isolated and cultured in either pellet or microwell systems. Upon induction of chondrogenesis, gene expression and extracellular matrix deposition were analysed. Results We found that chondrogenic pellets outperformed spheroids based on the expression levels of chondrogenic markers, such as SOX9, COL II, COLIXa2, COLXIa2, ACAN, VCAN, and the trio SOX5, SOX6, and SOX9. However, hypertrophic markers, such as COL X, RUNX2, COLI, and MMP13, were higher in chondrogenic pellets. DCN and BGN expression, along with increased COMP expression in the microwell spheroids, may reflect a role in matrix stabilisation and network organisation rather than chondrogenic differentiation. Histological analysis demonstrated a richer extracellular matrix deposition in the chondrogenic pellet culture, while the spheroids exhibited less calcification. Conclusions This study demonstrates the complexities of MSC’s chondrogenesis across different aggregate dimensions in balancing chondrogenesis and hypertrophy. Overall, these findings indicate that the culture system choice should reflect specific biological and translational aims, with each system offering complementary strengths.

The enthesis, the point where a tendon or ligament attaches to bone, is a graded fibrocartilaginous interface that poorly regenerates after injury. Here, we present a modular and scaffold-free strategy for engineering microtissues relevant to enthesis repair by fusing anterior cruciate ligament-derived spheroids with spheroids from osteogenically differentiated human mesenchymal stromal cells. We show that the maturation state of the constituent spheroids governs fusion dynamics and spatial organization, enabling the controlled formation of ligament-, fibrocartilage-, and bone-like regions within a single, radially/concentrically organized construct. Within 10 days, the fused tissues display locally distributed lineage-specific markers and type X collagen localized at the interface between the osteogenically-derived core and ligamentous shell. The latter is indicative of the de novo formation of a fibrocartilage-like region between these regions. The system is scalable by simply adjusting the spheroid number. It supports external mechanical stimulation via ultrasound. The acoustic cues further promote extracellular matrix deposition and tissue growth while maintaining structural integrity. This readily implementable heterotypic spheroid platform offers an in vitro model for studying enthesis mechanobiology, screening therapeutic compounds, and evaluating microscale biomaterials, with translational potential as injectable or bioprintable building blocks for enthesis repair.

The ability of the endometrium to accept and support embryo implantation is crucial, but factors influencing this process remain elusive. This method aims to obtain precise quantitative information on factors causally affecting the initial stages of embryo implantation. We developed a personalized implantation-on-chip platform using in vitro models of the endometrium (organoids) and the embryo (blastoids) to quantify functional embryo attachment. Here, we describe a microfluidic platform for precisely assessing functional receptivity of endometrial epithelium through blastoid adhesion. Endometrial organoids were expanded and transformed into epithelial monolayers within custom-made microfluidic chips. These chips were then infused with large numbers of blastoids (>100) per chip. Followed after 48 h of co-culture, blastoids were exposed to a controlled stepwise increasing flow rate (50, 100 and 400 μL/min), while the rate of adhered blastoids was precisely measured from image-based readouts. Our method offers a robust platform for studying endometrial epithelial receptivity and testing therapeutic interventions with potential impact for infertile patients.

Abstract Endocrine disruptors (EDs) are an exogenous group of compounds associated with thyroid malfunctioning in the human body. Nonetheless, there are currently no adequate in vivo or in vitro models for the preclinical testing of these compounds since both animal and two-dimensional (2D) cell-based models are not able to mimic thyroid physiological conditions from both functional and three-dimensional (3D) organization perspective. Recently, bioprinting technologies emerged as an innovative tool in the field of regenerative medicine and advanced 3D in vitro models that allow the creation of 3D well-organized structures able to mirror physiologically relevant tissue and organ architectures. In this study, we evaluated microfluidic bioprinting as a biofabrication technology to develop a 3D in vitro model of the thyroid gland. We studied the fundamental parameters to obtain a fine control over the bioprinted fibres for different biomaterials. Then, we assessed the possibility to bioprint single thyroid cells, thyroid spheroids and finally mouse embryonic stem cell-derived thyroid follicles. The different cell types maintained high viability and metabolic activity. The bioprinted thyroid model showed high expression of different early and late functional markers and to be responsive to ED exposure. These bioprinted thyroid constructs could provide a new set of advanced 3D in vitro models to test potential EDs and possible adverse outcomes that may be associated with their administration or exposure.

In this proof-of-concept study, we develop a novel 3D cell patterning and culture platform. The "Floor-Ceiling-Chip" (FC-Chip) simply consists of two opposing 2D substrates in the form of ion track-etched membranes, creating a pseudo-3D microenvironment for the cells between them. This allows the stimulation of both the dorsal and ventral sides of cells, thereby also eliminating the artificial polarization, for example, of stromal cells, observed in standard culture dishes and inserts. By providing the membranes with micropatterned cell-adhesive islands of varying geometries and sizes, the FC-Chip enables control over cell shape and alignment in a 3D environment. Analysis of fluorescence microscopic images reveals distinct cellular and nuclear morphology, along with perinuclear actin organization, in the on-chip cultures compared to cultures on traditional 2D substrates. Cells in the FC-Chip exhibit fewer focal adhesions, lower expression of lamin A/C, and less nuclear localization of the yes-associated protein 1. The chip demonstrates compatibility with standard biochemical assays and supports long-term cultures up to 10 days, expanding its potential applications. Overall, the early version of the FC-Chip presented here confirms the feasibility of a straightforward, accessible, and versatile future culture platform for the manipulation and modeling of cell morphology and organization in 3D.

BACKGROUND:Stem cell-based embryo models (SCBEMs) are clusters of pluripotent stem cells that can mimic morphological and functional aspects of early human embryos to different degrees. When cultured from human cells, SCBEMs offer technically scalable and amenable tools that can help refine, reduce, and, in the future, perhaps replace the use of animals and human embryos in fundamental and clinical research. These advantages propelled the development of SCBEMs, and several distinct types have been generated over the past decade, including gastruloids, axioloids, blastoids, and post-implantation-like embryoids. For purposes of governance, advisory reports distinguish between SCBEMs based on their presumed capacity to continuously undergo organized human development-referred to here as developmental potential. However, since functionally testing this potential by transferring human SCBEMs to a uterus would be unethical and is recommended to be prohibited, scientists lack clear or consistent ways to assess it. OBJECTIVE AND RATIONALE:This narrative review aims to tackle the question of how to assess developmental potential in SCBEMs by clarifying the different ways in which it can be and is being conceptualized. We achieve this by synthesizing insights from governance, science, and ethics. First, we examine how developmental potential is described in contemporary governance frameworks, and which aspects are emphasized. Next, we discuss biological markers for developmental potential and show how their scientific basis (in embryos, let alone SCBEMs) remains poorly understood. Then, we explore how the aspects considered relevant for assessments of developmental potential in governance and science may pre-emptively hinge on underlying conceptual interpretations and lead to differing normative implications. SEARCH METHODS:This narrative review combines insights from both the academic and grey literature on the (ethics of) embryo models. Original and review articles were selected from PubMed and Biorxiv with the main focus on articles published since 2015. Search terms included: embryo quality, in vitro fertilization, Gardner system, blastoid, gastruloid, embryo research, potentiality argument, developmental potential, transcriptomics, epigenetics, embryo metabolism, and related terms. Additional sources were identified through snowballing. This work focuses predominantly on human SCBEMs, but references to animal models are made. OUTCOMES:Comparison of the descriptions currently recommended for governance suggests at least three criteria that are used to assess developmental potential in SCBEMs: composition, organization, and interaction. Scientifically, developmental potential is multifaceted and only partly characterized, making it necessary to measure a broader range of aspects, using human embryos as benchmarks when possible. Since the range and significance of these aspects can be shaped by underlying accounts of developmental potential, contemporary advisory reports are examined to explore if and how they connote interpretations of developmental potential as possibility, probability, and predisposition. WIDER IMPLICATIONS:Categorization of the regulatory and scientific criteria currently used to assess developmental potential shows that they are underpinned by distinct interpretations of the concept, revealing tensions and questions for further inquiry. By synthesizing insights from governance, science, and ethics, this review thus aims to contribute to the responsible advancement of the SCBEM field and to support its coherent and transparent governance. REGISTRATION NUMBER:N/A.

Successful embryo implantation requires timely acquisition of endometrial receptivity, yet the epithelial mechanisms governing this transition remain poorly understood and difficult to study in humans. Current clinical assessments rely largely on transcriptomic markers, despite limited evidence that these predict functional implantation outcomes. Here, we present a human endometrium-on-chip model that enables controlled hormonal priming and quantitative measurement of blastoid attachment to patient-derived luminal epithelium. We show that hormonally primed epithelial monolayers maintain attachment competence across extended progesterone exposure, indicating a sustained permissive state rather than a sharply defined window of implantation. Single-cell RNA sequencing reveals a continuous maturation trajectory that is decoupled from functional adhesion. The model further recapitulates localized epithelial remodeling at blastoid contact sites. Together, this system provides a mechanistic framework to interrogate epithelial determinants of implantation, challenges marker-based definitions of receptivity, and offers a foundation for future diagnostic and personalized applications in medically assisted reproduction.

Abstract Endocrine disrupting chemicals (EDCs) are ubiquitous environmental contaminants capable of dysregulating the production of thyroid hormones. Traditional thyroid toxicological assays rely on 2D cell cultures and animal models, both of which fail to accurately recapitulate human thyroid physiology and provide limited mechanistic insight into EDC toxicity. To overcome these limitations, we report a novel thyroid-on-chip platform integrating mouse embryonic stem cell–derived thyroid organoids with advanced organ-on-chip (OoC) technology and downstream multi-omics analysis. The platform leverages a reversibly-sealed microphysiological flow battery (MFB) to allow scale up of dynamic organoid culture and controlled chemical exposure while reducing operational complexity. Upon EDC exposure, transcriptomic and proteomic analysis revealed new molecular signatures of thyroid disruption across four different EDC classes, even at very low EDC concentrations (1nM), validating the capacity of this system to mechanistically dissect EDC-induced responses. This represents an integrated platform consists of an advanced physiologically relevant assay framework for next-generation endocrine toxicity testing, bridging the gap between in vitro screening and in vivo thyroid physiology.

Abstract Bioengineers strive to recreate in vivo microenvironments in vitro to reduce our use of animal models and provide insights into human biology. While liver models show promise, sex differences in liver biology remain largely neglected in preclinical studies. Despite the 2014 EU mandate for the inclusion of women in clinical trials, decoupling of research data by sex is historically rare, with only 11% of papers disaggregating data by sex. This gap contributes to women being more susceptible to drug-induced liver injury (DILI) and being underserved in drug development, as well as to costly drug attrition levels. Here we present a novel approach to modelling sex differences in vitro. Human induced pluripotent stem cells (iPSCs) from both male (XY) and female (XX) donors, were differentiated into hepatocyte liver spheroids and exposed to in vivo -mimicking levels of testosterone, progesterone, and oestrogen in high-throughput microwell format. We successfully recapitulated sex-specific metabolic profiles and demonstrated significant differences in CYP1A2 and CYP3A4 drug metabolism and gene expression patterns consistent with reported in vivo observations, without compromising cell viability. These findings validate the utility of sex-differentiated microenvironments in early-stage research, offering a pathway to refine animal and clinical trials and improve therapeutic outcomes for all sexes.

Combining stem cell-derived multi-tissue constructs, such as non-integrated embryo models, with bioengineering technologies provide scalable tools for tissue engineering and drug testing. Here, we show the controlled initiation of organogenesis in XEn/EpiCs, a partial chemically-controllable mouse embryo model emulating aspects of E5.5 stage embryo, within milliwells. XEn/EpiCs spontaneously progress to develop a fluid-filled cavity, resembling a yolk sac encasing an embryonic compartment. Locally restricted inner regions of yolk sac-like (YS+) structures undergo cardiogenesis, displaying spontaneous contractions, and outer regions initiate vasculogenesis. Some vascularized YS+ structures develop blood islands composed of primitive erythrocytes, endothelial cells and mesenchyme. Further, we developed a continuous single-step culture using thermoformed milliwell platforms, enabling live tracking, pathway modulation, and in situ visualization of XEn/EpiCs towards organogenesis. Collectively, we demonstrate a multi-tissue vascularized embryo model devoid of trophoblast cells and external stimuli, capable of undergoing post-implantation morphogenesis and amenable to modulation within micro-engineered platforms for diverse applications.

Understanding developmental toxicity demands capturing the full spectrum of morphological diversity, or morphospace, that defines early human embryogenesis. Traditional toxicity testing methods, relying on animal models or simplified 2D cell cultures, inherently overlook the nuanced interplay between distinct developmental stages and cell lineages. Addressing this gap, we present an innovative high-throughput screening approach leveraging three complementary 3D stem cell-based embryo models: preimplantation stage blastoids, and early postimplantation stage epiblast-like (EPICs) and amnion-like clusters (AMNICs). Cultured in thermoformed microwell arrays, these models were systematically exposed to a library of 27 widely used compounds. Automated image-based analyses allowed for precise quantification of morphotoxic effects. Key findings include the identification of compounds such as ascorbic acid and valproic acid (VPA), which demonstrated distinct and opposing effects across the different embryo models, highlighting developmental stage-specific sensitivities. This integrative platform underscores the essential role of complementary embryo models to achieve robust and human-relevant developmental toxicity assessment.

Recently, microcavity arrays have been increasingly used to enable the controlled culture of 3D cellular aggregates, such as spheroids, organoids, and gastruloids. However, current fabrication techniques remain technically demanding and largely inaccessible. While micro(scale) thermoforming is already used for fabricating thin-walled microcavities in films from thermoplastic polymers, the process requires specialized equipment for the controlled application of forming temperature and pressure. Here, a new microfabrication method, referred to by us as "micro-cold-forming", is described. This method enables the simple, rapid, and inexpensive fabrication of microcavities (with diameters of 2 mm and depths ranging from 530 to 690 µm) in thin (25-30 µm) thermoplastic polymer films. The fabrication method is carried out at ambient or room temperature and only uses commercially available, easily affordable components. Here, we demonstrate that the microcavities can be applied for 3D cell culture by culturing adult human kidney organoids, called "tubuloids", in the microcavities. To demonstrate the ability of the microcavities to be applied for toxicity studies, the tubuloids were exposed to a high concentration of ascorbic acid. The new forming technique can provide widespread, low-barrier access to self-fabricated microcavities for researchers both in biological laboratories and in low-income countries.

Due to the inaccessibility of early human embryos for large, robust studies, many questions regarding the mechanisms of early embryogenesis remain. To address these questions, multiple research groups have developed human stem cell-based models of the pre-implantation blastocyst stage. These models, known as blastoids, mimic several key processes that natural blastocysts undergo as they prepare to implant into the uterine wall. One of the main advantages of blastoids is their scalability, making them suitable for both screenings and molecular studies. To leverage this advantage, we established a protocol for the parallel formation and culture of blastoids in thermoformed microwell arrays. Thin-walled thermoformed microwell platforms allow for uniform, large-scale generation of blastoids and enable in situ high-resolution imaging through both widefield and confocal microscopy. Here we present a step-by-step protocol for the culture of blastoids in thermoformed microwell platforms.

Cellular spheroids are considered a popular option for modeling healthy and diseased tissues in vitro and as injectable therapies. The formation and culture of spheroids can make use of different three-dimensional (3D) culture platforms, but the spheroids' analysis often has to rely on endpoint assays. In this study, we propose a microfluidic bioreactor to culture and nondestructively monitor human mesenchymal stem cell (hMSC) spheroids over time using non-Faradaic electr(ochem)ical impedance spectroscopy (EIS). For this, an array of porous microwells thermoformed from ion track-etched thin films and a pair of sensing electrodes from transparent indium tin oxide are integrated into the flow and culture chamber of the bioreactor. To measure the spheroid's electrical properties, the electrodes are connected to a frequency response analyzer (FRA), with a multiplexer in between to enable the operation of more than one bioreactor at the FRA at the same time. We find differences between the complex resistance/impedance and/or capacitance data of a reference condition without cells, a two-dimensional (2D) hMSC culture, hMSC spheroids, and hybrid spheroids aggregated from hMSCs and titanium or hydroxyapatite microparticles. We also found differences between different culture durations. These results suggest that our device can sense the presence and spatial arrangement of cells and micro(sized) biomaterials as a function of time.

In this study, a cell-mediated degradable alginate hydrogel system for organoid culture and amenable to biofabrication technologies is presented. Norbornene-functionalized alginate is crosslinked with a di-thiolated peptide sequence cleavable by matrix metalloproteinases and decorated with cysteine-terminated cell-adhesion peptide RGD, upon exposure to UV. Stiffness of the hydrogels can be controlled by tuning polymer and crosslinker concentrations. Pre-gel solutions are successfully bioprinted with a pneumatic extrusion-based system. The hydrogels are used to encapsulate a variety of sensitive cell types. Human endometrial organoids present high cell viability, grow in size over time, present spherical morphology, and express cell-cell contacts E-cadherin and proliferation marker Ki67. Encapsulated mouse embryonic stem cell-derived thyroid follicles produce thyroglobulin and T4. Mouse intestinal organoids adopt a proliferative phenotype. Vascularization inside the hydrogels is achieved using endothelial cells and supporting cells (single cell suspension and spheroids). Neurite outgrowth, both small and thick bundles, from encapsulated iPSC-derived neurospheres, demonstrates the reinnervation potential of the hydrogel. This polysaccharide hydrogel platform could be used as a defined, tunable, and ethical alternative to mouse sarcoma-extracted basement-membrane matrices.

In recent years, multiple efforts have been made to develop guidelines for research involving human embryo-like structures (ELS), also known as stem cell-based embryo models (SCBEMs). Policy recommendations have been proposed by advisory bodies in countries such as the Netherlands, Australia, France, Sweden, and the United Kingdom, as well as by expert committees of international professional organizations, including the International Society for Stem Cell Research (ISSCR) and the European Society of Human Reproduction and Embryology (ESHRE). A common ground across these guidelines is the distinction between ELS based on their foreseeable capacity for undergoing continuous human development—a normative recommendation that has given rise to (implicit or explicit) conceptual dichotomies like “integrated vs. non-integrated,” “complete vs. incomplete,” and “complex vs. simple”, among others. Some argue that this capacity confers upon ELS that possess it a moral status comparable to that of human embryos. Others suggest that, while it increases their moral value, it does not necessarily equate them to embryos. In short, there is consensus that ELS with a developmental potential akin to human embryos deserve some degree of moral consideration, but disagreement about the extent of that consideration. This underscores the need for accurate methods to assess and differentiate the developmental potential of specific types of ELS. From an experimental perspective, addressing this need is challenging. Developmental potential is difficult to quantify, and there is no “ideal” human embryo against which to benchmark, given the variability introduced by genetic, epigenetic, and environmental factors. Quantifiable standards are thus hard to establish, and the methods currently in use—such as visual assessments of blastocyst morphology—fail to provide a comprehensive picture of developmental potential. Philosophically, the challenge lies in qualifying developmental potential. Developmental potential may be considered morally valuable for two (non-mutually exclusive) reasons: (1) extrinsic—grounded in symbolic, relational, or instrumental value; and (2) intrinsic—based on properties or interests valued for their own sake. If the moral relevance of developmental potential stems from extrinsic factors, assessing it depends at least partly on empirical studies of how stakeholders—such as patients, scientists, and ethicists—perceive and assign meaning to ELS with such potential. This raises a further challenge: how can we ensure that these insights are interpreted and applied in a consistent, non-arbitrary way in ethical deliberation and policymaking? If alternatively (or additionally), developmental potential is considered morally relevant for intrinsic reasons, then identifying its biological markers becomes key. Yet interpretations of what constitutes developmental potential vary. An “active” view emphasizes internal parameters—such as organismic wholeness, self-organization, neural development, or organ formation—while a “passive” view focuses on external enabling factors, such as environmental cues or support systems necessary for further development. Different interpretations lead to the identification of different biological criteria, making it necessary to first clarify the specific interpretation(s) at stake. Clarifying the basis for considering some ELS morally more significant than others on account of their foreseeable developmental potential is also essential for responsibly translating the normative weight of this potential into national and international regulation. If developmental potential is regarded as intrinsically valuable, then ELS that possess it may warrant stronger protection than if their value were seen as merely extrinsic. The latter depends on context (i.e., is derivative), whereas the former does not (i.e., is non-derivative). Ultimately, how developmental potential is understood—and who determines its moral significance—will shape the governance of ELS research and its broader societal implications. Exploring the ethical bearing and biological markers of developmental potential is thus essential to determining how ELS can and should be used in (future) research and, therefore, the focus of this lecture.

Congenital abnormalities cause ≈3% of fetal defects and premature deaths in Europe, often due to maternal exposure to toxicants. To mitigate the ethical and logistical challenges of animal studies, stem cell-based models are being exploredthat offer scalable readouts at various stages of embryogenesis. However, most current in vitro models are limited in complexity, throughput, automation compatibility or real-time spatio-temporal read-outs. In this study, a scalable, automated platform capable of imaging and quantifying morphological features such as shape, size, texture, and marker intensity is presented. Using a microwell screening platform, XEn/EpiCs, a peri-implantation stage embryo model that mimics eXtraembryonic Endoderm and Epiblast co-development, is robustly generated and used to screen a library of 38 reported compounds. Unlike conventional cytotoxicity assays, this approach also evaluates development-disrupting morphological changes, termed "morphotoxicity", thereby offering complementary insights that may improve the prediction of developmental toxicity across cell types. This pilot study shows thathigh doses of compoundslike retinoic acid, caffeine, ampyrone, and dexamethasone, significantly disrupt XEn/EpiC development, causing morphotoxic effects with or without affecting cell viability. Together, thisstudy highlights the importance of complementing cytotoxicity assessments with morphotoxicity read-outs, emphasizing its potential to enhance the evaluation of teratogenic risks in toxicity tests.

In bone tissue engineering (TE) and regeneration, miniaturized, (sub)millimeter-sized bone models have become a popular trend since they bring about physiological biomimicry, precise orchestration of concurrent stimuli, and compatibility with high-throughput setups and high-content imaging. They also allow efficient use of cells, reagents, materials, and energy. In this review, we describe the state of the art of miniaturized in vitro bone models, or ‘mini-bones’, describing these models based on their characteristics of (multi)cellularity and engineered extracellular matrix (ECM), and elaborating on miniaturization approaches and fabrication techniques. We analyze the performance of ‘mini-bone’ models according to their applications for studying basic bone biology or as regeneration models, disease models, and screening platforms, and provide an outlook on future trends, challenges, and opportunities.