Timo Rademakers

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.

Cell spheroids have been exploited as fundamental engineering units applied as screening platforms or assembled as building blocks for tissue engineering applications. While spheroid encapsulation into hydrogels creates more reliable 3D models, it also brings several constraints, e.g., hydrogel swelling and dynamicity, shading, limitations on depth-resolution, and cell staining strategies for monitoring long real-time imaging. Hence, the objective of this work was to develop a post-imaging automated pipeline for the accurate tracking and measuring of spheroids encapsulated in 3D hydrogels. Using NIS Elements ARv5.30 (Nikon) software, we created a sequence of functions for enhancing spheroid borders, extending the depth of focus, reducing hydrogel shading, and identifying coordinates in an automated manner for time-lapse microscopy analysis up to 70 h. Additionally, we established a method for identifying and tracking migration trajectories of protruded cell clusters that detached from spheroids into the hydrogel. For a comparative hydrogel analysis, fluorescent beads were encapsulated in the ionically crosslinked xanthan gum–alginate (XG–Alg) and photocrosslinked methacrylate hyaluronic acid (HAMA). For pipeline validation, human mesenchymal stem cell spheroids were encapsulated in XG–Alg hydrogel. By employing our pipeline, a high dynamicity and intense swelling effect were detected within XG–Alg, while HAMA remained stable, without noticeable movements up to 60 h. Accurate imaging and tracking detected several spheroid morphological changes, including reversible spheroid-ellipsoid shapes, axis rotational motion, outermost layer movements, spheroid fusion, and a spheroid migration speed of approximately 1.3 µ m h ⁻¹ . Protruded cell clusters were detected in high numbers (83–173 per spheroid), migrating arbitrarily into the hydrogel (16°–311°), with an average speed of approximately 11.4 µ m h ⁻¹ . Our results indicate that this automated pipeline can facilitate the understanding of several cellular dynamic events with high accuracy and low manual interference, which are essential for scaling up tissue engineering and other advanced applications such as drug screening platforms.

Bioactive glasses (BGs) belong to a group of ceramic biomaterials having numerous applications due to their excellent biocompatibility and bioactivity. Depending on their composition, properties of BGs can be finely tuned. In this study, we investigated both angiogenic and osteogenic properties of a novel family of BGs from the SiO₂–CaO–Na₂O system. Three BGs were synthesised from calcite minerals and silica sands extracted from natural deposits. Silica sands used for the synthesis of each glass were obtained from different depths of the deposit, resulting in a different colour and elemental composition. The composition and structural properties of the obtained BGs were determined. Direct culture of human mesenchymal stromal cells (hMSCs) with BG particles at different concentrations was used to investigate the biocompatibility as well as the osteogenic and angiogenic properties of the BGs. In addition, BGs' effect on angiogenesis was further studied in a chick chorioallantoic membrane (CAM) model. Material characterisation confirmed the amorphous character of BGs. Investigated BGs were biocompatible and stimulated early upregulation of RUNX2, ALPL, COL1A1, OCN, and OPN. All BGs tested in a CAM model positively influenced the number, distribution, and branching of the blood vessels. Furthermore, our study revealed that the depth of sand deposit, at which the raw material was collected, had an impact on the osteogenic and angiogenic properties of the resulting glasses. On the one hand, silica sand collected at the deepest layer of the deposit, featuring a higher content of Fe₂O₃ and Al₂O₃, originated BGs with potent stimulative capacity of osteogenic and angiogenic gene expression. On the other hand, sand with high silica content and titanium ions resulted in a glass that better supported vessel structure. The BGs presented in this study showed the potential to promote osteogenesis and angiogenesis during bone tissue regeneration, and thus, they will be further studied as part of composite materials for the development of 3D implantable scaffolds.

Macrophage infiltration and accumulation in the atherosclerotic lesion are associated with plaque progression and instability. Depletion of macrophages from the lesion might provide valuable insights into plaque stabilization processes. Therefore, we assessed the effects of systemic and local macrophage depletion on atherogenesis. To deplete monocytes/macrophages we used atherosclerosis‐susceptible Apoe ^{− /−} mice, bearing a MaFIA (macrophage‐Fas‐induced‐apoptosis) suicide construct under control of the Csf1r (CD115) promotor, where selective apoptosis of Csf1r‐expressing cells was induced in a controlled manner, by administration of a drug, AP20187. Systemic induction of apoptosis resulted in a decrease in lesion macrophages and smooth‐muscle cells. Plaque size and necrotic core size remained unaffected. Two weeks after the systemic depletion of macrophages, we observed a replenishment of the myeloid compartment. Myelopoiesis was modulated resulting in an expansion of CSF1R ^lo myeloid cells in the circulation and a shift from Ly6c ^hi monocytes toward Ly6c ^int and Ly6c ^lo populations in the spleen. Local apoptosis induction led to a decrease in plaque burden and macrophage content with marginal effects on the circulating myeloid cells. Local, but not systemic depletion of Csf1r ⁺ myeloid cells resulted in decreased plaque burden. Systemic depletion led to CSF1R ^lo ‐monocyte expansion in blood, possibly explaining the lack of effects on plaque development.

Dynamic hydrogels are attractive platforms for tissue engineering and regenerative medicine, but there are limited polymer platforms amenable to the formation of these dynamic materials. Most architectures reported are based either on telechelic synthetic polymers or side-chain modified natural polymers; synthetic versions of side-chain modified polymers are noticeably absent. Dynamic hydrogels are key tools for their ability to mimic key extracellular (ECM) mechanical properties like strain stiffening and stress relaxation, while enabling enhanced processing characteristics like injectability, 3D printing, and self-healing. Currently, the limited chemical design space is hindering progress. To facilitate access to new classes of dynamic hydrogels, we report the straightforward synthesis of a water-soluble copolymer with a tunable fraction of pendent aldehyde groups (12–64%) using controlled radical polymerization, and their formation into hydrogel biomaterials with dynamic crosslinks. We found the polymer synthesis to be well-controlled with the determined reactivity ratios consistent with blocky gradient microarchitecture. Subsequently, we observed fast gelation kinetics with imine-type crosslinking. We were able to vary hydrogel stiffness from ≈ 2–20 kPa and tune the onset of strain-stiffening towards a biologically relevant regime (critical stress ≈ 10 Pa). Moreover, we used fluorescence resonance energy transfer (FRET) to demonstrate ligand displacement along the copolymer backbone via competitive binding and highlighted the cytocompatibility of our system using human dermal fibroblasts. The combination of highly tunable composition, stiffness, and strain-stiffening, in conjunction with temporal control over ligand exchange/presentation, positions these cytocompatible copolymers as a powerful synthetic platform for the rational design of next generation synthetic biomaterials.

Oxidative stress leads to a lower success rate of clinical islet transplantation. Here, FDA‐approved compounds are screened for their potential to decrease oxidative stress and to protect or enhance pancreatic islet viability and function. Studies are performed on in vitro “pseudoislet” spheroids, which are pre‐incubated with 1280 different compounds and subjected to oxidative stress. Cell viability and oxidative stress levels are determined using a high‐throughput fluorescence microscopy pipeline. Initial screening on cell viability results in 59 candidates. The top ten candidates are subsequently screened for their potential to decrease induced oxidative stress, and eight compounds efficient reduction of induced oxidative stress in both alpha and beta cells by 25–50%. After further characterization, the compound sulfisoxazole is found to be the most capable of reducing oxidative stress, also at short pre‐incubation times, which is validated in primary human islets, where low oxidative stress levels and islet function are maintained. This study shows an effective screening strategy with 3D cell aggregates based on cell viability and oxidative stress, which leads to the discovery of several compounds with antioxidant capacity. The top candidate, sulfisoxazole is effective after a 30 min pre‐incubation, maintains baseline islet function, and may help alleviate oxidative stress in pancreatic islets.

Patients with chronic kidney disease (CKD) have an increased risk for cardiovascular morbidity and mortality. Capillary rarefaction may be both one of the causes as well as a consequence of CKD and cardiovascular disease. We reviewed the published literature on human biopsy studies and conclude that renal capillary rarefaction occurs independently of the cause of renal function decline. Moreover, glomerular hypertrophy may be an early sign of generalized endothelial dysfunction, while peritubular capillary loss occurs in advanced renal disease. Recent studies with non-invasive measurements show that capillary rarefaction is detected systemically (e.g., in the skin) in individuals with albuminuria, as sign of early CKD and/or generalized endothelial dysfunction. Decreased capillary density is found in omental fat, muscle and heart biopsies of patients with advanced CKD as well as in skin, fat, muscle, brain and heart biopsies of individuals with cardiovascular risk factors. No biopsy studies have yet been performed on capillary rarefaction in individuals with early CKD. At present it is unknown whether individuals with CKD and cardiovascular disease merely share the same risk factors for capillary rarefaction, or whether there is a causal relationship between rarefaction in renal and systemic capillaries. Further studies on renal and systemic capillary rarefaction, including their temporal relationship and underlying mechanisms are needed. This review stresses the importance of preserving and maintaining capillary integrity and homeostasis in the prevention and management of renal and cardiovascular disease.

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.