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Engineering human brain organoids with floating scaffolds enhances the maturity and reproducibility of cortical tissue structure. Three-dimensional cell culture models have either relied on the self-organizing properties of mammalian cells 1 , 2 , 3 , 4 , 5 , 6 or used bioengineered constructs to arrange cells in an organ-like configuration 7 , 8 . While self-organizing organoids excel at recapitulating early developmental events, bioengineered constructs reproducibly generate desired tissue architectures. Here, we combine these two approaches to reproducibly generate human forebrain tissue while maintaining its self-organizing capacity. We use poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments as a floating scaffold to generate elongated embryoid bodies. Microfilament-engineered cerebral organoids (enCORs) display enhanced neuroectoderm formation and improved cortical development. Furthermore, reconstitution of the basement membrane leads to characteristic cortical tissue architecture, including formation of a polarized cortical plate and radial units. Thus, enCORs model the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration. Our data demonstrate that combining 3D cell culture with bioengineering can increase reproducibility and improve tissue architecture.

The authors have designed modular synthetic hydrogel networks for mouse and human intestinal stem cell cultures that support intestinal organoid formation. Synthetic matrices for organoid culture Epithelial organoids are being used in the laboratory to model organ development and function. So far these systems have relied on animal-derived matrices, which can be highly variable and are poorly defined, a problem that also makes them unsuitable for clinical application. Matthias Lutolf and colleagues have now designed modular synthetic hydrogen networks to support the formation of intestinal organoids from mouse and human intestinal stem cells. The authors produced dynamic matrices, initially optimal for intestinal stem cell expansion, which depends on high stiffness, and subsequently become permissive to intestinal differentiation and organoid formation through softening of their mechanical properties. Epithelial organoids recapitulate multiple aspects of real organs, making them promising models of organ development, function and disease 1 , 2 , 3 . However, the full potential of organoids in research and therapy has remained unrealized, owing to the poorly defined animal-derived matrices in which they are grown 4 . Here we used modular synthetic hydrogel networks 5 , 6 to define the key extracellular matrix (ECM) parameters that govern intestinal stem cell (ISC) expansion and organoid formation, and show that separate stages of the process require different mechanical environments and ECM components. In particular, fibronectin-based adhesion was sufficient for ISC survival and proliferation. High matrix stiffness significantly enhanced ISC expansion through a yes-associated protein 1 (YAP)-dependent mechanism. ISC differentiation and organoid formation, on the other hand, required a soft matrix and laminin-based adhesion. We used these insights to build a fully defined culture system for the expansion of mouse and human ISCs. We also produced mechanically dynamic matrices that were initially optimal for ISC expansion and subsequently permissive to differentiation and intestinal organoid formation, thus creating well-defined alternatives to animal-derived matrices for the culture of mouse and human stem-cell-derived organoids. Our approach overcomes multiple limitations of current organoid cultures and greatly expands their applicability in basic and clinical research. The principles presented here can be extended to identify designer matrices that are optimal for long-term culture of other types of stem cells and organoids.

Maintaining long-term euglycemia after intraportal islet transplantation is hampered by the considerable islet loss in the peri-transplant period attributed to inflammation, ischemia and poor angiogenesis. Here, we show that viable and functional islet organoids can be successfully generated from dissociated islet cells (ICs) and human amniotic epithelial cells (hAECs). Incorporation of hAECs into islet organoids markedly enhances engraftment, viability and graft function in a mouse type 1 diabetes model. Our results demonstrate that the integration of hAECs into islet cell organoids has great potential in the development of cell-based therapies for type 1 diabetes. Engineering of functional mini-organs using this strategy will allow the exploration of more favorable implantation sites, and can be expanded to unlimited (stem-cell-derived or xenogeneic) sources of insulin-producing cells. Islet transplantation is a feasible approach to treat type I diabetes, however inflammation and poor vascularisation impair long-term engraftment. Here the authors show that incorporating human amniotic epithelial cells into islet organoids improves engraftment and function of organoids, through enhanced revascularisation.

The development of neural circuits involves wiring of neurons locally following their generation and migration, as well as establishing long-distance connections between brain regions. Studying these developmental processes in the human nervous system remains difficult because of limited access to tissue that can be maintained as functional over time in vitro. We have previously developed a method to convert human pluripotent stem cells into brain region–specific organoids that can be fused and integrated to form assembloids and study neuronal migration. In contrast to approaches that mix cell lineages in 2D cultures or engineer microchips, assembloids leverage self-organization to enable complex cell–cell interactions, circuit formation and maturation in long-term cultures. In this protocol, we describe approaches to model long-range neuronal connectivity in human brain assembloids. We present how to generate 3D spheroids resembling specific domains of the nervous system and then how to integrate them physically to allow axonal projections and synaptic assembly. In addition, we describe a series of assays including viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics combined with calcium imaging and electrophysiological recordings to probe and manipulate the circuits in assembloids. The assays take 3–4 months to complete and require expertise in stem cell culture, imaging and electrophysiology. We anticipate that these approaches will be useful in deciphering human-specific aspects of neural circuit assembly and in modeling neurodevelopmental disorders with patient-derived cells.A protocol is described for generating human brain assembloids and performing viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics with calcium imaging and electrophysiological recordings to model neural circuits.

Time-lapse imaging systems for embryo incubation and selection might improve outcomes of in-vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) treatment due to undisturbed embryo culture conditions, improved embryo selection, or both. However, the benefit remains uncertain. We aimed to evaluate the effectiveness of time-lapse imaging systems providing undisturbed culture and embryo selection, and time-lapse imaging systems providing only undisturbed culture, and compared each with standard care without time-lapse imaging. We conducted a multicentre, three-parallel-group, double-blind, randomised controlled trial in participants undergoing IVF or ICSI at seven IVF centres in the UK and Hong Kong. Embryologists randomly assigned participants using a web-based system, stratified by clinic in a 1:1:1 ratio to the time-lapse imaging system for undisturbed culture and embryo selection (time-lapse imaging group), time-lapse imaging system for undisturbed culture alone (undisturbed culture group), and standard care without time-lapse imaging (control group). Women were required to be aged 18–42 years and men (ie, their partners) 18 years or older. Couples had to be receiving their first, second, or third IVF or ICSI treatment and could not participate if using donor gametes. Participants and trial staff were masked to group assignment, embryologists were not. The primary outcome was live birth. We performed analyses using the intention-to-treat principle and reported the main analysis in participants with primary outcome data available (full analysis set). The trial is registered on the International Trials Registry (ISRCTN17792989) and is now closed. 1575 participants were randomly assigned to treatment groups (525 participants per group) between June 21, 2018, and Sept 30, 2022. The live birth rates were 33·7% (175/520) in the time-lapse imaging group, 36·6% (189/516) in the undisturbed culture group, and 33·0% (172/522) in the standard care group. The adjusted odds ratio was 1·04 (97·5% CI 0·73 to 1·47) for time-lapse imaging arm versus control and 1·20 (0·85 to 1·70) for undisturbed culture versus control. The risk reduction for the absolute difference was 0·7 percentage points (97·5% CI –5·85 to 7·25) between the time-lapse imaging and standard care groups and 3·6 percentage points (–3·02 to 10·22) between the undisturbed culture and standard care groups. 79 serious adverse events unrelated to the trial were reported (n=28 in time-lapse imaging, n=27 in undisturbed culture, and n=24 in standard care). In women undergoing IVF or ICSI treatment, the use of time-lapse imaging systems for embryo culture and selection does not significantly increase the odds of live birth compared with standard care without time-lapse imaging. Barts Charity, Pharmasure Pharmaceuticals, Hong Kong OG Trust Fund, Hong Kong Health and Medical Research Fund, Hong Kong Matching Fund.

Balanced organogenesis requires the orchestration of multiple cellular interactions to create the collective cell behaviours that progressively shape developing tissues. It is currently unclear how individual, localized parts are able to coordinate with each other to develop a whole organ shape. Here we report the dynamic, autonomous formation of the optic cup (retinal primordium) structure from a three-dimensional culture of mouse embryonic stem cell aggregates. Embryonic-stem-cell-derived retinal epithelium spontaneously formed hemispherical epithelial vesicles that became patterned along their proximal–distal axis. Whereas the proximal portion differentiated into mechanically rigid pigment epithelium, the flexible distal portion progressively folded inward to form a shape reminiscent of the embryonic optic cup, exhibited interkinetic nuclear migration and generated stratified neural retinal tissue, as seen in vivo . We demonstrate that optic-cup morphogenesis in this simple cell culture depends on an intrinsic self-organizing program involving stepwise and domain-specific regulation of local epithelial properties. Stem cells self-generate retinal tissue Organogenesis relies on the orchestration of many cellular interactions to create the collective cell behaviours needed to shape developing tissues. Yoshiki Sasai and colleagues have developed a three-dimensional cell culture system in which floating clusters of mouse embryonic stem cells can successfully organize themselves into a layered structure resembling the optic cup, a pouch-like structure that develops into the inner and outer layers of the retina during embryogenesis. In further 3D culture, the optic cup forms fully stratified retinal tissue as seen in the postnatal eye. This approach might have important implications for stem-cell therapy for retinal repair. Organogenesis relies on the orchestration of many cellular interactions to create the collective cell behaviours that progressively shape developing tissues. Using a three-dimensional embryonic stem cell culture system, this study successfully generated neural retinal tissues that formed a fully stratified neural retinal structure with all the major components located in their proper spatial location as seen during optic-cup development in vivo . This approach might have important implications for stem cell therapy for retinal repair.

With advantageous features such as minimizing the cost, time, and sample size requirements, organ-on-a-chip (OOC) systems have garnered enormous interest from researchers for their ability for real-time monitoring of physical parameters by mimicking the in vivo microenvironment and the precise responses of xenobiotics, i.e., drug efficacy and toxicity over conventional two-dimensional (2D) and three-dimensional (3D) cell cultures, as well as animal models. Recent advancements of OOC systems have evidenced the fabrication of ‘multi-organ-on-chip’ (MOC) models, which connect separated organ chambers together to resemble an ideal pharmacokinetic and pharmacodynamic (PK-PD) model for monitoring the complex interactions between multiple organs and the resultant dynamic responses of multiple organs to pharmaceutical compounds. Numerous varieties of MOC systems have been proposed, mainly focusing on the construction of these multi-organ models, while there are only few studies on how to realize continual, automated, and stable testing, which still remains a significant challenge in the development process of MOCs. Herein, this review emphasizes the recent advancements in realizing long-term testing of MOCs to promote their capability for real-time monitoring of multi-organ interactions and chronic cellular reactions more accurately and steadily over the available chip models. Efforts in this field are still ongoing for better performance in the assessment of preclinical attributes for a new chemical entity. Further, we give a brief overview on the various biomedical applications of long-term testing in MOCs, including several proposed applications and their potential utilization in the future. Finally, we summarize with perspectives.

Traditional methods of cell growth and manipulation on 2-dimensional (2D) surfaces have been shown to be insufficient for new challenges of cell biology and biochemistry, as well as in pharmaceutical assays. Advances in materials chemistry, materials fabrication and processing technologies, and developmental biology have led to the design of 3D cell culture matrices that better represent the geometry, chemistry, and signaling environment of natural extracellular matrix. In this review, we present the status of state-of-the-art 3D cell-growth techniques and scaffolds and analyze them from the perspective of materials properties, manufacturing, and functionality. Particular emphasis was placed on tissue engineering and in vitro modeling of human organs, where we see exceptionally strong potential for 3D scaffolds and cell-growth methods. We also outline key challenges in this field and most likely directions for future development of 3D cell culture over the period of 5-10 years.

Life science research focuses on deciphering the biochemical mechanisms that regulate cell proliferation and function and largely depends on the use of tissue culture methods in which cells are grown on two-dimensional hard plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the complex three-dimensional (3D) architecture of a tissue or organ composed of various cell types, extracellular matrix (ECM) and interstitial fluids. Moreover, if we consider a cell as a perfectly defined volume, flattened cells have full access to the environment and limited cell-to-cell contact. However if the cell is a cube in a simple cuboidal epithelium, then its access to the lumen is limited to one face, with the opposite face facing the basal membrane and the remaining four faces lying in close contact with neighbouring cells. This is of great importance when considering the access of viruses and bacteria to the cell surface, the excretion of soluble factors or proteins or the signalling within or between cells. This short review discusses various cell culture approaches to improve the simulation of the 3D environment of cells.

The general approach to industrial production of monoclonal antibodies is fed-batch culture using Chinese Hamster Ovary (CHO) cells. Perfusion culture is also attracting attention as a next-generation culture method. In these culture methods, optimization of amino acid and glucose concentration in the culture medium is essential, and influences cell proliferation, viability, productivity, and monoclonal antibody quality. Further, the maintenance of optimal nutrient levels – by avoiding both depletion and accumulation – is crucial. This study aimed to develop a dynamic feeding strategy based on specific indicators to maintain optimal amino acid and glucose concentrations. Multivariate correlation analysis confirmed a strong relationship between nutrient consumption and viable cell density (VCD). Regression analysis was used to establish a regression model to estimate amino acid and glucose consumption based on VCD. Using this model, the nutrient composition of feed media for both fed-batch and perfusion cultures was adjusted, and a dynamic feeding strategy guided by VCD was evaluated. The observed nutrient concentration trends closely matched the model’s predictions, confirming that VCD is a reliable indicator for implementing dynamic feeding. In both fed-batch and perfusion cultures, the VCD-guided dynamic feeding strategy enables the maintenance of multiple amino acids and glucose at target concentrations.