three-dimensional, bone-like structures that mimic the physiological and structural properties of natural bone tissue. Bone organoids are created by differentiating induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs) into osteogenic lineages within a supportive scaffold, allowing for the self-organization and maturation of complex bone structures. In this study, organoids were implanted into critical-sized calvarial defect models in both wild-type and immunocompromised nude mouse models to delineate the role of immunity in orchestrating the bone regeneration process. It was discovered that immune cells play a crucial role in organoid integration and remodeling. A compromised host immune system hindered the organoid-mediated regeneration process. In wild-type mice, bone organoids significantly improved the healing of critical-sized bone defects. Overall, bone organoids represent a transformative tool in bone tissue engineering, offering a versatile and effective strategy for bone regeneration and the treatment of bone defects. Their ability to closely mimic natural bone properties and their potential for patient-specific applications underscore their importance in advancing regenerative medicine and improving clinical outcomes for patients with bone injuries and disorders.

a mechanically stiffer ECM. Various studies report on DC chemotaxis towards chemokine gradients, however, analyzing DC migration as a function of both chemotactic and stiffness gradients is critical to understanding the mechanisms of DC maturation and migration in TMEs. Here, we create a novel 2.5-dimensional GelMA migration platform with tunable agreeing and opposing chemokine and stiffness gradients. Using this model, we found that both unstimulated and stimulated DCs preferentially migrate towards softer ECMs lacking CCL21. In the presence of immobilized CCL21, however, stimulated CCR7-presenting DCs travel at significantly reduced velocities and travel smaller accumulated distances compared to CCR7-presenting DCs on matrices without CCL21. Directional migration, however, followed the opposite trend, with CCR7-presenting DCs traveling greater Euclidean distances on matrices incorporating immobilized CCL21 compared to matrices lacking the chemokine. Understanding the underlying mechanisms of these migration patterns is imperative for manipulating and guiding cell migration into TMEs. Here, we demonstrate the ability to vary migration cues in our platform and analyze resulting migration patterns to discover mechanisms of DC migration and maturation in TMEs.

the intricate mechanisms driving GBM progression remains elusive. Given the significant role of tumor niche signaling, we engineered a GBM tumor microenvironment to investigate how interactions between extracellular matrix ligands and matrix viscoelasticity impact GBM cell behavior. Integrating dynamic covalent bonds and cell-adhesive peptides into a hyaluronan-elastin-like protein (HELP) hydrogel, we designed a 3D biomaterial platform with adjustable stress relaxation rates. Two cell-adhesive peptides, mimicking ligands found in fibronectin and tenascin-C proteins, were introduced within the ELP backbone. Hyaluronan, fibronectin, and tenascin-C are all upregulated in the GMB extracellular matrix microenvironment. We formulated hydrogels with consistent stiffness (G' ~ 800 Pa) and adjustable stress relaxation rates (slow or fast) by altering the kinetics of the dynamic covalent crosslinks. HELP hydrogels maintained high viability (>90%) of encapsulated U87 cells, a GBM cell line. Slow stress-relaxing HELP gels induced formation of cell clusters in all ligand formulations, while fast stress-relaxing gels enhanced cell spreading. Furthermore, P-selectin expression, a cell adhesion molecule serving as a GBM invasion marker, was upregulated in slow stress-relaxation groups compared to fast stress-relaxation groups in all ligand formulations. This suggests that the matrix stress-relaxation rate may alter GBM invasiveness. In summary, HELP hydrogels with controlled biochemical and biomechanical properties modulate GBM cell behavior. While U87 cells remain viable and proliferative in all tested hydrogels, changes in P-selectin expression were influenced solely by the matrix stress-relaxation rate and not ligand identify. These findings highlight the potential of protein-engineered, viscoelastic biomaterials to advance GBM models and deepen our understanding of GBM progression.

similar to the human brain, providing a platform to explore the underlying biochemical and cellular responses preterm brain injury and two promising neurotherapeutics - azithromycin (Az) and erythropoietin (Epo). Objective: To examine microglial and transcriptomic signatures of combined Az and Epo neuroprotection in a postnatal day (P)14 extremely preterm-equivalent ferret ex vivo brain slice model of oxygen glucose deprivation (OGD). Methods/Approach: 300μm whole-hemisphere live ferret brain slices were obtained at P14. After 72h culturing in vitro, slices were subjected to 2h of OGD followed by treatment with Epo (3 IU/mL), Az (15mM), or Az+Epo for 24h. Cell death was determined using confocal microscopy of DAPI-stained pyknotic nuclei counts. Microglial morphology was assessed using a machine learning pipeline. Digital transcriptomics were analyzed using a ferret-specific NanoString nCounter panel of 255 genes. Results: Cell death was significantly reduced by Az+Epo, more so than either drug individually. Based on regional cell viability, Az+Epo displayed synergy according to multiple theoretical models except combination subthresholding. In regions where Az and Az+Epo did not have neuroprotective effects, the presence of ameboid microglial morphology was increasingly observed compared to OGD alone. Augmentation of inflammatory macrophage responses, and reversal of oxidative stress and apoptotic cellular responses were related to Az+Epo neuroprotection with emergent transcriptomic signatures that were synergistically regulated. Conclusions: In the extremely preterm-equivalent ferret brain, Az+Epo provide synergistic neuroprotection via mitigation of cell death pathways, morphological microglial changes, and transcriptomic responses, that are not seen with either drug individually. These pathways warrant further investigation as targets for neuroprotection in the extremely preterm brain.

functional tissues or organoids. We have developed a viable method to pattern encapsulated human induced pluripotent stem (hiPS) cells within a poly(ethylene glycol)-based hydrogel using digital light projection (DLP)-based bioprinting. We optimized the bioink formulation for encapsulating stem cells that can consistently maintain their stemness by developing a resultant hydrogel with a stiffness of 500 Pa and that has integrin binding sites preferred by stem cells. We bioprinted the hiPS cells to characterize their stemness and proliferation over the course of 7 days. On Day 7, the stem cell markers were still highly expressed, and the proliferation marker Ki-67 was still expressed. Notably, we did not need to add the ROCK inhibitor small molecule Y-27632 during bioprinting to achieve high viability. To further demonstrate the viability and function of the 3D-cultured bioprinted hiPS cells, we passaged the cells by degrading the matrix with collagenase and, after reseeding, observed hiPS cell colony formation, stemness, and proliferation. Further, we demonstrated that the 3D culture of hiPS cells can be differentiated into neuroepithelial and cardiomyocyte cells using standard commercial kits. Our results indicate that we have developed a hydrogel formulation compatible with light-based bioprinting to encapsulate human stem cells in a 3D matrix that enables stemness maintenance, proliferation, and directed differentiation. This work can immediately be applied to enable bioreactor scale up of stem cell production. Additionally, the ability to 3D pattern stem cells could enable the bottom-up engineering of multicellular tissue constructs.

as stenosis, branches, tortuosity, aneurysm, etc. cannot be recapitulated in a simple vascular model. Therefore, it is crucial to incorporate the architectural variations into the engineered vessels to better reflect the hemodynamics and subsequentially mechanobiology observed in vivo. Gravitational Lumen Patterning (GLP) is a biofabrication technique that has been employed in vascular organ-chips to embed the inner surfaces of the microfluidic channels with viscous extracellular matrix (ECM) proteins. GLP has demonstrated success in producing uniform cylindrical lumens in previous studies. However, a major limitation of the existing ECM-embedded vessel chips is the lack of spatial relevance to study diverse types of vessels. This study presents a new application of the GLP biofabrication technique in producing 3D complex vessels with stenosis, branch, aneurysms, and tortuous architectures that are embedded in collagen. GLP is executed by the integration of surface tension, gravity, and fluidic pressure forces between two fluids with significantly different levels of viscosity. The lumen patterning is ultimately governed by the shape and size of the external microfluidic channels through the local changes in pressure experienced by the two fluids, hence creating complex lumens with varying structures. We also reconstruct the vessel lumens using computer aided design and perform computational fluid dynamics simulations to predict the altered hemodynamics in the diseased regions then compare with the observed endothelial cell morphologies. These engineered vessels with various architectures can serve as platforms to eventually investigate vascular complications like aortic aneurysm, arterial tortuosity syndrome, atherosclerosis, and carotid artery disease etc., where vessel architecture plays a crucial role in disease onset and progression. The physiological architectures and spatial relevance of these vessels contribute to the increase in the predictive power of in vitro models.

the effects of toxins and drugs. Currently, the FDA as an organization has limited experience in evaluating preclinical data based on experimentation in 3D in-vitro models. Our research teams, funded by the Department of Defense(DoD), are currently developing strategies for generating stable and physiologically relevant human micro-engineered organ equivalents(MOEs) for majority of tissue types. Our group is more focused on MOE models for liver cells using Indium-Tin Oxide (ITO) as a scaffold for drug toxicity screening. Preliminary studies on this scaffold will be presented, along with self-assembled monolayer modifications and metabolomic data tracking the changes in the cells and plates over time using Nuclear Magnetic Resonance (NMR) Metabolomics. NMR spectroscopy is a non-invasive and nondestructive technique which gives a detailed insightful observation of intracellular responses and hepatotoxicity study using the passaged medium containing cellular exudates.

cell transplantation. Creating programmable behaviours using protein-based circuits have advantages such as compact delivery and robust context-independent performance compared to traditional transcriptional circuits. Previously, we described a generalizable platform called RELEASE (Retained Endoplasmic Cleavable Secretion) which enabled intercellular signalling through the removal of ER-retention motifs compatible with pre-existing protease-based circuits. However, to program complex expression profiles beyond simple off-to-on operations, multiple orthogonal proteases are required in multilayer cascades of signal processing prior to regulating secretion. These additional components reduce delivery options and decrease cascade efficiency. Rather, we sought a compact protein design that allowed for programed protein secretion using a single processing layer. To achieve this, we leverage endogenous scaffolding proteins known as 14-3-3 proteins to selectively bind and inhibit ER-retention motifs by blocking retrograde transport machinery. To best harness the combination of retrograde transport, protease cleavage, and steric inhibition, we designed a protein architecture which places a small 14-3-3 binder on the C-terminus of a RELEASE construct adjacent to an ER-retention motif. By introducing a protease cleavage site between these two domains, cleavage can be used to control protein secretion with NOT logic. By combining this architecture with the original RELEASE design, we have demonstrated all two-input Boolean logic, such as NIMPLY gating, with minimal construct delivery in a suite of protein designs. Furthermore, by altering cleavage efficiencies, we can also introduce quantitative processing such as bandpass and bandstop behaviors. With the ability to program new types of regulation into a suite of protein architectures, this platform expands the programmable capabilities of synthetic protein circuits in a compact manner and enables their delivery using pre-existing mRNA and viral vector methods.

the need for permanent systemic immunosuppression. Successful cell-based therapies for T1D require appropriate cell delivery and encapsulation systems that protect transplanted cells and enable them to function as the endocrine pancreas. For this, we developed the Neovascularized Implantable Cell Homing and Encapsulation (NICHE) platform, a subcutaneously implantable 3D-bioengineered device integrating direct vascularization and local immunosuppression. This study leveraged the pro-angiogenic and immunomodulatory properties of mesenchymal stem cells (MSCs) within NICHE to support islet engraftment. Methods: We explored sex-specific differences by conducting experiments in an immunocompetent and diabetic rat model accounting for both genders. We evaluated improvement in subcutaneous vascularization of NICHE loaded with MSCs. We characterized the local immune microenvironment after allogeneic islet transplantation with and without MSC co-transplantation via mass cytometry (CyTOF) and imaging mass cytometry (IMC). We further evaluated local and systemic immune responses through local cytokine quantification and flow cytometry of draining lymph node (dLN) and spleen. Results: We achieved subcutaneous vascularization in NICHE devices, with more functional blood vessels in NICHE loaded with MSCs. We characterized the immune cell populations infiltrating the NICHE local microenvironment after allogeneic islet transplant or islet-MSC co-transplant and identified early mechanisms of immunomodulation provided by MSCs. Notably, our results revealed gender-specific differences on both vascularization and immunomodulation. Conclusion: The NICHE platform is a novel and clinically translatable approach for islet transplantation. Here we leveraged MSCs to promote vascularization of NICHE and explored their immunomodulatory properties without immunosuppressant drugs. Our study aimed to characterize the immune response following allogeneic islet transplantation to better device local immunosuppression approaches. Future studies will focus on assessing the interplay of different immunosuppressants, uncovering optimal combinations and deepening the understanding of local immuno-delivery for clinical application.

samples indicate the presence of microorganism, making CO2 monitoring crucial for early detection of blood infection like sepsis. Traditional blood culture methods are often expensive, time-consuming, and prone to contamination. The lack of cost-effective and efficient diagnostic tools hampers timely and accurate diagnosis, leading to delayed treatments and higher mortality rates. This study therefore, addresses this critical gap by developing a semi-automated blood culture CO₂ monitoring device that is low-cost, efficient, and suitable for Low- or Middle-Income Countries (LMICs). The Semi-automated CO₂ Monitoring Device (SCMD) was developed using an MQ-2 gas sensor, powered by a 9V battery and a 5V regulator for the ATmega microcontroller. The device was tested with three positive sepsis samples and two negative sepsis samples in the University College Hospital (UCH) microbiology laboratory. Its performance was compared with traditional blood culture techniques, which are the gold standard, and the BACTEC blood culture bottle. The accuracy of the device and the testing time were evaluated. The SCMD detected the increased CO₂ produced by the growth of microorganisms in infected blood samples with over 90% accuracy and substantially reduced the turnaround times for detecting bacterial growth, potentially enabling quicker diagnosis and treatment of sepsis, by displaying test results in less than five minutes. Additionally, the device demonstrated a running time of up to 24 hours on a single battery charge, making it suitable for continuous use in healthcare settings. The Semi-automated CO₂ Monitoring Device offers a prompt solution to improve diagnostic capabilities and patient outcomes in resource-constrained environments. Integrating this device into existing healthcare infrastructure could significantly enhance the timely detection and treatment of bloodstream infections, ultimately saving lives.

address current medical needs and prepare us for a foreseeable pandemic. Thus, there is a pressing need to develop effective antimicrobials against systemic bacteremia infections caused by drug-resistant pathogens. Polymyxin B (PMB) is a clinically approved antibiotic with exceptional antimicrobial activity against multidrug-resistant gram-negative bacteria such as E. coli. Today, with the growing emergence of “superbugs” and a dry antibiotic discovery pipeline, PMB is increasingly used as a last-line therapy to treat systemic multidrug-resistant infections. However, PMB usage is strictly limited due to significant nephrotoxicity (up to 60%), which is the major dose-limiting factor preventing broader use of this highly bactericidal antibiotic. To improve the safety and efficacy of PMB, we developed a recombinant bacteriophage (phage) that binds lipopolysaccharide on gram-negative bacteria. By conjugating PMB to the phage to create “PMB-Phage”, a high concentration of PMB can be specifically delivered to bacterial surfaces. This has two major effects. First, PMB-Phage lowers the minimum inhibitory concentration of PMB by orders of magnitude, so less PMB is required to kill bacterial cells. For bacterial isolates that have moderate resistance to PMB, this effect actually renders them susceptible to PMB-Phage. Second, by reducing the required circulating concentration of drug and specifically targeting bacterial cells (not mammalian cells), PMB-Phage will greatly reduce the nephrotoxicity of PMB, enabling safe treatment for a broader patient population. PMB-Phage will thus simultaneously expand the number of bacterial strains susceptible to PMB and increase the safety of PMB. Preliminary studies support the efficacy and lack of toxicity of PMB-Phage when treating bloodstream infections in a mouse model. These advantages represent a new strategy to counter antimicrobial resistance by potentiating an existing antibiotic with a phage drug delivery vehicle.

applications. Functionalizing this polymer with other compounds could improve its properties or provide new features for that. This research aims to evaluate the effect of utilizing other additional compounds on physicochemical and biological properties of CMC-based hydrogel. In details, we have used CMC, and citric acid with the same amounts in all samples and polyethylene glycol and carboxylate β-cyclodextrin with different ratio. Different analyses (including fourier-transform infrared spectroscopy (FTIR), swelling and deswelling ratio, degradation ability, and water vapor transmission rate) were used to determine and compare physicochemical properties of the fabricated hydrogel. Then, ciprofloxacin, as an antibiotic, was loaded inside these hydrogels and the amounts of loading and released profile were compared with each other. In the final step, drug loaded samples were tests against 3T3 fibroblast cell line to see their effect on viability and migration ability of fibroblast cells.

superelastic properties allow it to deform during surgery and return to its shape after deployment, making it ideal for minimally invasive applications like brain aneurysms and heart stents. The coating's multilayer nanoparticle system creates a porous matrix, infused with a biocompatible lubricant to repel pathogens and cellular debris. We demonstrate the SLIPS coating’s adherence on TiNi substrates, showcasing omniphobic repellency and mechanical stability. This system reduces infection risk post-surgery, increases device patency, and improves flow within the conduit. Additionally, TiNi-SLIPS devices can be drug-loaded for treatments like antibiotics, anti-inflammatory drugs, or cancer molecules. We confirmed multilayer deposition stability on TiNi, with SEM images showing an amorphous, porous structure. The average particle size was 13.620 nm ± 2.431 nm (n=15) with 18.46% free space. Surface repellency was measured using static contact angle, contact angle hysteresis, and sliding angle with deionized water and DMSO. Non-SLIPS TiNi samples showed significant pinning, with high contact angle hysteresis and droplet removal failure after a 25° tilt. Deionized water had a 112.6° ± 10.0° contact angle and >36.6° ± 12.7° CAH. Post-lubrication with silicone oil (350 cP), the TiNi substrate had a 94.9° ± 7.3° contact angle, 4.2° ± 5.3° CAH, and 4.4° ± 5.2° sliding angle, confirming high repellency. Our initial tests have also demonstrated tunability of the drug delivery kinetics using various oil-infused viscosity and loading types. Our method applies SLIPS to a biocompatible, shape memory, and superelastic alloy, potentially reducing restenosis and thrombosis complications, and can be used in coronary and carotid stents.

collagen were examined to elucidate the influence of the hydrogel composition on the release profiles of proinsulin variants. HA- collagen-fibronectin hydrogels were synthesized according to a standardized protocol, with variations in collagen and fibronectin concentrations. Three proinsulin variants, F25D, F25D- K3, and F25D-K12, were used along with the hydrogels to conduct protein release assays over 24 hours using fluorescence endpoint measurements. Distinct release profiles were observed for each proinsulin variant. For F25D, hydrogels containing fibronectin at 0.125 mg/mL and collagen at 1.0 mg/mL exhibited the highest protein release, with a significant increase observed from 12 to 24 hours. Collagen, particularly at 1.0 mg/mL, showed the most considerable protein release across all variants, while fibronectin demonstrated comparatively lower release rates, especially at 0.25 mg/mL. The findings underscore the importance of hydrogel composition in modulating protein release kinetics. For instance, collagen emerged as a critical component in protein release, whereas fibronectin exhibited lower release rates. The results provide valuable insights for hydrogel-based drug delivery systems, emphasizing optimizing hydrogel compositions to achieve desired and realistic release profiles for real-life therapeutic applications. Further research might explore factors influencing protein release dynamics, facilitating more effective drug delivery strategies.

the design of therapeutic agents. Despite their pivotal roles, these strategies show inadequate efficacy, in part, due to the diversity of toxicants, leading to narrow-spectrum neutralization. Here, we developed of neuron cell membrane-based nanostructures. Cell membrane-based nanostructures represent a unique solution by mimicking susceptible host cells, intercepting harmful agents without knowing their molecular structure. We have used tetrodotoxin and botulinuum toxin as the model toxin. The nanostructures inhibited TTX-induced ion channel blockage and BoNT-mediated inhibition of synaptic vesicle recycling, which were evaluated by cell assay. In the mouse model of intoxication, the survival rate of mice was enhanced after treating with these nanostructures. Overall, by exploiting novel nanostructures, these nanostructures have potential in broad spectrum neutralization of a variety of pathological agents and offer enhanced therapeutic efficacy.

subcellular compartments other than the endosomes in order to exert activity. In nature, viruses can readily deliver their genetic material to the cytosol of host cells by triggering membrane fusion after endocytosis. For the influenza A virus, the hemagglutinin (HA) protein found on its surface fuses the viral envelope with the surrounding membrane at endosomal pH values. Biomimetic nanoparticles capable of endosomal escape were fabricated using a membrane coating derived from cells engineered to express HA on their surface. When evaluated in vitro, these virus-mimicking nanoparticles were able to deliver an mRNA payload to the cytosolic compartment of target cells, resulting in the successful expression of the encoded protein. When the mRNA-loaded nanoparticles were administered in vivo, protein expression levels were significantly increased in both local and systemic delivery scenarios. We therefore conclude that utilizing genetic engineering approaches to express viral fusion proteins on the surface of cell membrane-coated nanoparticles is a viable strategy for modulating the intracellular localization of encapsulated cargoes.

more effective antigen delivery to the immune system. Among these, nanotoxoids present a promising biomimetic platform, utilizing cell membrane coating technology to safely deliver otherwise toxic bacterial antigens in their native form for antivirulence vaccination. In this study, nanotoxoids formulated against staphylococcal α-hemolysin are embedded into a DNA-based hydrogel containing immunostimulatory CpG motifs to further boost their immunogenicity. The resulting nanoparticle–hydrogel composite is injectable and enhances the in vivo delivery of vaccine antigens while simultaneously stimulating nearby immune cells. This leads to elevated antibody production and stronger antigen-specific cellular immune responses. In murine models of pneumonia and skin infection caused by methicillin-resistant Staphylococcus aureus, mice vaccinated with the hybrid vaccine formulation are well-protected. This work underscores the benefits of combining nanoparticulate antigen delivery systems with immunostimulatory hydrogels into a single platform, which can be readily adapted for a wide range of infectious diseases.

disease treatment. Here, we reported the development and characterization of platelet membrane-derived nanodiscs (PLT-NDs) designed as platelet decoys for biological neutralization. PLT-NDs have shown the capability to neutralize anti-platelet autoantibodies, blocking their interaction with platelets. In a thrombocytopenia mouse model, PLT-NDs effectively bind with harmful anti-platelet antibodies, preventing platelet depletion and maintaining hemostasis. Additionally, PLT-NDs effectively neutralize bacterial virulence factors secreted by methicillin-resistant Staphylococcus aureus (MRSA). In a mouse model of MRSA infection, PLT-ND treatment significantly enhances survival rates. Biocompatibility and biosafety of PLT-NDs are confirmed through acute toxicity studies in mice. This work highlights the potential of PLT-NDs as innovative platelet mimics capable of neutralizing various biological threats, expanding the capabilities of platelet-mimicking nanomedicine by creating a distinctive disc-like nanostructure derived from natural platelet membranes.

analyzing macroscopic structure and microscopic morphology of bacterial biofilms. Few studies present procedures for electron microscopy of biofilms on non-planar substrates, such as the dental implantitself. This gap in electron microscopy procedure hinders the research of failed dental implants in clinical settings. We present a procedure for the sample preparation and electron microscopy of dental implants,developed using Streptococcus mutans, a common colonizer of the oral cavity, and MG 63 osteoblast-like cells, which provide insight into osseointegration. S. mutans bacteria formed a biofilm on the dental implantconsisting of rod-shaped cocci surrounded by extracellular polymeric substance (EPS). A fibroblast-like shape characterized MG 63 cell morphology, which tended to attach in monolayers. The dental implantswere cultured in two orientations, including right side up (RU) to mimic maxillary implants and upside down (UD) to represent mandibular dental implants. Implant orientation influences biofilm and cell spatialaccumulation emphasizing the role of local substrate features and gravity in modulating growth patterns. This work provides a sample preparation and electron microscopy procedure appropriate for dentalimplants, greatly aiding in future research of implant failure in clinical settings.

the encapsulated cells while enabling the exchange of nutrients, oxygen, or therapeutic agents. It also allows us to study cell responses to external stimuli such as light, temperature, or mechanical stress. The encapsulating materials are typically biocompatible and provide a supportive environment for cell survival and function. Various methods and engineering tools exist for creating an encapsulation system with cells, such as 3D bioprinting, microencapsulation, or microfluidics, depending on the desired size, morphology, and application. By combining microfluidics with photo-polymerization, a microfabrication technique known as stop-flow lithography (SFL) can produce precisely shaped micro-objects with high precision and control. This work focuses on creating complex geometrical objects using SFL for encapsulation of NIH-3T3 cell line carried out using different hydrogels (ColMA, HAMA, and dexHEMA). The impact of material on cell proliferation and viability will be presented with a focus on future mechanostimulation application.

improvement to current biomanufacturing practices. Traditional batch manufacturing of biopharmaceuticals is time-consuming and inefficient due to the intensive resource and labor requirements to complete the individual steps of the process development. Continuous manufacturing, incorporating process analytical technologies, automation, and real-time predictive analytics with process adjustments, has emerged as an alternative that would potentially reduce costs while improving production efficiency and product quality. While it has been consistently adopted in several industries including food and chemical production, continuous biomanufacturing is under development, experiencing several advancements in specific unit operations. With a particular focus on the mRNA drug modality, we demonstrate how real-time Raman spectral data acquisition and subsequent predictive analytics can be used to develop machine learning models to understand and adjust process parameters. Raman spectroscopy is a powerful tool that allows for the in-line implementation and analysis of biochemical content and concentration. Specifically, models developed from off-line Raman spectral analysis have been implemented within the upstream in vitro mRNA transcription unit operation. These hybrid models incorporate in-line spectral data acquired and further fitted to mechanistic models that describe the reaction, in order to enable real-time monitoring and control of the operation. The orchestration of process analytical technologies with data-driven models has demonstrated an increased understanding of this operation, as well as other downstream process development steps. However, significant challenges remain in accurately developing and implementing these technologies to enable real-time monitoring and control of the entire bioprocess.

in vivo-like cellular organization. One chanllenge is the lack of control over the initial formation of hPSC clusters during seeding, leading to variability in colony size and cell number — key morphogenetic factors known for the development of embryo- and organ-like structures.
Current bioengineering methods, such as microcontact printing, provide some control but require physical contact with the substrate, posing technical challenges when working with soft, delicate substrates that are required to mimic in vivo embryonic tissue mechanical environments.
In this study, we report a contactless technique that uses acoustic waves to guide free-floating single hPSCs into small, uniform cell clusters, regardless of substrate materials. A 3D-printed mask positioned above a planar circular acoustic transducer generates a gradient acoustic field that simulates Lamb waves. When a culture dish is placed on top, these waves create a pressure field directing cells to pressure minima, forming cell clusters.
These acoustically-patterned hPSC clusters self-organize into three-dimensional structures that resemble the peri-implantation epiblast. Different from those cultured on rigid glass surfaces, acoustically-patterned hPSCs cultured on a soft extracellular matrix layer that mimics the in vivo environment show cell differentiation, even without the addition of exogenous soluable factors. Detailed characterization further shows that the biomimetic soft culture surface could promote cell lineage diversification from the epiblast-like tissue, notably facilitating the differentiation of amnion- and primordial germ cell-like cells.
This innovative, contactless acoustic patterning approach offers a valuable tool for microscale cell patterning, a common strategy used in embryo and organ modeling from hPSCs. The combination of acoustic patterning with biomimetic substrates overcomes previous technical limitations and holds great promise for advancing in vitro modeling of human development.

identities in an emerging coordinate system. Despite its importance in human development, gastrulation remains difficult to study. Stem cell-based embryo models, including those that recapitulate different aspects of pre- and peri-gastrulation human development, are emerging as promising tools for studying human embryogenesis. However, it remains unclear whether existing human embryo models are capable of modeling the development of the trilaminar embryonic disc structure, a hallmark of human gastrulation. Here we report a transgene-free human embryo model derived solely from primed human pluripotent stem cells (hPSCs), which recapitulates various aspects of peri-gastrulation human development, including formation of trilaminar embryonic layers situated between dorsal amnion and ventral definitive yolk sac and primary hematopoiesis. We term this model the peri-gastrulation trilaminar embryonic disc (PTED) embryoid. The development of PTED embryoid does not follow natural developmental sequences of cell lineage diversification or spatial organization. Instead, it exploits both extrinsic control of tissue boundaries and intrinsic self-organizing properties and embryonic plasticity of the diverse peri-gastrulation-stage cell lineages, leading to the emergence of in vivo-like tissue organization and function at a global scale. Our lineage tracing study reveals that in PTED embryoids, embryonic and extraembryonic mesoderm cells, as well as embryonic and extraembryonic endoderm cells, share common progenitors emerging during peri-gastrulation development. Active hematopoiesis and blood cell generation are evident in the yolk sac-like structure of PTED embryoids. Together, PTED embryoids provide a promising and ethically less challenging model for studying self-organizing properties of peri-gastrulation human development.