Mind In Vitro Platforms: Versatile, Scalable, Robust, and Open Solutions to Interfacing with Living Neurons.

Motivated by the unexplored potential of in vitro neural systems for computing and by the corresponding need of versatile, scalable interfaces for multimodal interaction, an accurate, modular, fully customizable, and portable recording/stimulation solution that can be easily fabricated, robustly operated, and broadly disseminated is presented. This approach entails a reconfigurable platform that works across multiple industry standards and that enables a complete signal chain, from neural substrates sampled through micro-electrode arrays (MEAs) to data acquisition, downstream analysis, and cloud storage. Built-in modularity supports the seamless integration of electrical/optical stimulation and fluidic interfaces. Custom MEA fabrication leverages maskless photolithography, favoring the rapid prototyping of a variety of configurations, spatial topologies, and constitutive materials. Through a dedicated analysis and management software suite, the utility and robustness of this system are demonstrated across neural cultures and applications, including embryonic stem cell-derived and primary neurons, organotypic brain slices, 3D engineered tissue mimics, concurrent calcium imaging, and long-term recording. Overall, this technology, termed "mind in vitro" to underscore the computing inspiration, provides an end-to-end solution that can be widely deployed due to its affordable (>10× cost reduction) and open-source nature, catering to the expanding needs of both conventional and unconventional electrophysiology.

Artificial confocal microscopy for deep label-free imaging.

Widefield microscopy of optically thick specimens typically features reduced contrast due to "spatial crosstalk", in which the signal at each point in the field of view is the result of a superposition from neighbouring points that are simultaneously illuminated. In 1955, Marvin Minsky proposed confocal microscopy as a solution to this problem. Today, laser scanning confocal fluorescence microscopy is broadly used due to its high depth resolution and sensitivity, but comes at the price of photobleaching, chemical, and photo-toxicity. Here, we present artificial confocal microscopy (ACM) to achieve confocal-level depth sectioning, sensitivity, and chemical specificity, on unlabeled specimens, nondestructively. We equipped a commercial laser scanning confocal instrument with a quantitative phase imaging module, which provides optical path-length maps of the specimen in the same field of view as the fluorescence channel. Using pairs of phase and fluorescence images, we trained a convolution neural network to translate the former into the latter. The training to infer a new tag is very practical as the input and ground truth data are intrinsically registered, and the data acquisition is automated. The ACM images present significantly stronger depth sectioning than the input (phase) images, enabling us to recover confocal-like tomographic volumes of microspheres, hippocampal neurons in culture, and 3D liver cancer spheroids. By training on nucleus-specific tags, ACM allows for segmenting individual nuclei within dense spheroids for both cell counting and volume measurements. In summary, ACM can provide quantitative, dynamic data, nondestructively from thick samples, while chemical specificity is recovered computationally.

Cancer cell angiogenic capability is regulated by 3D culture and integrin engagement.

Three-dimensional culture alters cancer cell signaling; however, the underlying mechanisms and importance of these changes on tumor vascularization remain unclear. A hydrogel system was used to examine the role of the transition from 2D to 3D culture, with and without integrin engagement, on cancer cell angiogenic capability. Three-dimensional culture recreated tumor microenvironmental cues and led to enhanced interleukin 8 (IL-8) secretion that depended on integrin engagement with adhesion peptides coupled to the polymer. In contrast, vascular endothelial growth factor (VEGF) secretion was unaffected by 3D culture with or without substrate adhesion. IL-8 diffused greater distances and was present in higher concentrations in the systemic circulation, relative to VEGF. Implantation of a polymeric IL-8 delivery system into GFP bone marrow-transplanted mice revealed that localized IL-8 up-regulation was critical to both the local and systemic control of tumor vascularization in vivo. In summary, 3D integrin engagement within tumor microenvironments regulates cancer cell angiogenic signaling, and controlled local and systemic blockade of both IL-8 and VEGF signaling may improve antiangiogenic therapies.

Quenching Epigenetic Drug Resistance Using Antihypoxic Microparticles in Glioblastoma Patient-Derived Chips.

Glioblastoma (GBM) is one of the most intractable tumor types due to the progressive drug resistance upon tumor mass expansion. Incremental hypoxia inside the growing tumor mass drives epigenetic drug resistance by activating nongenetic repair of antiapoptotic DNA, which could be impaired by drug treatment. Hence, rescuing intertumor hypoxia by oxygen-generating microparticles may promote susceptibility to antitumor drugs. Moreover, a tumor-on-a-chip model enables user-specified alternation of clinic-derived samples. This study utilizes patient-derived glioblastoma tissue to generate cell spheroids with size variations in a 3D microchannel network chip (GBM chip). As the spheroid size increases, epigenetic drug resistance is promoted with inward hypoxia severance, as supported by the spheroid size-proportional expression of hypoxia-inducible factor-1a in the chip. Loading antihypoxia microparticles onto the spheroid surface significantly reduces drug resistance by silencing the expression of critical epigenetic factor, resulting in significantly decreased cell invasiveness. The results are confirmed in vitro using cell line and patient samples in the chip as well as chip implantation into a hypoxic hindlimb ischemia model in mice, which is an unprecedented approach in the field.

COVID-19 through Adverse Outcome Pathways: Building networks to better understand the disease - 3rd CIAO AOP Design Workshop.

On April 28-29, 2021, 50 scientists from different fields of expertise met for the 3rd online CIAO workshop. The CIAO project "Modelling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway (AOP) framework" aims at building a holistic assembly of the available scientific knowledge on COVID-19 using the AOP framework. An individual AOP depicts the disease progression from the initial contact with the SARS-CoV-2 virus through biological key events (KE) toward an adverse outcome such as respiratory distress, anosmia or multiorgan failure. Assembling the individual AOPs into a network highlights shared KEs as central biological nodes involved in multiple outcomes observed in COVID-19 patients. During the workshop, the KEs and AOPs established so far by the CIAO members were presented and posi­tioned on a timeline of the disease course. Modulating factors influencing the progression and severity of the disease were also addressed as well as factors beyond purely biological phenomena. CIAO relies on an interdisciplinary crowd­sourcing effort, therefore, approaches to expand the CIAO network by widening the crowd and reaching stakeholders were also discussed. To conclude the workshop, it was decided that the AOPs/KEs will be further consolidated, inte­grating virus variants and long COVID when relevant, while an outreach campaign will be launched to broaden the CIAO scientific crowd.

Material-based deployment enhances efficacy of endothelial progenitor cells.

Cell-based therapies are attractive for revascularizing and regenerating tissues and organs, but clinical trials of endothelial progenitor cell transplantation have not resulted in consistent benefit. We propose a different approach in which a material delivery system is used to create a depot of vascular progenitor cells in vivo that exit over time to repopulate the damaged tissue and participate in regeneration of a vascular network. Microenvironmental conditions sufficient to maintain the viability and outward migration of outgrowth endothelial cells (OECs) have been delineated, and a material incorporating these signals improved engraftment of transplanted cells in ischemic murine hindlimb musculature, and increased blood vessel densities from 260 to 670 vessels per mm(2), compared with direct cell injection. Further, material deployment dramatically improved the efficacy of these cells in salvaging ischemic murine limbs, whereas bolus OEC delivery was ineffective in preventing toe necrosis and foot loss. Finally, material deployment of a combination of OECs with another cell population commonly isolated from peripheral or cord blood, endothelial progenitor cells (EPCs) returned perfusion to normal levels in 40 days, and prevented toe and foot necrosis. Direct injection of an EPC/OEC combination was minimally effective in improving limb perfusion, and untreated limbs underwent autoamputation in 3 days. These results demonstrate that vascular progenitor cell utility is highly dependent on the mode of delivery, and suggest that one can create new vascular beds for a variety of applications with this material-controlled deployment of cells.

Morphologic and transcriptomic comparison of adipose- and bone-marrow-derived porcine stem cells cultured in alginate hydrogels.

Advances in bioengineering, material chemistry, and developmental biology have led to the design of three-dimensional (3D) culture systems that better resemble the surrounding structure and chemistry of the in situ niches of cells in tissues. This study was designed to characterize and compare porcine adipose-derived stem cells (ADSC) and bone-marrow-derived stem cells (BMSC) induced to differentiate toward osteogenic and adipogenic lineages in vitro by using a 3D alginate hydrogel. The morphology and gene expression of the two cell populations during differentiation were analyzed. Both ADSC and BMSC showed morphological evidence of osteogenic and adipogenic differentiation. Expression patterns of genes characteristic of the onset of osteogenic differentiation (ALP, COL1A1, SPARC, SPP1) were low at the beginning of culture and generally increased during the period of differentiation up to 28 days in culture. Expression of genes associated with adipogenic differentiation (ACSL1, ADFP, ADIPOQ, CD36, DBI, DGAT2, PPARG, SCD) was consistently increased in ADSC cultured in alginate hydrogel relative to the start of differentiation. However, adipogenic gene expression of BMSC cultured in alginate hydrogel was more limited when compared with that of ADSC. Evaluation of cell numbers (via the MTT staining assay) suggested a greater viability of BMSC under osteogenic conditions in alginate hydrogels than under adipogenic conditions, whereas ADSC had greater viability under adipogenic conditions than under osteogenic conditions. This study thus provides an important initial evaluation of ADSC and BMSC seeded and differentiated toward the osteogenic and adipogenic cell lineages in a 3D alginate hydrogel in vitro.

Microenvironmental regulation of biomacromolecular therapies.

There is currently great interest in molecular therapies to treat various diseases, and this has prompted extensive efforts to achieve target-specific and controlled delivery of bioactive macromolecules (for example, proteins, antibodies, DNA and small interfering RNA) through the design of smart drug carriers. By contrast, the influence of the microenvironment in which the target cell resides and the effect it might have on the success of biomacromolecular therapies has been under-appreciated. The extracellular matrix (ECM) component of the cellular niche may be particularly important, as many diseases and injury disrupt the normal ECM architecture, the cell adhesion to ECM, and the subsequent cellular activities. This Review will discuss the importance of the ECM and the ECM-cell interactions on the cell response to bioactive macromolecules, and suggest how this information could lead to new criteria for the design of novel drug delivery systems.

Engineering of biomaterials for tumor modeling.

Development of biomaterials mimicking tumor and its microenvironment has recently emerged for the use of drug discovery, precision medicine, and cancer biology. These biomimetic models have developed by reconstituting tumor and stroma cells within the 3D extracellular matrix. The models are recently extended to recapitulate the in vivo tumor microenvironment, including biological, chemical, and mechanical conditions tailored for specific cancer type and its microenvironment. In spite of the recent emergence of various innovative engineered tumor models, many of these models are still early stage to be adapted for cancer research. In this article, we review the current status of biomaterials engineering for tumor models considering three main aspects - cellular engineering, matrix engineering, and engineering for microenvironmental conditions. Considering cancer-specific variability in these aspects, our discussion is focused on pancreatic cancer, specifically pancreatic ductal adenocarcinoma (PDAC). In addition, we further discussed the current challenges and future opportunities to create reliable and relevant tumor models.

Integrin-adhesion ligand bond formation of preosteoblasts and stem cells in three-dimensional RGD presenting matrices.

Cell-interactive polymers have been widely used as synthetic extracellular matrices to regulate cell function and promote tissue regeneration. However, there is a lack of quantitative understanding of the cell-material interface. In this study, integrin-adhesion ligand bond formation of preosteoblasts and D1 stem cells with RGD presenting alginate matrices were examined using FRET and flow cytometry. Bond number increased with adhesion ligand density but did not change with RGD island spacing for both cell types. Integrin expression varied with cell type and substrate in 2D culture, but the integrin expression profiles of both cell types were similar when cultured in 3D RGD presenting substrates and distinct from 2D culture. In summary, combining a FRET technique to quantify bond formation with flow cytometry to elucidate integrin expression can define specific cell-material interactions for a given material system and may be useful for informing biomaterial design strategies for cell-based therapies.

Upregulation of bone cell differentiation through immobilization within a synthetic extracellular matrix.

There is a need for new therapeutic strategies to treat bone defects caused by trauma, disease or tissue loss. Injectable systems for cell transplantation have the advantage of allowing the use of minimally invasive surgical procedures, and thus for less discomfort to patients. In the present study, it is hypothesized that Arg-Gly-Asp (RGD)-coupled in a binary (low and high molecular weight) injectable alginate composition is able to influence bone cell differentiation in a three-dimensional (3D) structure. Viability, metabolic activity, cytoskeleton organization, ultrastructure and differentiation (alkaline phosphatase (ALP), von Kossa, alizarin red stainings and osteocalcin quantification) of immobilized cells were assessed. Cells within RGD-modified alginate microspheres were able to establish more interactions with the synthetic extracellular matrix as visualized by confocal laser scanning microscope and transmission electron microscopy imaging, and presented a much higher level of differentiation (more intense ALP and mineralization stainings and higher levels of osteocalcin secretion) when compared to cells immobilized within unmodified alginate microspheres. These findings demonstrate that peptides covalently coupled to alginate were efficient in influencing cell behavior within this 3D system, and may provide adequate preparation of osteoblasts for cell transplantation.

Regulating myoblast phenotype through controlled gel stiffness and degradation.

Mechanical stiffness and degradability are important material parameters in tissue engineering. The aim of this study was to address the hypothesis that these variables regulate the function of myoblasts cultured in 2-D and 3-D microenvironments. Development of cell-interactive alginate gels with tunable degradation rates and mechanical stiffness was established by a combination of partial oxidation and bimodal molecular weight distribution. Higher gel mechanical properties (13 to 45 kPa) increased myoblast adhesion, proliferation, and differentiation in a 2-D cell culture model. Primary mouse myoblasts were more highly responsive to this cue than the C2C12 myoblast cell line. Myoblasts were then encapsulated in gels varying in degradation rate to simultaneously investigate the effect of degradation and subsequent reduction of mechanical properties on cells in a 3-D environment. C2C12 cells in more rapidly degrading gels exhibited lower proliferation, as they exited the cell cycle to differentiate, compared to those in nondegradable gels. In contrast, mouse primary myoblasts illustrated significantly higher proliferation in degradable gels than in nondegradable gels, and exhibited minimal differentiation in either type of gel. Altogether, these studies suggest that a critical balance between material degradation rate and mechanical properties may be required to regulate formation of engineered skeletal muscle tissue, and that results obtained with the C2C12 cell line may not be predictive of the response of primary myoblasts to environmental cues. The principles delineated in these studies may be useful to tailor smart biomaterials that can be applied to many other polymeric systems and tissue types.

Mechanical strain regulates endothelial cell patterning in vitro.

Blood vessels of the vertebrate circulatory system typically exhibit tissue-specific patterning. However, the cues that guide the development of these patterns remain unclear. We investigated the effect of cyclic uniaxial strain on vascular endothelial cell dynamics and sprout formation in vitro in two-dimensional (2D) and three-dimensional (3D) culture systems under the influence of growth factors. Cells preferentially aligned and moved in the direction perpendicular to the major strain axis in monolayer culture, and mechanical strain also regulated the spatial location of cell proliferation in 2D cell culture. Cells in 3D cell culture could be induced to form sprouts by exposure to appropriate growth factor combinations (vascular endothelial growth factor and hepatocyte growth factor), and the strain direction regulated the directionality of this process. Moreover, cyclic uniaxial strain inhibited branching of the structures formed by endothelial cells and increased their thickness. Taken together, these data support the importance of external mechanical stimulation in the regulation of endothelial cell migration, proliferation, and differentiation into primitive vessels.

Quantitative assessment of scaffold and growth factor-mediated repair of critically sized bone defects.

An 8-mm rat segmental defect model was used to evaluate quantitatively the ability of longitudinally oriented poly(L-lactide-co-D,L-lactide) scaffolds with or without growth factors to promote bone healing. BMP-2 and TGF-beta3, combined with RGD-alginate hydrogel, were co-delivered to femoral defects within the polymer scaffolds at a dose previously shown to synergistically induce ectopic mineralization. A novel modular composite implant design was used to achieve reproducible stable fixation, provide a window for longitudinal in vivo micro-CT monitoring of 3D bone ingrowth, and allow torsional biomechanical testing of functional integration. Sequential micro-CT analysis showed that bone ingrowth increased significantly between 4 and 16 weeks for the scaffold-treated defects with or without growth factors, but no increase with time was observed in empty defect controls. Treatment with scaffold alone improved defect stability at 16 weeks compared to nontreatment, but did not achieve bone union or restoration of mechanical function. Augmentation of scaffolds with BMP-2 and TGF-beta3 significantly increased bone formation at both 4 and 16 weeks compared to nontreatment, but only produced bone bridging of the defect region in two of six cases. Histological evaluation indicated that bone formed first at the periphery of the scaffolds, followed by more limited mineral deposition within the scaffold interior, suggesting that the cells participating in the initial healing response were primarily derived from periosteum. This study introduces a challenging segmental defect model that facilitates quantitative evaluation of strategies to repair critically sized bone defects. Healing of the defect region was improved by implanting structural polymeric scaffolds infused with growth factors incorporated within RGD-alginate. However, functional integration of the constructs appeared limited by continued presence of slow-degrading scaffolds and suboptimal dose or delivery of osteoinductive signals.

Multi-scale modeling to predict ligand presentation within RGD nanopatterned hydrogels.

The adhesion ligand RGD has been coupled to various materials to be used as tissue culture matrices or cell transplantation vehicles, and recent studies indicate that nanopatterning RGD into high-density islands alters cell adhesion, proliferation, and differentiation. However, elucidating the impact of nanopattern parameters on cellular responses has been stymied by a lack of understanding of the actual ligand presentation within these systems. We have developed a multi-scale predictive modeling approach to characterize the adhesion ligand nanopatterns within an alginate hydrogel matrix. The models predict the distribution of ligand islands, the spacing between ligands within an island and the fraction of ligands accessible for cell binding. These model predictions can be used to select pattern parameter ranges for experiments on the effects of individual parameters on cellular responses. Additionally, our technique could also be applied to other polymer systems presenting peptides or other signaling molecules.

Alginate hydrogels as biomaterials.

[Image: see text] Alginate hydrogels are proving to have a wide applicability as biomaterials. They have been used as scaffolds for tissue engineering, as delivery vehicles for drugs, and as model extracellular matrices for basic biological studies. These applications require tight control of a number of material properties including mechanical stiffness, swelling, degradation, cell attachment, and binding or release of bioactive molecules. Control over these properties can be achieved by chemical or physical modifications of the polysaccharide itself or the gels formed from alginate. The utility of these modified alginate gels as biomaterials has been demonstrated in a number of in vitro and in vivo studies.Micro-CT images of bone-like constructs that result from transplantation of osteoblasts on gels that degrade over a time frame of several months leading to improved bone formation.

Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution.

Degradability is often a critical property of materials utilized in tissue engineering. Although alginate, a naturally derived polysaccharide, is an attractive material due to its biocompatibility and ability to form hydrogels, its slow and uncontrollable degradation can be an undesirable feature. In this study, we characterized gels formed using a combination of partial oxidation of polymer chains and a bimodal molecular weight distribution of polymer. Specifically, alginates were partially oxidized to a theoretical extent of 1% with sodium periodate, which created acetal groups susceptible to hydrolysis. The ratio of low MW to high MW alginates used to form gels was also varied, while maintaining the gel forming ability of the polymer. The rate of degradation was found to be controlled by both the oxidation and the ratio of high to low MW alginates, as monitored by the reduction of mechanical properties and corresponding number of crosslinks, dry weight loss, and molecular weight decrease. It was subsequently examined whether these modifications would lead to reduced biocompatibility by culturing C2C12 myoblast on these gels. Myoblasts adhered, proliferated, and differentiated on the modified gels at a comparable rate as those cultured on the unmodified gels. Altogether, this data indicates these hydrogels exhibit tunable degradation rates and provide a powerful material system for tissue engineering.

Designing alginate hydrogels to maintain viability of immobilized cells.

Hydrogel-forming materials have been widely utilized as an immobilization matrix and transport vehicle for cells. Success in these applications is dependent upon maintaining cell viability through the gel preparation process. We hypothesized that the high viscosity of pre-gelled solutions typically used in these applications may decrease cell viability due to the high shear forces required to mix cells with these solutions. Further, we proposed this harmful effect could be mediated by decreasing the molecular weight (Mw) of the polymer used to form the gel, while maintaining its gel-forming ability. To investigate this hypothesis, alginate was used as model system, as this copolymer consists of cross-linkable guluronic acid (G) blocks and non-cross-linkable blocks. Decreasing the Mw of alginate using irradiation (e.g., irradiating at dose of 2 Mrad) decreased the low shear viscosity of 2% (w/w) pre-gelled solutions from 1000 to 4 cP, while maintaining high elastic moduli, once cross-linked to form a gel. Importantly, the immobilization of cells with these polymer hydrogels increased cell viability from 40% to 70%, as compared to using high Mw polymer chains to form the gels. Furthermore, the solids concentration of gels formed with the low Mw alginate could be raised to further increase the moduli of gels without significantly deteriorating the viability of immobilized cells. This was likely due to the limited increase in the viscosity of these solutions. This material design approach may be useful with a variety of synthetic or naturally occurring block copolymers used to immobilize cells.

The effects of poly(ethyleneimine) (PEI) molecular weight on reinforcement of alginate hydrogels.

Alginate hydrogels are widely used for cell encapsulation and transplantation, and they are frequently surface reinforced with secondary polymers to enhance their mechanical rigidity and stability. We hypothesized that the molecular weight (MW) of the polymer utilized to reinforce alginate would be an important factor in their stability, particularly when the gel network was homogeneously reinforced with the polymer. This hypothesis was investigated with alginate hydrogels cross-linked with Ca2+, and reinforced throughout the bulk of the gel with poly(ethyleneimine) (PEI) having different MWs. Interactions between the two polymers became significant following gelation, leading to higher elastic moduli (E) than gels with no PEI. The decrease in E of gels incubated in isotonic salt solutions over time, utilized as an indication of gel break down, was ameliorated with an increase in the MW of the PEI. In addition, the dependencies of the moduli and viscoelasticity on the temperature also became smaller with the use of high MW PEI. This is likely due to the limited mobility of high MW PEI, leading to a higher energy for dissociation. The stable interactions between the alginate and PEI prevented alterations of the pore structure in the gels, and slowed the deterioration of gel properties even under continuous agitation in a bioreactor. The results of this study will likely be useful in designing alginate encapsulation strategies for various applications.

Tracheal reconstruction using tissue-engineered cartilage.

OBJECTIVES: To determine whether rabbit cartilage can be tissue engineered using a polyglycolic acid (PGA) construct composed of PGA mesh, autologous chondrocytes, and alginate covalently linked with the cell adhesion sequence arginine-glycine-aspartic acid (RGD), and to investigate the feasibility of reconstructing tracheal defects using the PGA construct in conjunction with a bioabsorbable intratracheal stent. METHODS: Nineteen New Zealand White rabbits were used. Nine rabbits underwent subcutaneous implantation of 3 different PGA construct combinations: (1) PGA, autologous chondrocytes, and RGD-modified alginate; (2) PGA, autologous chondrocytes, and unmodified alginate; and (3) PGA and RGD-modified alginate. The remaining 10 animals underwent anterior tracheal reconstruction using fascia lata grafts and the complete PGA construct (PGA, autologous chondrocytes, and RGD-modified alginate). At the time of tracheal reconstruction, a poly-l-lactic acid intratracheal stent was placed in 5 of these latter animals. Rates of tracheal stenosis and mortality were compared with those of historical control animals. Histologic analysis was performed on the PGA constructs. RESULTS: In the subcutaneous implants, the PGA constructs made with chondrocytes (with and without RGD) demonstrated mature cartilage formation in 7 (78%) of the 9 animals. No cartilage was seen in PGA constructs made without chondrocytes. Two of the 10 animals that underwent tracheal reconstruction with the complete PGA construct survived to 20 weeks and demonstrated patent airways, 1 with a stent and 1 without a stent (80% overall mortality). Histologic analysis showed mature cartilage formation at the tracheal reconstruction site. Historical control animals that underwent reconstruction with fascia lata alone demonstrated the lowest overall mortality. CONCLUSIONS: Cartilage can be tissue engineered in rabbits using PGA mesh embedded with alginate-encapsulated autologous chondrocytes. It is also possible to reconstruct tracheal defects with this method of cartilage engineering, although the mortality rate in this study is high.