Engineering tissue barrier models on hydrogel microfluidic platforms

Tissue barriers play a crucial role in human physiology by establishing tissue compartmentalization and regulating organ homeostasis. At the interface between the extracellular matrix (ECM) and flowing fluids, epithelial and endothelial barriers are responsible for solute and gas exchange. In the past decade, microfluidic technologies and organ-on-chip devices became popular as in vitro models able to recapitulate these biological barriers. However, in conventional microfluidic devices, cell barriers are primarily grown on hard polymeric membranes within polydimethylsiloxane (PDMS) channels that do not mimic the cell¿ECM interactions nor allow the incorporation of other cellular compartments such as stromal tissue or vascular structures. To develop models that accurately account for the different cellular and acellular compartments of tissue barriers, researchers have integrated hydrogels into microfluidic setups for tissue barrier-on-chips, either as cell substrates inside the chip, or as self-contained devices. These biomaterials provide the soft mechanical properties of tissue barriers and allow the embedding of stromal cells. Combining hydrogels with microfluidics technology provides unique opportunities to better recreate in vitro the tissue barrier models including the cellular components and the functionality of the in vivo tissues. Such platforms have the potential of greatly improving the predictive capacities of the in vitro systems in applications such as drug development, or disease modeling. Nevertheless, their development is not without challenges in their microfabrication. In this review, we will discuss the recent advances driving the fabrication of hydrogel microfluidic platforms and their applications in multiple tissue barrier models

Micro/nanofluidic and lab-on-a-chip devices for biomedical applications

Micro/Nanofluidic and lab-on-a-chip devices have been increasingly used in biomedical research [1]. Because of their adaptability, feasibility, and cost-efficiency, these devices can revolutionize the future of preclinical technologies. Furthermore, they allow insights into the performance and toxic effects of responsive drug delivery nanocarriers to be obtained, which consequently allow the shortcomings of two/three-dimensional static cultures and animal testing to be overcome and help to reduce drug development costs and time [2–4]. With the constant advancements in biomedical technology, the development of enhanced microfluidic devices has accelerated, and numerous models have been reported. Given the multidisciplinary of this Special Issue (SI), papers on different subjects were published making a total of 14 contributions, 10 original research papers, and 4 review papers. The review paper of Ko et al. [1] provides a comprehensive overview of the significant advancements in engineered organ-on-a-chip research in a general way while in the review presented by Kanabekova and colleagues [2], a thorough analysis of microphysiological platforms used for modeling liver diseases can be found. To get a summary of the numerical models of microfluidic organ-on-a-chip devices developed in recent years, the review presented by Carvalho et al. [5] can be read. On the other hand, Maia et al. [6] report a systematic review of the diagnosis methods developed for COVID-19, providing an overview of the advancements made since the start of the pandemic. In the following, a brief summary of the research papers published in this SI will be presented, with organs-on-a-chip, microfluidic devices for detection, and device optimization having been identified as the main topics.info:eu-repo/semantics/publishedVersio

Developing of an organ on chip device as novel in vitro platform to study organ mechanobiology: Peristalsis on a chip.

Developing of an organ on chip device as novel in vitro platform to study organ mechanobiology: Peristalsis on a chip. Knowing the mechanical properties of the gastrointestinal (GI) tract appears to be important for understanding the molecular and cellular responses to mechanical stimuli on physiological processes such as foods, xenobiotic or drugs digestion/absorption. These processes are mediated by various intestinal cells such as epithelial cells, interstitial cells, smooth muscle cells, and neurocytes. The loss or dysfunction of specific cells or mechanical strength of cell bowel wall directly results in GI tract disease. Reversing the abnormal status of pathogenic cells has been considered crucial to treatment of gut diseases. Gut bioengineered models have been developing for the purpose to replace the damaged tissues and to provide three-dimensional platforms that mimic the in vivo environment to study drug development, absorption and toxicity. Nevertheless, the need to develop more complex models in vitro to study mechanical stress is growing. In this perspective, this project will allow us to get an automatized microfluidic gut platform to evaluate the pathophysiology of the small intestine through the study of the shear stress of the bolus on the epithelial cells layer at the lumen side of the healthy or diseased 3D intestine models. To this aim, the major goals of this project are the the design and fabrication of complex and innovative microfluidic device provided with an integrated PDMS membrane designed to mimic the crypt-villus axis in order to promote the differentiation of the intestinal epithelium and the establishment of peristaltic motion by means of an automatized and controlled elettrovalve system. The platform was used to estimate the intestinal transport properties of the bolus and the physiological condition of the shear stress under peristaltic motion. An important feature of the device, is the possibility to induce a fluid flow both at the basolateral and the lumen side of the intestinal epithelium, therefore the possibility to introduce integrated electrodes in the apical side and basoteral side in order to be enable continuous monitoring of cells behaviour and differentiation through TransEpithelial Electrical Resistance measurements. The effect of PDMS membrane morphology, peristaltic motion and shear stress on intestinal epithelial cell differentiation, mucus production and molecules adsorption process has been evaluated. The development of the Peristalsis on chip device could be reduce the poorly predictive preclinical evaluation generated by the phylogenetic distance between laboratory animals and humans, the discrepancy between current in vitro systems and the human body, and the restrictions of in silico modelling

Generation and large-scale expansion of highly functional hPSC-derived hepatocytes for Cellular therapies and bioengineered livers: the unknown role of human microbiome

Los hepatocitos diferenciados a partir de células madre pluripotentes inducidas humanas (hiPSC), también conocidos como hepatocyte-like cells (HLC), proporcionan una cantidad sin precedentes de posibilidades para el desarrollo de terapias celulares y, con ello, el tratamiento de una gran variedad de enfermedades hepáticas. Sin embargo, más de quince años de investigación en este campo han sido insuficientes para obtener una terapia médica avanzada a base de HLC. Los fenotipos inmaduros que presentan estas células, la falta de protocolos fiables y reproducibles para su producción a gran escala, así como la incertidumbre con respecto a su capacidad para injertarse en andamios (o scaffolds), tanto in vitro como in vivo, son algunas de las razones que dificultan su aplicación clínica. Por lo tanto, el objetivo de esta tesis ha sido el desarrollo de estrategias eficientes para generar HLC maduras y funcionales en cantidades clínicamente relevantes para su uso en terapia celular y medicina regenerativa. Para ello, se ha desarrollado un método integrado que combina plataformas de fabricación avanzada con estrategias inspiradas en procesos naturales, estudiando el efecto del microbioma intestinal humano en la maduración y la preservación de las funciones de las HLC.Recapitular in vitro el proceso de maduración fisiológica del hígado supone un gran reto, ya que dura aproximadamente dos años desde el nacimiento e implica la inducción de una amplia gama de vías metabólicas y de desintoxicación. Investigaciones recientes sobre el desarrollo del hígado han revelado que la maduración y el desarrollo de la función hepática podrían estar altamente asociados con el establecimiento y la diversificación del microbioma intestinal.En el Capítulo 1, discutimos las moléculas microbianas y los mecanismos moleculares que constituyen esta interacción.En el Capítulo 2, investigamos los efectos del secretoma de la microbiota intestinal sobre la funcionalidad (o maduración) de las hPSC-HLC mediante el tratamiento de estas células, generadas utilizando diferentes estrategias en 2D o 3D, con un secretoma microbiano formulado in vitro en forma de medio condicionado. Nuestros resultados muestran que las HLC expuestas al medio condicionado presentan una mayor expresión de marcadores hepáticos (p.ej. HNF4A, CYP1B1, -3A4, -2C9, -2D6,16-2E1, CPS1, PPARA, TLR1, -2, -5, -6, etc.); conservan la actividad basal de CYP3A4 y/o mostraban metabolismo inducible CYP3A4; mejoran la expresión de ALB; y aumentan la secreción de las proteínas hepáticas plasmáticas ALB y A1AT, en comparación con HLC no tratadas con el medio condicionado.Dado que las terapias celulares requieren una alta producción celular, en los Capítulos 3 y 4 desarrollamos bioprocesos escalables para la producción de HLC en forma de agregados celulares tridimensionales.En el Capítulo 3, implementamos una estrategia integrada para la producción a gran escala de hiPSC‐HLC, que combina la expansión y diferenciación en 3D de las hiPSC usando biorreactores de tanque agitado en modo de perfusión. Mediante este protocolo, las hiPSC son capaces de crecer en agregados 3D y las HLC resultantes expresan marcadores hepáticos típicos y exhiben características funcionales de los hepatocitos, incluyendo el almacenamiento de glucógeno y la capacidad de metabolización de fármacos. Además, la incorporación de un sensor de capacitancia en el sistema del biorreactor nos ha permitido demostrar por primera vez el potencial de la espectroscopia dieléctrica para monitorizar la expansión y diferenciación de hiPSC en los biorreactores de tanque agitado. Los resultados que obtuvimos mostraron una buena correlación entre la permitividad celular medida on-line y el biovolumen de los agregados medido por métodos estándar off-line.Con el objetivo de mejorar la expansión celular y el rendimiento de la diferenciación, en el Capítulo 4 optimizamos el bioproceso al mantener la concentración de oxígeno disuelto en niveles bajos (4% O2) durante la fase de especificación hepática. Hemos validado esta optimización para dos líneas celulares de hiPSC mejorando el rendimiento de producción de HLC (hasta 3.2x106 células/mL) y la eficiencia de diferenciación (> 80% células Albumina+) en comparación con condiciones no controladas (0.6×106 células/mL). Un análisis transcriptómico detallado de las HLC en diferentes etapas de maduración muestra que las hiPSC-HLC maduras difieren aproximadamente un 35% en su transcriptoma completo con respecto a hiPSC-HCL inmaduras. Estas diferencias incluyen vías relacionadas con el injerto celular, teniendo implicaciones en la capacidad celular para injertarse en andamios, ya que solo las HLC maduras pueden adherirse, proliferar y mantener su funcionalidad después de 14 días de cultivo.17Por último, en el Capítulo 5, presentamos una discusión general de los logros y conclusiones principales del trabajo realizado y delineamos futuras perspectivas a investigar.En conclusión, este estudio representa un importante primer paso hacia la generación de HLC derivadas de hiPSC más maduras y funcionales que, esperamos, harán que las terapias con células madre hepáticas sean una realidad tangible para los pacientes con enfermedad hepática en etapa terminal. También abre un nuevo cambio de paradigma en la bioingeniería de células madre y lo vincula con un campo inesperado, el microbioma humano.<br /

Characterizing Migratory Signaling Pathways Of Transplantable Retinal Progenitor Cells And Photoreceptor Precursor Cells Toward Restoration Of Degenerative Retina \u27 A Systems Biology Approach

A common feature of several heterogeneous diseases that result in retinal degeneration (RD) is photoreceptor loss, leading to an irreversible decline in visual function [15-17]. There are no cell replacement treatments available for RD diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Although many RD cases are of a genetic origin, a promising strategy to treat diseased phenotypes is by replacing lost photoreceptor cells, for synaptic integration and restoration of visual function. To advance photoreceptor-replacement strategies as a practical therapy, in light of highly restricted integration rates reported across studies, this body of research focused on defining the molecular mechanisms facilitating migration of transplantable photoreceptor precursors in the retinal microenvironment. To accomplish this work we utilized bioinformatics, bioengineering and molecular biologic techniques for a systems level approach. Guided by classic neuronal migration models, we hypothesized that transplanted photoreceptor precursors navigate to specific retinal lamina in part due to cell surface receptor expression and in response to spatially gradated directional ligand cues provided by the host retinal microenvironment. Given the neural origin of the mammalian retinal system, we also predicted that these chemotactic receptor-ligand pairs trigger intracellular signaling events in migrating photoreceptors analogous to canonical migration pathways exhibited by neuronal precursors. For a comprehensive account of these motility-deterministic biochemical interactions, we first performed in silico bioinformatics modeling of PPC transplantation into light-damaged retina by matching microarray datasets between PPC receptors and ligands in the light-damaged retinal microenvironment. We then refined the gene expression network data to focus on motility deterministic interactions at the interface of the PPC cell-surface receptors and extracellular ligands of the damaged retina. Our in silico network modeling generated a library of ligand-receptor pairs associated with cellular movement specific for this retinal transplantation paradigm and the intracellular signaling pathways induced by candidate chemotactic ligands. Working from predicted interactions of in silico paired PPC receptors and retinal ligands, we then performed cell migration analysis to evaluate whether exposure to stromal derived factor-1α (SDF-1α) would guide the motility of PPCs and RPCs in vitro. We also assessed the chemotactic effects of epidermal growth factor (EGF) on RPCs. Cell surface expression of C-X-C chemokine receptor type 4 (CXCR4) receptors on PPCs and RPCs, and EGF receptor expression on RPCs were verified via immunocytochemical staining and validated by Western blot analysis. Boyden chamber analysis was used as an initial high-throughput screen to verify the motogenic effects of the ligands on PPCs and RPCs. We determined that RPC motility was optimally stimulated in these chambers by EGF concentrations in the range of 20-400ng/ml, with decreased stimulation at higher concentrations, suggesting concentration-dependence of EGF-induced motility. Both RPCs and PPCs also demonstrated a concentration-dependent chemotactic response to an optimal SDF-1α concentration of 100ng/ml. Using bioinformatics downstream signaling pathway analysis of the EGF and SDF-1α ligands in a retina-specific gene network, we predicted a chemotactic function for EGF involving the MAPK and JAK-STAT intracellular signaling pathways. Based on targeted inhibition studies, we show that ligand binding, phosphorylation of EGFR and activation of the intracellular STAT3 and PI3Kinase signaling pathways are necessary to drive RPC motility. The JAK-STAT pathway was also implicated in transducing similar motogenic effects on PPCs with SDF-1α induction. To test our hypothesis of the gradated nature of ECM ligand effects on both ontogenetic retinal cell types, we employed engineered microfluidic devices to generate quantifiable steady-state gradients of EGF and SDF-1α coupled with live-cell tracking, and analyzed the dynamics of individual RPC and PPC motility. Microfluidic analysis, including center of mass and maximum accumulated distance, revealed that EGF induced motility is chemokinetic in EGFR expressing RPCs with optimal activity observed in response to low concentration gradients. On the other hand, PPCs and RPCs exhibited significant chemotaxis towards the source of SDF-1α with longer accumulated Euclidean distances and Center of Mass (COM) compared to controls. We also ascertained that receptor mediated signaling was requisite for ligand-induced motility by using the CXCR4 inhibitor, AMD 3100, to antagonize the SDF-1α receptor. CXCR4 receptor inhibition resulted in decreases of PPC and RPC movement in uniform and steady state gradients for a number of migration indices measured. To advance translational application of the characterized chemotactic signaling potential of transplantable photoreceptor precursors, we performed computational drug analysis of our newly identified motility-deterministic networks, to develop a library of FDA approved drugs and small molecules predicted to potentially influence the expression of target motility signaling mechanisms in photoreceptor progenitor cells. Using the Expression2Kinases software and LINCS drug computational algorithm, we were able to identify pharmacological drug targets that modulate the biochemical activity of transcriptional regulatory genes which govern the expression of candidate receptor protein targets, and provide preliminary results validating the up-regulatory effect of candidate drug aminophenazone on SDF-1α receptor CXCR4 expression. Results from this study demonstrate the applicability of our systems level in silico modeling of matched transplantable cell surface-receptors and transplantation site ligands to predict molecular signaling guiding migration. Verification of in silico predictions, using molecular and microfluidic analysis provide important data for defining cell response properties to specific ligands present during transplantation into the retinal microenvironment. The drug computational analysis provides a translational perspective to our in silico modeling paradigms extending its applicability. Future studies will validate the functionality of resolved ligand-receptor pairs from our in silico library and characterize down-stream signaling guiding motility and homing. This systems level paradigm can effectively be applied to defining the molecular basis of transplantable cell migration in vivo toward improved efficiency for repair of retina and other neural tissue types

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin