Live reporting for hypoxia : Hypoxia sensor–modified mesenchymal stem cells as in vitro reporters

Natural oxygen gradients occur in tissues of biological organisms and also in the context of three-dimensional (3D) in vitro cultivation. Oxygen diffusion limitation and metabolic oxygen consumption by embedded cells produce areas of hypoxia in the tissue/matrix. However, reliable systems to detect oxygen gradients and cellular response to hypoxia in 3D cell culture systems are still missing. In this study, we developed a system for visualization of oxygen gradients in 3D using human adipose tissue–derived mesenchymal stem cells (hAD-MSCs) modified to stably express a fluorescent genetically engineered hypoxia sensor HRE-dUnaG. Modified cells retained their stem cell characteristics in terms of proliferation and differentiation capacity. The hypoxia-reporter cells were evaluated by fluorescence microscopy and flow cytometry under variable oxygen levels (2.5%, 5%, and 7.5% O2). We demonstrated that reporter hAD-MSCs output is sensitive to different oxygen levels and displays fast decay kinetics after reoxygenation. Additionally, the reporter cells were encapsulated in bulk hydrogels with a variable cell number, to investigate the sensor response in model 3D cell culture applications. The use of hypoxia-reporting cells based on MSCs represents a valuable tool for approaching the genuine in vivo cellular microenvironment and will allow a better understanding of the regenerative potential of AD-MSCs. © 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LL

The Effect of Scaffold Modulus on the Morphology and Remodeling of Fetal Mesenchymal Stem Cells

Hydrogel materials have been successfully used as matrices to explore the role of biophysical and biochemical stimuli in directing stem cell behavior. Here, we present our findings on the role of modulus in guiding bone marrow fetal mesenchymal stem cell (BMfMSC) fate determination using semi-synthetic hydrogels made from PEG-fibrinogen (PF). The BMfMSCs were cultivated in the PF for up to 2 weeks to study the influence of matrix modulus (i.e., cross-linking density of the PF) on BMfMSC survival, morphology and integrin expression. Both two-dimensional (2D) and three-dimensional (3D) culture conditions were employed to examine the BMfMSCs as single cells or as cell spheroids. The hydrogel modulus affected the rate of BMfMSC metabolic activity, the integrin expression levels and the cell morphology, both as single cells and as spheroids. The cell seeding density was also found to be an important parameter of the system in that high densities were favorable in facilitating more cell-to-cell contacts that favored higher metabolic activity. Our findings provide important insight about design of a hydrogel scaffold that can be used to optimize the biological response of BMfMSCs for various tissue engineering applications

Effects of antibiotic treatment and phagocyte infiltration on development of Pseudomonas aeruginosa biofilm—Insights from the application of a novel PF hydrogel model in vitro and in vivo

Background and purposeBacterial biofilm infections are major health issues as the infections are highly tolerant to antibiotics and host immune defenses. Appropriate biofilm models are important to develop and improve to make progress in future biofilm research. Here, we investigated the ability of PF hydrogel material to facilitate the development and study of Pseudomonas aeruginosa biofilms in vitro and in vivo.MethodsWild-type P. aeruginosa PAO1 bacteria were embedded in PF hydrogel situated in vitro or in vivo, and the following aspects were investigated: 1) biofilm development; 2) host immune response and its effect on the bacteria; and 3) efficacy of antibiotic treatment.ResultsMicroscopy demonstrated that P. aeruginosa developed typical biofilms inside the PF hydrogels in vitro and in mouse peritoneal cavities where the PF hydrogels were infiltrated excessively by polymorphonuclear leukocytes (PMNs). The bacteria remained at a level of ~106 colony-forming unit (CFU)/hydrogel for 7 days, indicating that the PMNs could not eradicate the biofilm bacteria. β-Lactam or aminoglycoside mono treatment at 64× minimal inhibitory concentration (MIC) killed all bacteria in day 0 in vitro biofilms, but not in day 1 and older biofilms, even at a concentration of 256× MIC. Combination treatment with the antibiotics at 256× MIC completely killed the bacteria in day 1 in vitro biofilms, and combination treatment in most of the cases showed significantly better bactericidal effects than monotherapies. However, in the case of the established in vivo biofilms, the mono and combination antibiotic treatments did not efficiently kill the bacteria.ConclusionOur results indicate that the bacteria formed typical biofilms in PF hydrogel in vitro and in vivo and that the biofilm bacteria were tolerant against antibiotics and host immunity. The PF hydrogel biofilm model is simple and easy to fabricate and highly reproducible with various application possibilities. We conclude that the PF hydrogel biofilm model is a new platform that will facilitate progress in future biofilm investigations, as well as studies of the efficacy of new potential medicine against biofilm infections

Dynamic mechanical conditioning regulates the development of cell-seeded collagen constructs in vitro : implications for tissue-engineered blood vessels

Ph.D.Robert M. Nere

Hydrogels for therapeutic cardiovascular angiogenesis

10.1016/j.addr.2015.07.003ADVANCED DRUG DELIVERY REVIEWS9631-3

Injectable PEGylated fibrinogen cell-laden microparticles made with a continuous solvent- and oil-free preparation method

Prova tipográficaA new methodology is reported for the continuous, solvent- and oil-free production of photopolymerizable microparticles containing encapsulated human dermal fibroblasts. A precursor solution of cells in photoreactive poly(ethylene glycol) (PEG)-fibrinogen (PF) polymer was transported through a transparent injector exposed to light irradiation before being atomized in a jet-in-air nozzle. Shear rheometry data indicated the crosslinking kinetics of each PF/cell solution, which was then used to determine the amount of irradiation required to partially polymerize the mixture just prior to atomization. The partially polymerized drops of PF/cells fell into a gelation bath for further crosslinking until fully polymerized hydrogel microparticles were formed. As the drops of solution leave the air-in-jet nozzle, their viscosity was designed to be sufficiently high so as to prevent rapid mixing and/or dilution in the gelation bath, but without undergoing complete gelation in the nozzle. Several parameters of this system were varied to control the size and polydispersity of the microparticles, including the cell density, the flow rate and the air pressure in the nozzle. The system was capable of producing cell-laden microparticles with an average diameter of between 88.1 to 347.1 Î¼m, and a dispersity of between 1.1 and 2.4, depending on the parameters chosen. Varying the precursor flow rate and/or cell density was beneficial in controlling the size and polydispersity of the microparticles; all microparticles exhibited very high cell viability, which was not affected by these parameters. In conclusion, this dropwise photopolymerization methodology for preparing cell-laden microparticles is an attractive alternative to existing techniques that use harsh solvents/oils and offer limited control over particle size and polydispersity.This research was partially supported by the Singapore National Research Foundation under CREATE program: The Regenerative Medicine Initiative in Cardiac Restoration Therapy (START). This work was also partially supported by EC-IP FP7 grant Biodesign and the Portuguese Foundation for Science and Technology (FCT) in the scope of project PTDC/CTM-BIO/1814/2012 and grant SFRH/BD/71396/2010

Dispositivo lab-on-chip per studiare la migrazione cellulare in sistemi tridimensionali e relativo metodo di utilizzo

L’invenzione si basa sulla realizzazione di un sistema innovativo Lab-on-Chip, che noi abbiamo chiamato “3D-cell migration chip” (3DCM-chip), per studiare la migrazione cellulare di cellule in un sistema tridimensionale a base di idrogel in grado di mimare la matrice extracellulare (ECM). Può essere utilizzato per mimare il microambiente cellulare e fare studi di co-colture cellulari per creare sistemi complessi 3D e valutare effetti di "farmaci" molecolari e cellulari sulla migrazione ed invasività cellulare

In vivo restoration of dystrophin expression in mdx mice using intra-muscular and intra-arterial injections of hydrogel microsphere carriers of exon skipping antisense oligonucleotides

Duchenne muscular dystrophy (DMD) is a genetic disease caused by a mutation in the X-linked Dytrophin gene preventing the expression of the functional protein. Exon skipping therapy using antisense oligonucleotides (AONs) is a promising therapeutic strategy for DMD. While benefits of AON therapy have been demonstrated, some challenges remain before this strategy can be applied more comprehensively to DMD patients. These include instability of AONs due to low nuclease resistance and poor tissue uptake. Delivery systems have been examined to improve the availability and stability of oligonucleotide drugs, including polymeric carriers. Previously, we showed the potential of a hydrogel-based polymeric carrier in the form of injectable PEG-fibrinogen (PF) microspheres for delivery of chemically modified 2'-O-methyl phosphorothioate (2OMePs) AONs. The PF microspheres proved to be cytocompatible and provided sustained release of the AONs for several weeks, causing increased cellular uptake in mdx dystrophic mouse cells. Here, we further investigated this delivery strategy by examining in vivo efficacy of this approach. The 2OMePS/PEI polyplexes loaded in PF microspheres were delivered by intramuscular (IM) or intra-femoral (IF) injections. We examined the carrier biodegradation profiles, AON uptake efficiency, dystrophin restoration, and muscle histopathology. Both administration routes enhanced dystrophin restoration and improved the histopathology of the mdx mice muscles. The IF administration of the microspheres improved the efficacy of the 2OMePS AONs over the IM administration. This was demonstrated by a higher exon skipping percentage and a smaller percentage of centered nucleus fibers (CNF) found in H&E-stained muscles. The restoration of dystrophin expression found for both IM and IF treatments revealed a reduced dystrophic phenotype of the treated muscles. The study concludes that injectable PF microspheres can be used as a carrier system to improve the overall therapeutic outcomes of exon skipping-based therapy for treating DMD