Fluorescent hiPSC-derived MYH6-mScarlet cardiomyocytes for real-time tracking, imaging, and cardiotoxicity assays.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) hold great potential in the cardiovascular field for human disease modeling, drug development, and regenerative medicine. However, multiple hurdles still exist for the effective utilization of hiPSC-CMs as a human-based experimental platform that can be an alternative to the current animal models. To further expand their potential as a research tool and bridge the translational gap, we have generated a cardiac-specific hiPSC reporter line that differentiates into fluorescent CMs using CRISPR-Cas9 genome editing technology. The CMs illuminated with the mScarlet fluorescence enable their non-invasive continuous tracking and functional cellular phenotyping, offering a real-time 2D/3D imaging platform. Utilizing the reporter CMs, we developed an imaging-based cardiotoxicity screening system that can monitor distinct drug-induced structural toxicity and CM viability in real time. The reporter fluorescence enabled visualization of sarcomeric disarray and displayed a drug dose-dependent decrease in its fluorescence. The study also has demonstrated the reporter CMs as a biomaterial cytocompatibility analysis tool that can monitor dynamic cell behavior and maturity of hiPSC-CMs cultured in various biomaterial scaffolds. This versatile cardiac imaging tool that enables real time tracking and high-resolution imaging of CMs has significant potential in disease modeling, drug screening, and toxicology testing.

Optical Projection Tomography Technique for Image Texture and Mass Transport Studies in Hydrogels Based on Gellan Gum.

The microstructure and permeability are crucial factors for the development of hydrogels for tissue engineering, since they influence cell nutrition, penetration, and proliferation. The currently available imaging methods able to characterize hydrogels have many limitations. They often require sample drying and other destructive processing, which can change hydrogel structure, or they have limited imaging penetration depth. In this work, we show for the first time an alternative nondestructive method, based on optical projection tomography (OPT) imaging, to characterize hydrated hydrogels without the need of sample processing. As proof of concept, we used gellan gum (GG) hydrogels obtained by several cross-linking methods. Transmission mode OPT was used to analyze image microtextures, and emission mode OPT to study mass transport. Differences in hydrogel structure related to different types of cross-linking and between modified and native GG were found through the acquired Haralick's image texture features followed by multiple discriminant analysis (MDA). In mass transport studies, the mobility of FITC-dextran (MW 20, 150, 2000 kDa) was analyzed through the macroscopic hydrogel. The FITC-dextran velocities were found to be inversely proportional to the size of the dextran as expected. Furthermore, the threshold size in which the transport is affected by the hydrogel mesh was found to be 150 kDa (Stokes' radii between 69 and 95 Å). On the other hand, the mass transport study allowed us to define an index of homogeneity to assess the cross-linking distribution, structure inside the hydrogel, and repeatability of hydrogel production. As a conclusion, we showed that the set of OPT imaging based material characterization methods presented here are useful for screening many characteristics of hydrogel compositions in relatively short time in an inexpensive manner, providing tools for improving the process of designing hydrogels for tissue engineering and drugs/cells delivery applications.

Chemical modification strategies for viscosity-dependent processing of gellan gum.

Recently, the hydrogel-forming polysaccharide gellan gum (GG) has gained popularity as a versatile biomaterial for tissue engineering purposes. Here, we examine the modification strategies suitable for GG to overcome processing-related limitations. We emphasize the thorough assessment of the viscoelastic and mechanical properties of both precursor solutions and final hydrogels. The investigated modification strategies include purification, oxidation, reductive chain scission, and blending. We correlate polymer flow and hydrogel forming capabilities to viscosity-dependent methods including casting, injection and printing. Native GG and purified NaGG are shear thinning and feasible for printing, being similar in gelation and compression behavior. Oxidized GGox possesses reduced viscosity, higher toughness, and aldehydes as functional groups, while scissored GGsciss has markedly lower molecular weight. To exemplify extrudability, select modification products are printed using an extrusion-based bioprinter utilizing a crosslinker bath. Our robust modification strategies have widened the processing capabilities of GG without affecting its ability to form hydrogels.

Optical projection tomography as a quantitative tool for analysis of cell morphology and density in 3D hydrogels.

Assessing cell morphology and function, as well as biomaterial performance in cell cultures, is one of the key challenges in cell biology and tissue engineering (TE) research. In TE, there is an urgent need for methods to image actual three-dimensional (3D) cell cultures and access the living cells. This is difficult using established optical microscopy techniques such as wide-field or confocal microscopy. To address the problem, we have developed a new protocol using Optical Projection Tomography (OPT) to extract quantitative and qualitative measurements from hydrogel cell cultures. Using our tools, we demonstrated the method by analyzing cell response in three different hydrogel formulations in 3D with 1.5 mm diameter samples of: gellan gum (GG), gelatin functionalized gellan gum (gelatin-GG), and Geltrex. We investigated cell morphology, density, distribution, and viability in 3D living cells. Our results showed the usability of the method to quantify the cellular responses to biomaterial environment. We observed that an elongated morphology of cells, thus good material response, in gelatin-GG and Geltrex hydrogels compared with basic GG. Our results show that OPT has a sensitivity to assess in real 3D cultures the differences of cellular responses to the properties of biomaterials supporting the cells.

Design of modular gellan gum hydrogel functionalized with avidin and biotinylated adhesive ligands for cell culture applications.

This article proposes the coupling of the recombinant protein avidin to the polysaccharide gellan gum to create a modular hydrogel substrate for 3D cell culture and tissue engineering. Avidin is capable of binding biotin, and thus biotinylated compounds can be tethered to the polymer network to improve cell response. The avidin is successfully conjugated to gellan gum and remains functional as shown with fluorescence titration and electrophoresis (SDS-PAGE). Self-standing hydrogels were formed using bioamines and calcium chloride, yielding long-term stability and adequate stiffness for 3D cell culture, as confirmed with compression testing. Human fibroblasts were successfully cultured within the hydrogel treated with biotinylated RGD or biotinylated fibronectin. Moreover, human bone marrow stromal cells were cultured with hydrogel treated with biotinylated RGD over 3 weeks. We demonstrate a modular and inexpensive hydrogel scaffold for cell encapsulation that can be equipped with any desired biotinylated cell ligand to accommodate a wide range of cell types.

Screening of Hydrogels for Human Pluripotent Stem Cell-Derived Neural Cells: Hyaluronan-Polyvinyl Alcohol-Collagen-Based Interpenetrating Polymer Network Provides an Improved Hydrogel Scaffold.

There is a clear need for novel in vitro models, especially for neuronal applications. Development of in vitro models is a multiparameter task consisting of cell-, biomaterial-, and environment-related parameters. Here, three different human origin neuronal cell sources are studied and cultured in various hydrogel 3D scaffolds. For the efficient evaluation of complex results, an indexing method for data is developed and used in principal component analysis (PCA). It is found that no single hydrogel is superior to other hydrogels, and collagen I (Col1) and hyaluronan-poly(vinyl alcohol) (HA1-PVA) gels are combined into an interpenetrating network (IPN) hydrogel. The IPN gel combines cell supportiveness of the collagen gel and stability of the HA1-PVA gel. Moreover, cell adhesion is studied in particular and it is found that adhesion of neurons differs from that observed for fibroblasts. In conclusion, the HA1-PVA-col1 hydrogel is a suitable scaffold for neuronal cells and supports adhesion formation in 3D.

Bioactive glass ions induce efficient osteogenic differentiation of human adipose stem cells encapsulated in gellan gum and collagen type I hydrogels.

BACKGROUND: Due to unmet need for bone augmentation, our aim was to promote osteogenic differentiation of human adipose stem cells (hASCs) encapsulated in gellan gum (GG) or collagen type I (COL) hydrogels with bioactive glass (experimental glass 2-06 of composition [wt-%]: Na2O 12.1, K2O 14.0, CaO 19.8, P2O5 2.5, B2O3 1.6, SiO2 50.0) extract based osteogenic medium (BaG OM) for bone construct development. GG hydrogels were crosslinked with spermidine (GG-SPD) or BaG extract (GG-BaG). METHODS: Mechanical properties of cell-free GG-SPD, GG-BaG, and COL hydrogels were tested in osteogenic medium (OM) or BaG OM at 0, 14, and 21 d. Hydrogel embedded hASCs were cultured in OM or BaG OM for 3, 14, and 21 d, and analyzed for viability, cell number, osteogenic gene expression, osteocalcin production, and mineralization. Hydroxyapatite-stained GG-SPD samples were imaged with Optical Projection Tomography (OPT) and Selective Plane Illumination Microscopy (SPIM) in OM and BaG OM at 21 d. Furthermore, Raman spectroscopy was used to study the calcium phosphate (CaP) content of hASC-secreted ECM in GG-SPD, GG-BaG, and COL at 21 d in BaG OM. RESULTS: The results showed viable rounded cells in GG whereas hASCs were elongated in COL. Importantly, BaG OM induced significantly higher cell number and higher osteogenic gene expression in COL. In both hydrogels, BaG OM induced strong mineralization confirmed as CaP by Raman spectroscopy and significantly improved mechanical properties. GG-BaG hydrogels rescued hASC mineralization in OM. OPT and SPIM showed homogeneous 3D cell distribution with strong mineralization in BaG OM. Also, strong osteocalcin production was visible in COL. CONCLUSIONS: Overall, we showed efficacious osteogenesis of hASCs in 3D hydrogels with BaG OM with potential for bone-like grafts.

Polyethylene Terephthalate Textiles Enhance the Structural Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have the potential to serve as a model for human cardiomyocytes. However, hiPSC-CMs are still considered immature. CMs differentiated from hiPSCs more resemble fetal than adult cardiomyocytes. Putative factors enhancing maturation include in vitro culture duration, culture surface topography, and mechanical, chemical, and electrical stimulation. Stem cell-derived cardiomyocytes are traditionally cultured on glass surfaces coated with extracellular matrix derivatives such as gelatin. hiPSC-CMs are flat and round and their sarcomeres are randomly distributed and unorganized. Morphology can be enhanced by culturing cells on surfaces providing topographical cues to the cells. In this study, a textile based-culturing method used to enhance the maturation status of hiPSC-CMs is presented. Gelatin-coated polyethylene terephthalate (PET)-based textiles were used as the culturing surface for hiPSC-CMs and the effects of the textiles on the maturation status of the hiPSC-CMs were assessed. The hiPSC-CMs were characterized by analyzing their morphology, sarcomere organization, expression of cardiac specific genes, and calcium handling. We show that the topographical cues improve the structure of the hiPSC-CMs in vitro. Human iPSC-CMs grown on PET textiles demonstrated improved structural properties such as rod-shape structure and increased sarcomere orientation.

Mechanically Biomimetic Gelatin-Gellan Gum Hydrogels for 3D Culture of Beating Human Cardiomyocytes.

To promote the transition of cell cultures from 2D to 3D, hydrogels are needed to biomimic the extracellular matrix (ECM). One potential material for this purpose is gellan gum (GG), a biocompatible and mechanically tunable hydrogel. However, GG alone does not provide attachment sites for cells to thrive in 3D. One option for biofunctionalization is the introduction of gelatin, a derivative of the abundant ECM protein collagen. Unfortunately, gelatin lacks cross-linking moieties, making the production of self-standing hydrogels difficult under physiological conditions. Here, we explore the functionalization of GG with gelatin at biologically relevant concentrations using semiorthogonal, cytocompatible, and facile chemistry based on hydrazone reaction. These hydrogels exhibit mechanical behavior, especially elasticity, which resembles the cardiac tissue. The use of optical projection tomography for 3D cell microscopy demonstrates good cytocompatibility and elongation of human fibroblasts (WI-38). In addition, human-induced pluripotent stem cell-derived cardiomyocytes attach to the hydrogels and recover their spontaneous beating in 24 h culture. Beating is studied using in-house-built phase contrast video analysis software, and it is comparable with the beating of control cardiomyocytes under regular culture conditions. These hydrogels provide a promising platform to transition cardiac tissue engineering and disease modeling from 2D to 3D.

Composite Hydrogels Using Bioinspired Approach with in Situ Fast Gelation and Self-Healing Ability as Future Injectable Biomaterial.

Biopolymers are attractive candidates to fabricate biocompatible hydrogels, but the low water solubility of most of them at physiological pH has hindered their applications. To prepare a water-soluble derivative of chitosan (WSC) biopolymer, it was grafted with a small anionic amino acid, l-glutamic acid, using a single-step 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide coupling reaction. This resulted in a zwitterion-tethered structure onto the polymer backbone. The degree of substitution range was 13-16 ± 1.25%, which was controlled by varying the feeding reagent ratios. Differential scanning calorimetry- and X-ray diffraction-based analysis confirmed a transition from  amorphous into a moderately amorphous/crystalline morphology after amino acid grafting, which made the derivative water-soluble at physiological pH. Composite hydrogels gelated within 60 s when using this WSC together with benzaldehyde-terminated 4-arm poly(ethylene glycol) as cross-linker. The compressive modulus of these hydrogels could be easily tuned between 4.0 ± 1.0 and 31 ± 2.5 kPa, either by changing the cross-linker concentration or total solid content in the final gel. The gels were injectable at the lowest cross-linker as well as total solid content, due to the enhanced elastic behavior. These hydrogels showed biodegradability during a 1 month incubation period in phosphate-buffered saline with weight remaining of 60 ± 1.5 and 44 ± 1.45% at pHs 7.4 and 6.5, respectively. The cytocompatibility of the gels was tested using the fibroblast cell line (i.e., WI-38), which showed good cell viability on the gel surface. Therefore, these hydrogels could be an important injectable biomaterial for delivery purpose in the future.

The production of injectable hydrazone crosslinked gellan gum-hyaluronan-hydrogels with tunable mechanical and physical properties.

Gellan gum (GG) has been proposed for use in tissue engineering (TE) due to its structural and functional similarities with alginate. The most traditional crosslinking methods of GG, ionical and photocrosslinking, have downsides such as loss of stability or phototoxicity, which can limit their use in certain applications. In this study, an alternative hydrazone crosslinking method is introduced. Hydrazone crosslinking is a simple method that produces no toxic reagents or side-products. The method enables the fabrication of injectable hydrogels. GG was combined with hyaluronan (HA) to improve some properties such as cell attachment. The mechanical and physical properties of GG-HA hydrogels were controlled by changing the molecular weight, the degree of modification, and the ratio of polymer components. GG-HA hydrogels showed ionic nature of deswelling in the presence of cations enabling the control of physical properties in different solution environments. Due to the non-linear elastic behavior of hydrogels and tissues, the stiffness as a function of strain was represented instead of solely giving the second-order elastic constants. The stiffness of GG-HA hydrogels was similar to that of soft tissues at small strains.

Bioamine-crosslinked gellan gum hydrogel for neural tissue engineering.

Neural tissue engineering and three-dimensional in vitro tissue modeling require the development of biomaterials that take into account the specified requirements of human neural cells and tissue. In this study, an alternative method of producing biomimetic hydrogels based on gellan gum (GG) was developed by replacing traditional crosslinking methods with the bioamines spermidine and spermine. These bioamines were proven to function as crosslinkers for GG hydrogel at +37 °C, allowing for the encapsulation of human neurons. We studied the mechanical and rheological properties of the formed hydrogels, which showed biomimicking properties comparable to naïve rabbit brain tissue under physiologically relevant stress and strain. Human pluripotent stem cell-derived neuronal cells demonstrated good cytocompatibility in the GG-based hydrogels. Moreover, functionalization of GG hydrogels with laminin resulted in cell type-specific behavior: neuronal cell maturation and neurite migration.