Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels.

Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.

Grafting Density-Dependent Phase Transition Mechanism of Thermoresponsive Poly(glycidyl ether) Brushes: A Comprehensive QCM-D Study.

Thermoresponsive coatings that exhibit "switchable" protein- and cell-adhesive properties are frequently used for the fabrication of cell sheets. Among other architectures, polymer brush coatings have shown to be especially viable due to their distinct phase transition behavior, which can be tailored via a manifold of adjustable brush characteristics, such as the (co)monomer composition, polymer chain length, and grafting density. Brush coatings based on poly(glycidyl ether)s (PGEs) have shown to efficiently mediate cell sheet fabrication when tethered to various tissue culture substrates. Herein, we report the phase transition of self-assembled PGE brushes with respect to polymer molecular weight (M: 10 and 22 kDa) and grafting density (0.07-0.5 chains nm-2) on gold model substrates studied by quasi-static QCM-D temperature ramp measurements. The brush grafting density can be tuned via the applied grafting conditions, and all brushes investigated feature broad phase transition regimes (ΔT ∼15 °C) with volume phase transition temperatures (VPTTs) close to the cloud point temperatures (CPTs) of the PGEs in solution. We further demonstrate that brush coatings with a low grafting density (0.07-0.12 chains nm-2) exhibit a continuous brush-to-mushroom transition, whereas brushes with medium grafting densities (0.3-0.5 chains nm-2) undergo a brush-to-brush transition comprising vertical phase separation during the phase transition progress. These insights help to understand the transition behavior of thin, thermoresponsive brushes prepared via grafting-to strategies and contribute to their rational design for improved functional surfaces.

3D organ models-Revolution in pharmacological research?

3D organ models have gained increasing attention as novel preclinical test systems and alternatives to animal testing. Over the years, many excellent in vitro tissue models have been developed. In parallel, microfluidic organ-on-a-chip tissue cultures have gained increasing interest for their ability to house several organ models on a single device and interlink these within a human-like environment. In contrast to these advancements, the development of human disease models is still in its infancy. Although major advances have recently been made, efforts still need to be intensified. Human disease models have proven valuable for their ability to closely mimic disease patterns in vitro, permitting the study of pathophysiological features and new treatment options. Although animal studies remain the gold standard for preclinical testing, they have major drawbacks such as high cost and ongoing controversy over their predictive value for several human conditions. Moreover, there is growing political and social pressure to develop alternatives to animal models, clearly promoting the search for valid, cost-efficient and easy-to-handle systems lacking interspecies-related differences. In this review, we discuss the current state of the art regarding 3D organ as well as the opportunities, limitations and future implications of their use.

Teburu-Open source 3D printable bioreactor for tissue slices as dynamic three-dimensional cell culture models.

Three-dimensional tissue cultures are important models for the study of cell-cell and cell-matrix interactions, as well as, to investigate tissue repair and reconstruction pathways. Therefore, we designed a reproducible and easy to handle printable bioreactor system (Teburu), that is applicable for different approaches of pathway investigation and targeted tissue repair using human tissue slices as a three-dimensional cell culture model. Here, we definitively describe Teburu as a controlled environment to reseed a 500-µm thick decellularized human liver slice using human mesenchymal stroma cells. During a cultivation period of eight days, Teburu, as a semi-open and low consumption system, was capable to maintain steady pH and oxygenation levels. Its combination with additional modules delivers an applicability for a wide range of tissue engineering approaches under optimal culture conditions.

Switchable Oligo(glycidyl ether) Acrylate Bottlebrushes "Grafted-from" Polystyrene Surfaces: A Versatile Strategy toward Functional Cell Culture Substrates.

Thermoresponsive brushes based on linear poly(glycidyl ether)s (PGEs) have already shown to be functional coatings for cell sheet fabrication. In here, we introduce a method to functionalize polystyrene (PS) tissue culture substrates with thermoresponsive coatings comprising glycidyl ether-based bottlebrush architectures. Utilizing the UV-induced "grafting-from" approach, thermoresponsive oligo(glycidyl ether) acrylate (OGEA) macromonomers were polymerized from PS substrates under bulk conditions. Applying ellipsometry, water contact angle (CA), and atomic force microscopy (AFM) measurements, we found that OGEA coatings exhibit a complex, gel-like structure comprising nanosized roughness and exhibit a temperature-dependent phase transition in water through the reversible hydration of OGEA bottlebrush side chains. To assess the utility of the coatings as functional substrates for cell sheet fabrication, human dermal fibroblast (HDF) adhesion and detachment were investigated. By adjusting the bottlebrush properties via the grafting procedure and coating structure, we were able to harvest confluent HDF sheets from functionalized PS substrates in a temperature-triggered, controlled manner. As the first report on surface-grafted bottlebrushes comprising thermoresponsive side chains with molecular weight of up to 1 kDa, this study demonstrates the potential of OGEA-based coatings for cell sheet fabrication.

Endothelial, smooth muscle and fibroblast cell sheet fabrication from self-assembled thermoresponsive poly(glycidyl ether) brushes.

In this study, we introduce a platform to fabricate human dermal fibroblast (HDF), human aortic smooth muscle cell (HAoSMC) and human umbilical vein endothelial cell (HUVEC) sheets using thermoresponsive poly(glycidyl ether) coatings. Copolymer brushes based on glycidyl methyl ether (GME) and ethyl glycidyl ether (EGE) were self-assembled onto polystyrene (PS) culture substrates via the physical adsorption of a hydrophobic, photoreactive benzophenone anchor block based on the monomer 4-[2-(2,3-epoxypropoxy)ethoxy]benzophenone (EEBP). The directed self-assembly of well-defined, end-tethered poly(GME-ran-EGE)-block-poly(EEBP) (PGE) brushes was achieved via the selective, EEBP-driven adsorption of the asymmetric block copolymer from dilute aqueous solution below its cloud point temperature (CPT). Subsequently, the PGE brush layers were covalently immobilized onto the PS surfaces by irradiation with UV light and characterized by ellipsometry, static water contact angle (CA) measurements and atomic force microscopy (AFM). We found that, by decreasing the temperature from 37 to 20 °C, the coatings undergo a pancake-to-brush transition, which triggers cell sheet detachment. In addition, cell culture parameters were optimized to allow proper adhesion and controlled detachment of confluent HDF, HAoSMC and HUVEC sheets, which can be applied in vascular tissue engineering.

Generation of a 3D Liver Model Comprising Human Extracellular Matrix in an Alginate/Gelatin-Based Bioink by Extrusion Bioprinting for Infection and Transduction Studies.

Bioprinting is a novel technology that may help to overcome limitations associated with two-dimensional (2D) cell cultures and animal experiments, as it allows the production of three-dimensional (3D) tissue models composed of human cells. The present study describes the optimization of a bioink composed of alginate, gelatin and human extracellular matrix (hECM) to print human HepaRG liver cells with a pneumatic extrusion printer. The resulting tissue model was tested for its suitability for the study of transduction by an adeno-associated virus (AAV) vector and infection with human adenovirus 5 (hAdV5). We found supplementation of the basic alginate/gelatin bioink with 0.5 and 1 mg/mL hECM provides desirable properties for the printing process, the stability of the printed constructs, and the viability and metabolic functions of the printed HepaRG cells. The tissue models were efficiently transduced by AAV vectors of serotype 6, which successfully silenced an endogenous target (cyclophilin B) by means of RNA interference. Furthermore, the printed 3D model supported efficient adenoviral replication making it suitable to study virus biology and develop new antiviral compounds. We consider the approach described here paradigmatic for the development of 3D tissue models for studies including viral vectors and infectious viruses.

Poly(glycidyl ether)-Based Monolayers on Gold Surfaces: Control of Grafting Density and Chain Conformation by Grafting Procedure, Surface Anchor, and Molecular Weight.

For a meaningful correlation of surface coatings with their respective biological response reproducible coating procedures, well-defined surface coatings, and thorough surface characterization with respect to layer thickness and grafting density are indispensable. The same applies to polymeric monolayer coatings which are intended to be used for, e.g., fundamental studies on the volume phase transition of surface end-tethered thermoresponsive polymer chains. Planar gold surfaces are frequently used as model substrates, since they allow a variety of straightforward surface characterization methods. Herein we present reproducible grafting-to procedures performed with thermoresponsive poly(glycidyl ether) copolymers composed of glycidyl methyl ether (GME) and ethyl glycidyl ether (EGE). The copolymers feature different molecular weights (2 kDa, 9 kDa, 24 kDa) and are equipped with varying sulfur-containing anchor groups in order to achieve adjustable grafting densities on gold surfaces and hence control the tethered polymers' chain conformation. We determined "wet" and "dry" thicknesses of these coatings by QCM-D and ellipsometry measurements and deduced anchor distances and degrees of chain overlap of the polymer chains assembled on gold. Grafting under cloud point conditions allowed for higher degrees of chain overlap compared to grafting from a good solvent like ethanol, independent of the used sulfur-containing anchor group for polymers with low (2 kDa) and medium (9 kDa) molecular weights. By contrast, the achieved grafting densities and thus chain overlaps of surface-tethered polymers with high (24 kDa) molecular weights were identical for both grafting methods. Monolayers prepared from an ethanolic solution of poly(glycidyl ether)s equipped with sterically demanding disulfide-containing anchors revealed the lowest degrees of chain overlap. The ratio of the radius of gyration to the anchor distance (2 Rg/l) of the latter coating was found to be lower than 1.4, indicating that the assembly was rather in the mushroom-like than in the brush regime. Polymer chains with thiol-containing anchors of different alkyl chain lengths (C11SH vs C4SH) formed assemblies with comparable degrees of chain overlap with 2 Rg/l values above 1.4 and are thus in the brush regime. Molecular weights influenced the achievable degree of chain overlap on the surface. Coatings prepared with the medium molecular weight polymer (9 kDa) resulted in the highest chain packing density. Control of grafting density and thus chain overlap in different regimes (brush vs mushroom) on planar gold substrates are attainable for monolayer coatings with poly(GME-ran-EGE) by adjusting the polymer's molecular weight and anchor group as well as the conditions for the grafting-to procedure.

Dendritic Polyglycerol Sulfate Inhibits Microglial Activation and Reduces Hippocampal CA1 Dendritic Spine Morphology Deficits.

Hyperactivity of microglia and loss of functional circuitry is a common feature of many neurological disorders including those induced or exacerbated by inflammation. Herein, we investigate the response of microglia and changes in hippocampal dendritic postsynaptic spines by dendritic polyglycerol sulfate (dPGS) treatment. Mouse microglia and organotypic hippocampal slices were exposed to dPGS and an inflammogen (lipopolysaccharides). Measurements of intracellular fluorescence and confocal microscopic analyses revealed that dPGS is avidly internalized by microglia but not CA1 pyramidal neurons. Concentration and time-dependent response studies consistently showed no obvious toxicity of dPGS. The adverse effects induced by proinflammogen LPS exposure were reduced and dendritic spine morphology was normalized with the addition of dPGS. This was accompanied by a significant reduction in nitrite and proinflammatory cytokines (TNF-α and IL-6) from hyperactive microglia suggesting normalized circuitry function with dPGS treatment. Collectively, these results suggest that dPGS acts anti-inflammatory, inhibits inflammation-induced degenerative changes in microglia phenotype and rescues dendritic spine morphology.

Hydrogel-Integrated Millifluidic Systems: Advancing the Fabrication of Mucus-Producing Human Intestinal Models.

The luminal surface of the intestinal epithelium is protected by a vital mucus layer, which is essential for lubrication, hydration, and fostering symbiotic bacterial relationships. Replicating and studying this complex mucus structure in vitro presents considerable challenges. To address this, we developed a hydrogel-integrated millifluidic tissue chamber capable of applying precise apical shear stress to intestinal models cultured on flat or 3D structured hydrogel scaffolds with adjustable stiffness. The chamber is designed to accommodate nine hydrogel scaffolds, 3D-printed as flat disks with a storage modulus matching the physiological range of intestinal tissue stiffness (~3.7 kPa) from bioactive decellularized and methacrylated small intestinal submucosa (dSIS-MA). Computational fluid dynamics simulations were conducted to confirm a laminar flow profile for both flat and 3D villi-comprising scaffolds in the physiologically relevant regime. The system was initially validated with HT29-MTX seeded hydrogel scaffolds, demonstrating accelerated differentiation, increased mucus production, and enhanced 3D organization under shear stress. These characteristic intestinal tissue features are essential for advanced in vitro models as they critically contribute to a functional barrier. Subsequently, the chamber was challenged with human intestinal stem cells (ISCs) from the terminal ileum. Our findings indicate that biomimicking hydrogel scaffolds, in combination with physiological shear stress, promote multi-lineage differentiation, as evidenced by a gene and protein expression analysis of basic markers and the 3D structural organization of ISCs in the absence of chemical differentiation triggers. The quantitative analysis of the alkaline phosphatase (ALP) activity and secreted mucus demonstrates the functional differentiation of the cells into enterocyte and goblet cell lineages. The millifluidic system, which has been developed and optimized for performance and cost efficiency, enables the creation and modulation of advanced intestinal models under biomimicking conditions, including tunable matrix stiffness and varying fluid shear stresses. Moreover, the readily accessible and scalable mucus-producing cellular tissue models permit comprehensive mucus analysis and the investigation of pathogen interactions and penetration, thereby offering the potential to advance our understanding of intestinal mucus in health and disease.

Thrombogenicity assessment of perfusable tissue engineered constructs: a systematic review.

Vascular surgery faces a critical demand for novel vascular grafts that are biocompatible and thromboresistant. This urgency particularly applies to bypass operations involving small caliber vessels. In the realm of tissue engineering, the development of fully vascularized organs holds great promise as a solution to organ shortage for transplantation. To achieve this, it is imperative to (re-)construct a biocompatible and non-thrombogenic vascular network within these organs. In this systematic review, we identify, classify and discuss basic principles and methods used to perform in vitro/ex vivo dynamic thrombogenicity testing of perfusable tissue engineered organs and tissues. We conducted a pre-registered systematic review of studies published in the last 23 years according to PRISMA-P Guidelines, comprising a systematic data extraction, in-depth analysis and risk of bias assessment of 116 included studies. We identified shaking (n=28), flow loop (n=17), ex vivo (arterio-venous shunt, n=33) and dynamic in vitro models (n=38) as main approaches for thrombogenicity assessment. This comprehensive review unveils a prevalent lack of standardization and serves as a valuable guide in the design of standardized experimental setups.

Chemistry matters: A side-by-side comparison of two chemically distinct methacryloylated dECM bioresins for vat photopolymerization.

Decellularized extracellular matrix (dECM) is an excellent natural source for 3D bioprinting materials due to its inherent cell compatibility. In vat photopolymerization, the use of dECM-based bioresins is just emerging, and extensive research is needed to fully exploit their potential. In this study, two distinct methacryloyl-functionalized, photocrosslinkable dECM-based bioresins were prepared from digested porcine liver dECM through functionalization with glycidyl methacrylate (GMA) or conventional methacrylic anhydride (MA) under mild conditions for systematic comparison. Although the chemical modifications did not significantly affect the structural integrity of the dECM proteins, mammalian cells encapsulated in the respective hydrogels performed differently in long-term culture. In either case, photocrosslinking during 3D (bio)printing resulted in transparent, highly swollen, and soft hydrogels with good shape fidelity, excellent biomimetic properties and tunable mechanical properties (~ 0.2-2.5 kPa). Interestingly, at a similar degree of functionalization (DOF ~ 81.5-83.5 %), the dECM-GMA resin showed faster photocrosslinking kinetics in photorheology resulting in lower final stiffness and faster enzymatic biodegradation compared to the dECM-MA gels, yet comparable network homogeneity as assessed via Brillouin imaging. While human hepatic HepaRG cells exhibited comparable cell viability directly after 3D bioprinting within both materials, cell proliferation and spreading were clearly enhanced in the softer dECM-GMA hydrogels at a comparable degree of crosslinking. These differences were attributed to the additional hydrophilicity introduced to dECM via methacryloylation through GMA compared to MA. Due to its excellent printability and cytocompatibility, the functional porcine liver dECM-GMA biomaterial enables the advanced biofabrication of soft 3D tissue analogs using vat photopolymerization-based bioprinting.

Synthesis and biological evaluation of radio and dye labeled amino functionalized dendritic polyglycerol sulfates as multivalent anti-inflammatory compounds.

Herein we describe a platform technology for the synthesis and characterization of partially aminated, (35)S-labeled, dendritic polyglycerol sulfate (dPG(35)S amine) and fluorescent dPGS indocarbocyanine (ICC) dye conjugates. These polymer conjugates, based on a biocompatible dendritic polyglycerol scaffold, exhibit a high affinity to inflamed tissue in vivo and represent promising candidates for therapeutic and diagnostic applications. By utilizing a one-step sequential copolymerization approach, dendritic polyglycerol (Mn ≈ 4.5 kDa) containing 9.4% N-phthalimide protected amine functionalities was prepared on a large scale. Sulfation and simultaneous radio labeling with (35)SO3 pyridine complex, followed by cleavage of the N-phthalimide protecting groups, yielded dPG(35)S amine as a beta emitting, inflammation specific probe with free amino functionalities for conjugation. Furthermore, efficient labeling procedures with ICC via iminothiolane modification and subsequent "Michael" addition of the maleimide functionalized ICC dye, as well as by amide formation via NHS derivatized ICC on a dPGS amine scaffold, are described. The dPGS-ICC conjugates were investigated with respect to their photophysical properties, and both the radiolabeled and fluorescent compounds were comparatively visualized in histological tissue sections (radio detection and fluorescence microscopy) of animals treated with dPGS. Furthermore, cellular uptake of dPGS-ICC was found in endothelial cord blood (HUVEC) and the epithelial lung cells (A549). The presented synthetic routes allow a reproducible, controlled synthesis of dPGS amine on kilogram scale applying a one-pot batch reaction process. dPGS amine can be used for analysis via radioactivity or fluorescence, thereby creating a new platform for inflammation specific, multimodal imaging purposes using other attachable probes or contrast agents.

Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation.

Adhesive interactions of leukocytes and endothelial cells initiate leukocyte migration to inflamed tissue and are important for immune surveillance. Acute and chronic inflammatory diseases show a dysregulated immune response and result in a massive efflux of leukocytes that contributes to further tissue damage. Therefore, targeting leukocyte trafficking may provide a potent form of anti-inflammatory therapy. Leukocyte migration is initiated by interactions of the cell adhesion molecules E-, L-, and P-selectin and their corresponding carbohydrate ligands. Compounds that efficiently address these interactions are therefore of high therapeutic interest. Based on this rationale we investigated synthetic dendritic polyglycerol sulfates (dPGS) as macromolecular inhibitors that operate via a multivalent binding mechanism mimicking naturally occurring ligands. dPGS inhibited both leukocytic L-selectin and endothelial P-selectin with high efficacy. Size and degree of sulfation of the polymer core determined selectin binding affinity. Administration of dPGS in a contact dermatitis mouse model dampened leukocyte extravasation as effectively as glucocorticoids did and edema formation was significantly reduced. In addition, dPGS interacted with the complement factors C3 and C5 as was shown in vitro and reduced C5a levels in a mouse model of complement activation. Thus, dPGS represent an innovative class of a fully synthetic polymer therapeutics that may be used for the treatment of inflammatory diseases.

Efficient material-induced activation of monocyte-derived dendritic cells releasing surface molecules, matrix metalloproteinases, and growth factors needed for regenerative tissue remodeling.

New experimental approaches for tissue repair have recently been proposed and include the application of natural or synthetic biomaterials and immune cells. Herein, fully synthetic poly(glycidyl ether) (PGE) copolymer coatings are evaluated as bioinstructive materials for the in vitro culture and intrinsic activation of human immune cells. Immature monocyte-derived dendritic cells (moDCs) are exposed to PGE brush and gel coatings of varying copolymer composition, wettability, and deformability immobilized on polystyrene culture dishes. Compared to moDCs cultured on standard tissue culture-treated polystyrene, activation marker levels on the cell surface are strongly enhanced on PGE substrates. Thereby, moDCs undergo a distinct morphological change and reach levels of activation comparable to those achieved by toll-like receptor (TLR) ligand liposaccharide (LPS), specifically for the expression of costimulatory molecules CD86 and CD40 as well as human leukocyte antigen (HLA)-DR. In addition, PGE coatings induce a significantly enhanced level of programmed cell death ligands 1 and 2 (PD-L1/-L2) on the moDC surface, two molecules crucially involved in maintaining immune tolerance. In addition, an increased release of matrix metalloproteinases MMP-1 and MMP-7, as well as transforming growth factor (TGF)-ß1 and epidermal growth factor (EGF) was observed in moDCs cultured on PGE substrates. As fully synthetic biomaterials, PGE coatings demonstrate intrinsic functional competence in instructing immature human moDCs for phenotypic activation in vitro, accompanied by the secretion of bioactive molecules, which are known to be crucial for tissue regeneration. Hence, PGE coatings hold strong potential for immune-modulating implant coatings, while PGE-activated moDCs are promising candidates for future clinical cell-based immunoengineering therapies.

Rise of tissue- and species-specific 3D bioprinting based on decellularized extracellular matrix-derived bioinks and bioresins.

Thanks to its natural complexity and functionality, decellularized extracellular matrix (dECM) serves as an excellent foundation for creating highly cell-compatible bioinks and bioresins. This enables the bioprinted cells to thrive in an environment that closely mimics their native ECM composition and offers customizable biomechanical properties. To formulate dECM bioinks and bioresins, one must first pulverize and/or solubilize the dECM into non-crosslinked fragments, which can then be chemically modified as needed. In bioprinting, the solubilized dECM-derived material is typically deposited and/or crosslinked in a layer-by-layer fashion to build 3D hydrogel structures. Since the introduction of the first liver-derived dECM-based bioinks, a wide variety of decellularized tissue have been employed in bioprinting, including kidney, heart, cartilage, and adipose tissue among others. This review aims to summarize the critical steps involved in tissue-derived dECM bioprinting, starting from the decellularization of the ECM to the standardized formulation of bioinks and bioresins, ultimately leading to the reproducible bioprinting of tissue constructs. Notably, this discussion also covers photocrosslinkable dECM bioresins, which are particularly attractive due to their ability to provide precise spatiotemporal control over the gelation in bioprinting. Both in extrusion printing and vat photopolymerization, there is a need for more standardized protocols to fully harness the unique properties of dECM-derived materials. In addition to mammalian tissues, the most recent bioprinting approaches involve the use of microbial extracellular polymeric substances in bioprinting of bacteria. This presents similar challenges as those encountered in mammalian cell printing and represents a fascinating frontier in bioprinting technology.

Surface functionalization of poly(ether imide) membranes with linear, methylated oligoglycerols for reducing thrombogenicity.

Materials for biomedical applications are often chosen for their bulk properties. Other requirements such as a hemocompatible surface shall be fulfilled by suitable chemical functionalization. Here we show, that linear, side-chain methylated oligoglycerols (OGMe) are more stable to oxidation than oligo(ethylene glycol) (OEG). Poly(ether imide) (PEI) membranes functionalized with OGMes perform at least as good as, and partially better than, OEG functionalized PEI membranes in view of protein resistance as well as thrombocyte adhesion and activation. Therefore, OGMes are highly potent surface functionalizing molecules for improving the hemocompatibility of polymers.

"Fuzzy hair" promotes cell sheet detachment from thermoresponsive brushes already above their volume phase transition temperature.

Thermoresponsive poly(glycidyl ether) (PGE) brushes have shown to be viable substrates for the culture and temperature-triggered detachment of confluent cell sheets. Surface-tethered PGEs with a cloud point temperature (TCP) around ~30 °C exhibit phase transitions well-centered within the physiological range (20-37 °C), which makes them ideal candidates for cell sheet fabrication. However, PGEs with TCPs at ~20 °C also afford the detachment of various types of cell sheets, even at room temperature (20-23 °C), i.e., above the polymers' TCPs. In this study, we investigate the phase transition of PGE brushes tethered to polystyrene (PS) culture substrates with varying grafting density and TCP to arrive at a mechanistic understanding of their functionality in cell sheet fabrication. Using quartz crystal microbalance with dissipation (QCM-D) monitoring, we demonstrate that brushes fabricated from PGEs with TCPs at ~20 °C display volume phase transition temperatures (VPTTs) well below room temperature. Although the investigated coatings obviously do not exhibit marked thermal switching in terms of brush hydration and layer thickness, their physical properties at the brush-water interface, as ascertained by QCM-D and AFM measurements, undergo subtle changes upon cooling from 37 °C to room temperature which is sufficient to promote cell sheet detachment. Thus, it appears that discreet rehydration of the outmost brush layer, resembling "fuzzy hair" at the brush-water interface, renders the surfaces less protein- and cell-adhesive at room temperature. This minor structural change of the interface allows for the reliable detachment of human dermal fibroblast sheets already at 20 °C well above the VPTT of the brushes.

In vitrovascularization of hydrogel-based tissue constructs via a combined approach of cell sheet engineering and dynamic perfusion cell culture.

The bioengineering of artificial tissue constructs requires special attention to their fast vascularization to provide cells with sufficient nutrients and oxygen. We addressed the challenge ofin vitrovascularization by employing a combined approach of cell sheet engineering, 3D printing, and cellular self-organization in dynamic maturation culture. A confluent cell sheet of human umbilical vein endothelial cells (HUVECs) was detached from a thermoresponsive cell culture substrate and transferred onto a 3D-printed, perfusable tubular scaffold using a custom-made cell sheet rolling device. Under indirect co-culture conditions with human dermal fibroblasts (HDFs), the cell sheet-covered vessel mimic embedded in a collagen gel together with additional singularized HUVECs started sprouting into the surrounding gel, while the suspended cells around the tube self-organized and formed a dense lumen-containing 3D vascular network throughout the gel. The HDFs cultured below the HUVEC-containing cell culture insert provided angiogenic support to the HUVECs via molecular crosstalk without competing for space with the HUVECs or inducing rapid collagen matrix remodeling. The resulting vascular network remained viable under these conditions throughout the 3 week cell culture period. This static indirect co-culture setup was further transferred to dynamic flow conditions, where the medium perfusion was enabled via two independently addressable perfusion circuits equipped with two different cell culture chambers, one hosting the HDFs and the other hosting the HUVEC-laden collagen gel. Using this system, we successfully connected the collagen-embedded HUVEC culture to a dynamic medium flow, and within 1 week of the dynamic cell culture, we detected angiogenic sprouting and dense microvascular network formation via HUVEC self-organization in the hydrogel. Our approach of combining a 3D-printed and cell sheet-covered vascular precursor that retained its sprouting capacity together with the self-assembling HUVECs in a dynamic perfusion culture resulted in a vascular-like 3D network, which is a critical step toward the long-term vascularization of bioengineeredin vitrotissue constructs.

Flow-induced glycocalyx formation and cell alignment of HUVECs compared to iPSC-derived ECs for tissue engineering applications.

The relevance of cellular in vitro models highly depends on their ability to mimic the physiological environment of the respective tissue or cell niche. Static culture conditions are often unsuitable, especially for endothelial models, since they completely neglect the physiological surface shear stress and corresponding reactions of endothelial cells (ECs) such as alignment in the direction of flow. Furthermore, formation and maturation of the glycocalyx, the essential polysaccharide layer covering all endothelial surfaces and regulating diverse processes, is highly dependent on applied fluid flow. This fragile but utterly important macromolecular layer is hard to analyze, its importance is often underestimated and accordingly neglected in many endothelial models. Therefore, we exposed human umbilical vein ECs (HUVECs) and human induced pluripotent stem cell-derived ECs (iPSC-ECs) as two relevant EC models in a side-by-side comparison to static and physiological dynamic (6.6 dyn cm-2) culture conditions. Both cell types demonstrated an elongation and alignment along the flow direction, some distinct changes in glycocalyx composition on the surface regarding the main glycosaminoglycan components heparan sulfate, chondroitin sulfate or hyaluronic acid as well as an increased and thereby improved glycocalyx thickness and functionality when cultured under homogeneous fluid flow. Thus, we were able to demonstrate the maturity of the employed iPSC-EC model regarding its ability to sense fluid flow along with the general importance of physiological shear stress for glycocalyx formation. Additionally, we investigated EC monolayer integrity with and without application of surface shear stress, revealing a comparable existence of tight junctions for all conditions and a reorganization of the cytoskeleton upon dynamic culture leading to an increased formation of focal adhesions. We then fabricated cell sheets of EC monolayers after static and dynamic culture via non-enzymatic detachment using thermoresponsive polymer coatings as culture substrates. In a first proof-of-concept we were able to transfer an aligned iPSC-EC sheet to a 3D-printed scaffold thereby making a step in the direction of vascular modelling. We envision these results to be a valuable contribution to improvements of in vitro endothelial models and vascular engineering in the future.