Mesenchymal stromal cells (MSCs) are widely used in regenerative medicine and cell engineering; however, conventional two-dimensional culture systems fail to reproduce the physiological microenvironment of cells and limit the preservation of their functional state. Objective. This study aimed to investigate the effect of gelatin concentration in the core of alginate core–shell capsules on the viability, metabolic activity and spatial organisation of human bone marrow-derived MSCs. Materials and Methods. Alginate core–shell capsules were fabricated using coaxial electrospraying with an alginate shell and hydrogel core containing different gelatin concentrations (0, 2.5, 5 and 7.5% w/v). Rheological properties of the core and shell materials were analysed by rotational rheometry. Cell viability was assessed using Live/Dead fluorescence staining, metabolic activity by Alamar Blue assay, and spatial organisation by light and fluorescence microscopy. Capsules were cultured under physiological conditions for 17 days and subsequently transferred to ambient temperature storage. Results. All capsule formulations supported high MSC viability during long-term culture. However, the functional state of the cells strongly depended on the core composition. Capsules containing 2.5% gelatin demonstrated the most pronounced increase in metabolic activity and the formation of interconnected, network-like cellular structures. Increasing gelatin concentration to 5–7.5% resulted in reduced metabolic activity and the formation of compact cell aggregates. Following storage at ambient temperature (22 °C), metabolic activity decreased in all groups, whereas cell viability remained stable. Conclusions. Gelatin concentration within the capsule core is a key parameter regulating MSC functional behaviour in three-dimensional culture systems. Alginate core–shell capsules represent a promising platform for short-term storage and transportation of cells while maintaining their viability and structural organisation

The advancement of cell-populated bioengineered constructs (BECs) has created an ever-increasing demand for effective cryopreservation strategies that guarantee long-term storage without compromising structural integrity or biological function. By allowing constructs to be stored until required, cryopreservation helps align tissue construct unrestricted availability with patient needs and enables regenerative treatments to be initiated on demand. Through the prism of biomaterial properties, this review delves into the historical development and recent progress in the in toto (whole cell-biomaterial construct) cryopreservation of engineered artificial tissues. Particular emphasis is placed on elastic fibrous matrices, alginate-based solid and core-shell constructs, and bulk 3D micro and macroporous scaffolds, complemented by practical examples from the authors’ own research. Beyond these aspects, the review explores methods for recording freezing patterns within cell-free BECs to better characterize thermal events and establish reproducible frameworks with minimal construct-to-construct variability. Finally, this work surveys key regulatory considerations and translational barriers that must be overcome to achieve Good Manufacturing Practice (GMP)-compliant, clinically viable cryopreservation protocols for cell-populated BECs.

IntroductionCochlear implant electrodes are frequently ensheathed by connective tissue as a consequence of surgical trauma and foreign body response, which can impair electrical stimulation. In this study, fiber mats fabricated by electrospinning with three different spinning times (15, 30 and 45 min) were evaluated for their suitability as surface modifications for cochlear implant electrodes to reduce connective tissue-induced insulation after implantation.MethodsTheir biocompatibility was assessed through incubation with an extract solution generated from the fiber mat using two different cell types and methods. The influence of the three different polymeric structures on electrical conductivity was examined by coating SEM-holders and measuring changes in impedance. In addition, the wetting behavior of the fiber mats was monitored over a period of 3 weeks. Cell proliferation was further investigated by directly seeding NIH/3T3 eGFP fibroblasts onto the fiber mats and analyzing their growth in a period of 7 days.Results and DiscussionThe fiber mats were considered biocompatible, although incubation with 100% extract resulted in decreased cell viability in NIH/3T3 fibroblasts. Impedance values increased with increasing fiber mat thickness. Wetting behavior was independent of mat thickness, occurring primarily within the first 24 h and reaching a plateau after 1–2 weeks of immersion in physiological sodium chloride solution. Although cell proliferation was reduced on the fiber mats compared to the conventional cell culture substrate after the 7 days of incubation, cell growth was delayed rather than completely inhibited. Overall, surface modification of cochlear implant electrodes with electrospun fiber mats shows potential to mitigate electrode insulation caused by connective tissue formation.

Over the past decades, cryobiology has become increasingly integrated across a variety of domains. With broad intersection, this discipline has recently also emerged as a cornerstone for advancing and clinically translating tissue-engineered constructs (TECs). This review provides a comprehensive overview of recent advances at the intersection of cryobiology and tissue engineering. Vitrification of both scaffold-based and scaffold-free TECs as well as approaches of its upscaling including benefits of polymers and technical devices are comprehensibly discussed. The development of biologically inspired nanoscale materials is outlined as an integral part of this research. Success in vitrification of organoids is also discussed. Developments in controlled-rate slow-cooling/freezing protocols are then examined, with particular attention to xeno-free and Me2SO-free cryoprotective systems that enhance cell viability and biocompatibility. Preclinical studies utilizing cryopreserved TECs in animal models are further outlined as key milestones toward clinical translation. Furthermore, this review introduces emerging synergistic approaches that make TECs more adaptable to cryopreservation by incorporating cryoprotective agents (CPAs), nanoparticles, cold-responsive polymers and ice recrystallization/devitrification inhibitors at the scaffold design stage. Finally, cryobioprinting as an emerging approach that unites cryobiology and tissue engineering, offering new opportunities for the fabrication, storage, and on-demand deployment of viable tissue constructs is reviewed. Overall, the overviewed experimental evidence underscores the transformative role of cryobiology in driving recent advances in the field of tissue engineering and fostering innovative and forward-looking strategies in this field. Ultimately, a closer convergence of cryobiology, tissue engineering, and transplantation science will be essential to advance TECs toward scalable, off-the-shelf availability.

Blood exhibits complex flow behavior governed by red blood cell (RBC) deformation, aggregation, and confinement effects, which are difficult to reproduce in vitro at single-cell level under confinement. Existing blood mimicking fluids (BMFs) primarily replicate bulk rheology but fail to capture microscale single-cell mechanics relevant to microcirculation. Here, we present a particulate blood mimicking fluid (BMF) composed of monodisperse hydrogel-based artificial erythrocytes (ARBC) with a physiological diameter of 9 μm, biconcave geometry, and plasma-phase-dependent mechanical properties. ARBCs are generated using a cross-flow microfluidic fabrication approach, enabling reproducible fabrication and integration into well-defined plasma-phase analogues. Adjustment of the surrounding plasma-phase analogue enabled modulation of particle swelling, elasticity, and interparticle interactions. Under confined microchannel flow, particles exhibited velocity-dependent deformations from disc-like to bullet-like morphologies, reproducing the characteristic trend observed for human RBCs. Depending on the plasma-phase composition, measured deformation indices overlapped with those obtained for RBCs under comparable confinement conditions. By combining physiological geometry, elasticity, and controllable plasma-phase properties, this platform provides a standardized model system for studying microscale hemorheology and for validating deformation-based lab-on-a-chip technologies.

Scaffolds' production for hard to soft tissues recently become of great interest, as bone-tendon insertion tissue engineering, where injuries mainly occur. Interfacial tissue engineering aims at developing grafts to mimic the gradients of those tissues as far as composition, mechanical properties and structures are concerned. Additive manufacturing can offer solutions to meet these requirements, but still requires to improve processes to achieve such gradients in a few steps. In this study, we developed a 3D-printed collector to combine gap-spinning and micropatterning. We were able to manufacture a scaffold (60 mm long, 5 mm wide) with a smooth gradient of 5 mm long from honeycomb structure to aligned fibers (promoting bone and tendon fate, respectively) in a single step. We estimated a gradient in Young modulus from 20 MPa to 30 MPa from the bone to the tendon side. Deformation tracking permitted to highlight significant difference of local strains between both areas, which could then impact cells' response. Murine stem cells C3H10T1/2 were then seeded at both scaffold parts and cultivated without any growth factors in stretching conditions. Alkaline phosphatase staining and tenomodulin immunostaining suggested the effect of stretching to cells' behavior between the bone and tendon area, compared to static condition. However, benefit of topographical and mechanical cues only cannot be fully established to foster cells to specific fate probably due to the limits of this cell line. This novel collector system however permitted to produce a relevant scaffold to study interfaces where a topographical gradient might be needed.

Hippophae rhamnoides is a cosmopolitan shrub that has attracted the attention of many scientists worldwide, not only for its therapeutic properties and metabolite richness but also for its ability to sustain freezing down to -40 °C. Endowed with the capacity to withstand harsh conditions, this plant controls freezing processes to cope with seasonal exposure to sub-zero temperatures. Intriguingly, H. rhamnoides is reported to harbour intrinsic ice-nucleating agents (INAs) active at temperatures above -5 °C. Moreover, its fruits provide a habitat for diverse populations of bacteria represented by genera that might exogenously initiate ice formation on vegetative and reproductive parts. Inspired by the multifaceted cold adaptation features H. rhamnoides, in this work, we have examined freezing patterns of its aqueous crude leaf homogenates (CLHs) in admixture with cryoprotective agents (CPAs) known to promote supercooling: antifreeze protein (AFP) type III, sucrose, trehalose, and dimethyl sulfoxide (Me2SO). For this, we employed infrared thermography, cryomicroscopy, and differential scanning calorimetry (DSC). In order to identify the possible microbial contributions to H. rhamnoides INAs, bulk and filtered CLHs underwent microbiological studies. Analogous to Snomax™ (SM), the addition of CLH to disaccharides increased crystallization duration and reduced both the degree of supercooling and freezing time compared to the respective controls. Findings from experiments involving filtered and heat-treated CLHs suggest the presence of soluble ice nucleators that may originate from the plants themselves, as well as from ice-nucleating bacteria that are active at warmer sub-zero temperatures. Using conventional aerobic cultivation on chromogenic solid media, we obtained three bacterial isolates from bulk H. rhamnoides homogenates. Using 16S rRNA sequencing, the isolates were identified as representatives of the Erwiniaceae family. The sequence of one isolate clustered with those of Pantoea agglomerans (formerly known as Enterobacter agglomerans or Erwinia herbicola), a well-known bacterial ice-nucleating species. Interestingly, two isolates were clustered together with the recently delineated species Duffyella gerundensis. In summary, the investigation into the freezing characteristics of leaves derived from H. rhamnoides is of potential environmental and cryobiological utility.

The treatment of degenerative pathologies affecting articular cartilage remains a significant clinical challenge. Non-invasive biophysical stimuli, such as electric fields, have demonstrated potential as therapeutic tools for cartilage tissue restoration. Previous studies have reported that electric fields enhance chondrocyte proliferation and the synthesis of key extracellular matrix components, such as glycosaminoglycans. However, inconsistencies in experimental designs have led to variable findings. This study examines the effects of capacitively coupled electric fields on chondrocytes cultured in gelatin hydrogels. Alternating voltages of 50 V (7.7 mV/cm) and 100 V (8.7 mV/cm) at a frequency of 60 kHz were applied for 21 days. Cell quantification and glycosaminoglycan analysis were performed on both stimulated and control samples. On day 7, exposure to the electric field resulted in a significant reduction in cell proliferation by 24.7% and 39.2% at 7.7 mV/cm and 8.7 mV/cm, respectively (p < 0.05). However, stimulation at 8.7 mV/cm led to a 35.7% increase in glycosaminoglycan synthesis compared to the control group (p < 0.05). These findings indicate that electric field stimulation can modulate the synthesis of essential extracellular matrix components, such as glycosaminoglycans, in hyaline cartilage. This highlights the potential of electric fields as a promising strategy to enhance outcomes in articular cartilage tissue engineering, particularly in hydrogel-based therapeutic approaches.

The development of accurate blood-mimicking fluids (BMFs) is essential for in vitro studies of blood contacting medical devices. Experimental data typically relies on single-phase glycerin/water solutions as substitutes to visualize simplified blood flow. These models are accurate only at high shear rates, limiting their applicability at lower shear rates. In this study, we investigated three potential BMFs, each composed of poly(sodium acrylate-co-acrylamide) hydrogel microparticles (beads) as artificial erythrocytes. Microbeads were produced using microfluidic systems (MFS) and were suspended in three plasma-like solutions: 10% and 50% (v/v) glycerol/water solutions and a Dextran40/CaCl2 solution. The BMFs were evaluated for their rheological and mechanical properties, including particle elasticity, sedimentation behavior, and shear flow analysis, to assess their suitability for mimicking blood. Rheometric measurements were performed at room temperature using a plate-plate configuration, measuring viscosity and shear stress for shear rates of 5-500 s-1. Atomic force microscopy (AFM) measurements were conducted to assess their mechanical response. The Dextran40/CaCl2-based BMF was identified as the most promising, demonstrating rheological and mechanical properties that closely align with those of human blood. This research offers a refined approach to developing blood analogs that better simulate the mechanical response and flow characteristics of blood for the validation and development of blood contacting medical devices.

This study investigates methodological variability across various expert laboratories worldwide, with regards to characterizing the mechanical properties of biological tissues. Two testing rounds were conducted on the specific use case of uniaxial tensile testing of porcine aorta. In the first round, 24 labs were invited to apply their established methods to assess inter-laboratory variability. This revealed significant methodological diversity and associated variability in the stress-stretch results, underscoring the necessity for a standardized approach. In the second round, a consensus protocol was collaboratively developed and adopted by 19 labs in an attempt to minimize variability. This involved standardized sample preparation and uniformity in testing protocol, including the use of a common cutting and thickness measurement tool. Despite protocol harmonization, significant variability persisted across labs, which could not be solely attributed to inherent biological differences in tissue samples. These results illustrate the challenges in unifying testing methods across different research settings, underlining the necessity for further refinement of testing practices. Enhancing consistency in biomechanical experiments is pivotal when comparing results across studies, as well as when using the resulting material properties for in silico simulations in medical research.

Magnetic hydrogels are emerging as promising biomaterials for tissue engineering due to their ability to provide structural support and controlled drug release. In this study, gelatin-hyaluronic acid hydrogels (70:30) were synthesized and functionalized with Divi-Divi (Libidibia coriaria) extract (1% w/v) and magnetic nanoparticles (Fe3O4@SiO2, 1% w/v). The extract was obtained through an aqueous extraction method and characterized for its bioactive properties. Rheological analysis showed that the incorporation of magnetic nanoparticles increased hydrogel stiffness, whereas Divi-divi extract did not significantly affect the mechanical properties. Controlled release experiments were conducted using a 30 mT magnetic field at 100 kHz for 30 minutes in PBS, resulting in a release of approximately 62 μg/ml of the extract, as detected by UV spectrophotometry. These results demonstrate the potential of these hydrogels as magnetically responsive drug delivery systems for biomedical applications. Future studies will optimize the release kinetics and evaluate their biocompatibility in vitro.

Understanding the tribological behavior of blood-lubricated interfaces is considered relevant for the design of heart pumps, heart valves or for the understanding of the blood flow in very narrow blood vessels. Tribological studies with real biological materials are challenging, e.g., due to their limited stability or potential risk of infections. To overcome these challenges, artificial materials are used in this study to mimic real blood and the biological interfaces. As for the lubricant, a hydrogel-based artificial blood with a glycerol-water solution, as a continuous phase, is used. Various compositions of artificial blood are investigated and compared with real plasma as well as platelet-rich plasma as lubricants. Soft biological interfaces are represented by a glass-ball-on-three-elastomeric pins setup. Results from tribological model system measurements on the different lubricants are shown in the form of Stribeck curves and possible lubricating mechanisms are discussed. Results from complementary shear rheological measurements of the blood fluids are shown and discussed.