The neuroregenerative effects of topical decorin on the injured mouse cornea.

BACKGROUND: The cornea is innervated with a rich supply of sensory nerves that play important roles in ocular surface health. Any injury or pathology of the corneal nerves increases the risk of dry eye disease and infection. This study aims to evaluate the therapeutic potential of topical decorin to improve corneal nerve regeneration in a mouse model of sterile epithelial abrasion injury. METHODS: Bilateral central corneal epithelial abrasions (2-mm, Alger Brush) were performed on young C57BL/6 J mice to remove the corneal sensory nerves. Decorin, or vehicle, was applied topically, three times per day for 1 week or every 2 h for 6 h. Spectral-domain optical coherence tomography was performed to measure the abrasion area and corneal thickness. Wholemount immunofluorescence staining was used to assess sensory nerve regeneration (ß-tubulin III) and immune cell density (CD45, Iba1, CD11c). To investigate the specific role of dendritic cells (DCs), Cx3cr1gfp/gfp mice, which spontaneously lack resident corneal epithelial DCs, were also investigated. The effect of prophylactic topical administration of recombinant human decorin (applied prior to the abrasion) was also investigated. Nerve tracing (NeuronJ software) was performed to compare recovery of basal nerve axons and superficial nerve terminals in the central and peripheral cornea. RESULTS: At 6 h after injury, topical decorin application was associated with greater intraepithelial DC recruitment but no change in re-epithelialisation or corneal thickness, compared to the vehicle control. One week after injury, sub-basal nerve plexus and superficial nerve terminal density were significantly higher in the central cornea in the decorin-treated eyes. The density of corneal stromal macrophages in the decorin-treated eyes and their contralateral eyes was significantly lower compared to saline-treated corneas. No significant improvement in corneal nerve regeneration was observed in Cx3cr1gfp/gfp mice treated with decorin. CONCLUSIONS: Decorin promotes corneal epithelial nerve regeneration after injury. The neuroregenerative effect of topical decorin was associated with a higher corneal DC density during the acute phase, and fewer macrophages at the study endpoint. The corneal neuroregenerative effects of decorin were absent in mice lacking intraepithelial DCs. Together, these findings support a role for decorin in DC-mediated neuroregeneration following corneal abrasion injury.

Evaluating the swelling, erosion, and compaction properties of cellulose ethers.

Swelling, erosion, deformation, and consolidation properties can affect the performance of cellulose ethers, the most commonly used matrix former in hydrophilic sustained tablet formulations. The present study was designed to comparatively evaluate the swelling, erosion, compression, compaction, and relaxation properties of the cellulose ethers in a comprehensive study using standardised conditions. The interrelationship between various compressional models and the inherent deformation and consolidation properties of the polymers on the derived swelling and erosion parameters are consolidated. The impact of swelling (Kw) on erosion rates (KE) and the inter-relationship between Heckel and Kawakita plasticity constants was also investigated. It is evident from the findings that the increases in both substitution and polymer chain length led to higher Kw, but a lower KE; this was also true for all particle size fractions regardless of polymer grade. Smaller particle size and high substitution levels tend to increase the relative density of the matrix but reduce porosity, yield pressure (Py), Kawakita plasticity parameter (b-1) and elastic relaxation. Both KW versus KE (R2 = 0.949-0.980) and Py versus. b-1 correlations (R2 = 0.820-0.934) were reasonably linear with regards to increasing hydroxypropyl substitution and molecular size. Hence, it can be concluded that the combined knowledge of swelling and erosion kinetics in tandem with the in- and out-of-die compression findings can be used to select a specific polymer grade and further to develop and optimize formulations for oral controlled drug delivery applications.

Biologically Analogous Calcium Phosphate Tubes from a Chemical Garden.

Calcium phosphate (CaPO4) tubes with features comparable to mineralized biological microstructures, such as Haversian canals, were grown from a calcium gel/phosphate solution chemical garden system. A significant difference in gel mass in response to high and low solute phosphate equivalent environments existed within 30 min of solution layering upon gel (p = 0.0067), suggesting that the nature of advective movement between gel and solution is dependent on the solution concentration. The transport of calcium cations (Ca2+) and phosphate anions (PO43-) was quantified and changes in pH were monitored to explain the preferential formation of tubes within a PO43- concentration range of 0.5-1.25 M. Ingress from the anionic solution phase into the gel followed by the liberation of Ca2+ ions from the gel was found to be essential for acquiring self-assembled tubular CaPO4 structures. Tube analysis by scanning electron microscopy (SEM), X-ray diffraction (XRD), and micro X-ray florescence (µ-XRF) revealed hydroxyapatite (HA, Ca10(PO4)6(OH)2) and dicalcium phosphate dihydrate (DCPD, CaHPO4·2H2O) phases organized in a hierarchical manner. Notably, the tubule diameters ranged from 100 to 150 µm, an ideal size for the permeation of vasculature in biological hard tissue.

Vesicles in Nature and the Laboratory: Elucidation of Their Biological Properties and Synthesis of Increasingly Complex Synthetic Vesicles.

The important role of vesicles in many aspects of cell function is well-recognized, but only recently have sophisticated imaging techniques begun to reveal their ubiquity in nature. While we further our understanding of the biological properties of vesicles and their physiological functions, increasingly elegant artificial vesicles are being developed for a wide range of technological applications and basic research. Herein, we examine the state of the art of biological and synthetic vesicles and place their biological features in the context of recent synthetic developments, thus providing a unique overview of these complex and rapidly developing fields. The challenges and opportunities associated with future biological and synthetic studies of vesicles are also presented.

Modification of gellan gum with nanocrystalline hydroxyapatite facilitates cell expansion and spontaneous osteogenesis.

Nanocomposites composed of hydrogels and calcium phosphates are of great interest in the development of bone graft replacements since they may have a structural and compositional resemblance to bone. Culture beads formed from such materials could be used in stirred tank culture and thereby enable cell expansion in a sufficiently efficient manner to allow for the generation of enough large number of cells for large-scale bone reconstruction. Although combinations of materials such as alginate, collagens, and various calcium phosphates have been investigated as culture beads, these materials are unsuitable for application since they have been shown to rapidly degrade in physiological conditions and enable relatively little tailoring of mechanical properties. In this study, gellan gum-nano sized hydroxyapatite (nHA) composites, which have been shown to be resistant to degradation and easily modified with respect to modulus, were formulated and characterized as regards their ability to enable cell attachment and proliferation. It was shown that the addition of 5 wt% of nHA to the culture beads enabled cell attachment and that an increase in nHA concentration to up to 25 wt% enhanced the rate of cell proliferation. Most importantly, it was demonstrated that the addition of nHA to the cell culture beads enabled the formation of nodules in culture of MC3T3-E1 cells and strikingly stimulated the osteogenic differentiation of bone marrow stromal cells in the absence of osteogenic media when compared with tissue culture plastic (TCP) with the same condition. Biotechnol. Bioeng. 2016;113: 1568-1576. © 2016 Wiley Periodicals, Inc.

Imaging the hard/soft tissue interface.

Interfaces between different tissues play an essential role in the biomechanics of native tissues and their recapitulation is now recognized as critical to function. As a consequence, imaging the hard/soft tissue interface has become increasingly important in the area of tissue engineering. Particularly as several biotechnology based products have made it onto the market or are close to human trials and an understanding of their function and development is essential. A range of imaging modalities have been developed that allow a wealth of information on the morphological and physical properties of samples to be obtained non-destructively in vivo or via destructive means. This review summarizes the use of a selection of imaging modalities on interfaces to date considering the strengths and weaknesses of each. We will also consider techniques which have not yet been utilized to their full potential or are likely to play a role in future work in the area.

Low temperature aqueous precipitation of needle-like nanophase hydroxyapatite.

The use of tissue engineered biodegradable porous scaffolds has become an important focus of the biomedical research field. The precursor materials used to form these structures play a vital role in their overall performance thus making the study and synthesis of these selected materials imperative. The authors present a comparison and characterisation of hydroxyapatite (HA), a popular calcium phosphate (CaP) biomaterial, synthesised by an aqueous precipitation (AP) method. The influence of various reaction conditions on the phase, crystallinity, particle size as well as morphology, molecular structure, potential in-vivo bioactivity and cell viability were assessed by XRD, SEM and TEM, FTIR, a simulated body fluid (SBF) test and a live/dead assay using MC3T3 osteoblast precursor cells, respectively. Naturally carbonated nanoparticles of HA with typically needle-like morphology were synthesised by the reported AP method. Initial pH was found to influence the crystallisation process and determine the CaP phase formed as well as the resultant particle and crystallite sizes. A marked change in particle morphology was also observed above pH 9. The use of toluene as a replacement solvent for water up to 60% was found to reduce the crystallinity of as-synthesised HA. This has marked influence on the effect of ethanolamine (5 wt%), which was found to improve HA crystallinity. SEM and EDS were used to confirm the growth of carbonated apatite on the surface of HA pellets immersed in SBF for up to 28 days. Cell culture results revealed viable cells on all samples where pH was controlled and maintained at 10-11 during precipitation, including those that used ethanolamine and toluene in preparation. When the initial alkali pH was not maintained non-viable cells were observed on HA substrates.

A bio-hybrid tactile sensor incorporating living artificial skin and an impedance sensing array.

The development of a bio-hybrid tactile sensor array that incorporates a skin analogue comprised of alginate encapsulated fibroblasts is described. The electrical properties are modulated by mechanical stress induced during contact, and changes are detected by a ten-channel dual-electrode impedance sensing array. By continuously monitoring the impedance of the sensor array at a fixed frequency, whilst normal and tangential loads are applied to the skin surface, transient mechanotransduction has been observed. The results demonstrate the effectiveness and feasibility of the preliminary prototype bio-hybrid tactile sensor.

Achieving net-zero in the dry eye disease care pathway.

Climate change is a threat to human health and wellbeing across the world. In recent years, there has been a surge in awareness of this crisis, leading to many countries and organisations setting "net-zero" targets. This entails minimising carbon emissions and neutralising remaining emissions by removing carbon from the atmosphere. At the 2022 United Nations Climate Change Conference (COP27), commitments to transition away from fossil fuels and augment climate targets were underwhelming. It is therefore imperative for public and private sector organisations to demonstrate successful implementation of net-zero and set a precedent for the global political consensus. As a top 10 world employer, the United Kingdom National Health Service (NHS) has pledged to reach net-zero by 2045. The NHS has already taken positive steps forward, but its scale and complexity as a health system means stakeholders in each of its services must highlight the specifications for further progress. Dry eye disease is a chronic illness with an estimated global prevalence of 29.5% and an environmentally damaging care pathway. Moreover, environmental damage is a known aggravator of dry eye disease. Worldwide management of this illness generates copious amounts of non-recyclable waste, utilises inefficient supply chains and involves recurrent follow-up appointments and prescriptions. By mapping the dry eye disease care pathway to environmental impact, in this review we will highlight seven key areas in which reduced emissions and pollution could be targeted. Examining these approaches for improved environmental sustainability is critical in driving the transformation needed to preserve our health and wellbeing.

Fabrication of gradient hydrogels using a thermophoretic approach in microfluidics.

The extracellular matrix presents spatially varying physical cues that can influence cell behavior in many processes. Physical gradients within hydrogels that mimic the heterogenous mechanical microenvironment are useful to study the impact of these cues on cellular responses. Therefore, simple and reliable techniques to create such gradient hydrogels are highly desirable. This work demonstrates the fabrication of stiffness gradient Gellan gum (GG) hydrogels by applying a temperature gradient across a microchannel containing hydrogel precursor solution. Thermophoretic migration of components within the precursor solution generates a concentration gradient that mirrors the temperature gradient profile, which translates into mechanical gradients upon crosslinking. Using this technique, GG hydrogels with stiffness gradients ranging from 20 to 90 kPa over 600µm are created, covering the elastic moduli typical of moderately hard to hard tissues. MC3T3 osteoblast cells are then cultured on these gradient substrates, which exhibit preferential migration and enhanced osteogenic potential toward the stiffest region on the gradient. Overall, the thermophoretic approach provides a non-toxic and effective method to create hydrogels with defined mechanical gradients at the micron scale suitable forin vitrobiological studies and potentially tissue engineering applications.

Trabecular meshwork cell differentiation in response to collagen and TGFß-2 spatial interactions.

Primary open angle glaucoma (POAG) is currently the most prevalent cause of irreversible blindness globally. To date, there are few in vitro models that can faithfully recapitulate the complex architecture of the trabecular meshwork (TM) and the specialized trabecular meshwork cell (TMC) characteristics that are local to structurally opposing regions. This study aimed to investigate the parameters that govern TMC phenotype by adapting the extracellular matrix structure to mimic the juxtacanalicular tissue (JCT) region of the TM. Initially, TMC phenotypic characteristics were investigated within type I collagen matrices of controlled fiber density and anisotropy, generated through confined plastic compression (PC). Notably, PC-collagen presented biophysical cues that induced JCT cellular characteristics (elastin, α-ß-Crystallin protein expression, cytoskeletal remodeling and increased mesenchymal and JCT-specific genetic markers). In parallel, a pathological mesenchymal phenotype associated with POAG was induced through localized transforming growth factor -beta 2 (TGFß-2) exposure. This resulted in a profile of alternative mesenchymal states (fibroblast/smooth muscle or myofibroblast) displayed by the TMC in vitro. Overall, the study provides an advanced insight into the biophysical cues that modulate TMC fate, demonstrating the induction of a JCT-specific TMC phenotype and transient mesenchymal characteristics that reflect both healthy or pathological scenarios. STATEMENT OF SIGNIFICANCE: Glaucoma is the most prevalent cause of blindness, with a lack of efficacy within current drug candidates. Reliable trabecular meshwork (TM) in vitro models will be critical for enhancing the fields understanding of healthy and disease states for pre-clinical testing. To date, trabecular meshwork cells (TMCs) display heterogeneity throughout the hierarchical TM, however our understanding into recapitulating these phenotypes in vitro, remains elusive. This study hypothesizes the importance of specific matrix/growth factor spatial stimuli in governing TMC phenotype. By emulating certain biophysical/biochemical in vivo parameters, we introduce an advanced profile of distinct TMC phenotypic states, reflecting healthy and disease scenarios. A notion that has not be stated prior and a fundamental consideration for future TM 3D in vitro modelling.

Expression of E-cadherin by CD8+ T cells promotes their invasion into biliary epithelial cells.

The presence of CD8+ T cells in the cytoplasm of biliary epithelial cells (BEC) has been correlated with biliary damage associated with primary biliary cholangitis (PBC). Here, we characterise the mechanism of CD8+ T cell invasion into BEC. CD8+ T cells observed within BEC were large, eccentric, and expressed E-cadherin, CD103 and CD69. They were also not contained within secondary vesicles. Internalisation required cytoskeletal rearrangements which facilitated contact with BEC. Internalised CD8+ T cells were observed in both non-cirrhotic and cirrhotic diseased liver tissues but enriched in PBC patients, both during active disease and at the time of transplantation. E-cadherin expression by CD8+ T cells correlated with frequency of internalisation of these cells into BEC. E-cadherin+ CD8+ T cells formed ß-catenin-associated interactions with BEC, were larger than E-cadherin- CD8+ T cells and invaded into BEC more frequently. Overall, we unveil a distinct cell-in-cell structure process in the liver detailing the invasion of E-cadherin+ CD103+ CD69+ CD8+ T cells into BEC.

Brushite cement additives inhibit attachment to cell culture beads.

Brushite-forming calcium phosphate cements are of great interest as bone replacement materials because they are resorbable in physiological conditions. Cell-attached culture beads formed from this material could be of great use for cell therapy. Despite a significant amount of work on optimizing the physicochemical properties of these materials, there are very few studies that have evaluated the capacity of the materials to facilitate cell adhesion. In this study, we have formed resorbable calcium phosphate (brushite) culture beads and for the first time we showed that cell attachment to the surface of the brushite cement (BC) could be inhibited by the presence of an intermediate dicalcium phosphate-citrate complex, formed in the cement as a result of using citric acid, a retardant and viscosity modifier used in many cement formulations. The BC beads formed from the mixture of ß-TCP/orthophosphoric acid using citric acid did not allow cell attachment without further treatment. Ageing of BC beads in serum-free Dulbecco's Modified Eagle's Medium (DMEM) solution at 37°C for 1 week greatly enhanced the cell adhesion capacity of the material. Scanning electron microscopy, X-ray diffraction (XRD), and confocal Raman microspectrometry indicated the increased capacity for cell adhesion was due to the changes in phase composition of BC. XRD patterns collected before and after ageing in aqueous solution and a high initial mass loss, suggest the formation of a dicalcium phosphate-citrate complex within the matrix. Since compacts formed from brushite powder supported cell attachment, it was hypothesized that the dicalcium phosphate-citrate complex prevented attachment to the cement surface.

Triggered metabolism of adenosine triphosphate as an explanation for the chemical heterogeneity of heterotopic ossification.

Heterotopic ossification (HO), the pathological formation of bone in soft tissues, is a debilitating condition, as well as one of the few instances of de novo bone formation in adults. Chemical mapping of HO tissue showed distinct islands of calcium phosphate within phosphate-deficient, calcium-rich regions, suggesting a transition to apatitic bone mineral from a non-phosphatic precursor. The transition of amorphous calcium carbonate (ACC), a generally suggested bone-mineral precursor, in physiological conditions was thus investigated. Here, we show that adenosine triphosphate (ATP), present in high amounts in forming bone, stabilised ACC for weeks in physiological conditions and that enzymatic degradation of ATP triggered rapid crystallisation into apatite, through an amorphous calcium phosphate phase. It is suggested that this localised enzymatic degradation could explain the chemical heterogeneity seen in HO and may also represent a pathway to physiological bone mineralisation.

Hydrogels and Bioprinting in Bone Tissue Engineering: Creating Artificial Stem-Cell Niches for In Vitro Models.

Advances in bioprinting have enabled the fabrication of complex tissue constructs with high speed and resolution. However, there remains significant structural and biological complexity within tissues that bioprinting is unable to recapitulate. Bone, for example, has a hierarchical organization ranging from the molecular to whole organ level. Current bioprinting techniques and the materials employed have imposed limits on the scale, speed, and resolution that can be achieved, rendering the technique unable to reproduce the structural hierarchies and cell-matrix interactions that are observed in bone. The shift toward biomimetic approaches in bone tissue engineering, where hydrogels provide biophysical and biochemical cues to encapsulated cells, is a promising approach to enhancing the biological function and development of tissues for in vitro modeling. A major focus in bioprinting of bone tissue for in vitro modeling is creating dynamic microenvironmental niches to support, stimulate, and direct the cellular processes for bone formation and remodeling. Hydrogels are ideal materials for imitating the extracellular matrix since they can be engineered to present various cues whilst allowing bioprinting. Here, recent advances in hydrogels and 3D bioprinting toward creating a microenvironmental niche that is conducive to tissue engineering of in vitro models of bone are reviewed.

Mature primary human osteocytes in mini organotypic cultures secrete FGF23 and PTH1-34-regulated sclerostin.

Introduction: For decades, functional primary human osteocyte cultures have been crucially needed for understanding their role in bone anabolic processes and in endocrine phosphate regulation via the bone-kidney axis. Mature osteocyte proteins (sclerostin, DMP1, Phex and FGF23) play a key role in various systemic diseases and are targeted by successful bone anabolic drugs (anti-sclerostin antibody and teriparatide (PTH1-34)). However, cell lines available to study osteocytes produce very little sclerostin and low levels of mature osteocyte markers. We have developed a primary human 3D organotypic culture system that replicates the formation of mature osteocytes in bone. Methods: Primary human osteoblasts were seeded in a fibrinogen / thrombin gel around 3D-printed hanging posts. Following contraction of the gel around the posts, cells were cultured in osteogenic media and conditioned media was collected for analysis of secreted markers of osteocyte formation. Results: The organoids were viable for at least 6 months, allowing co-culture with different cell types and testing of bone anabolic drugs. Bulk RNAseq data displayed the developing marker trajectory of ossification and human primary osteocyte formation in vitro over an initial 8- week period. Vitamin D3 supplementation increased mineralization and sclerostin secretion, while hypoxia and PTH1-34 modulated sclerostin. Our culture system also secreted FGF23, enabling the future development of a bone-kidney-parathyroid-vascular multi-organoid or organ-on-a-chip system to study disease processes and drug effects using purely human cells. Discussion: This 3D organotypic culture system provides a stable, long-lived, and regulated population of mature human primary osteocytes for a variety of research applications.

Development of Neovasculature in Axially Vascularized Calcium Phosphate Cement Scaffolds.

Augmenting the vascular supply to generate new tissues, a crucial aspect in regenerative medicine, has been challenging. Recently, our group showed that calcium phosphate can induce the formation of a functional neo-angiosome without the need for microsurgical arterial anastomosis. This was a preclinical proof of concept for biomaterial-induced luminal sprouting of large-diameter vessels. In this study, we investigated if sprouting was a general response to surgical injury or placement of an inorganic construct around the vessel. Cylindrical biocement scaffolds of differing chemistries were placed around the femoral vein. A contrast agent was used to visualize vessel ingrowth into the scaffolds. Cell populations in the scaffold were mapped using immunohistochemistry. Calcium phosphate scaffolds induced 2.7-3 times greater volume of blood vessels than calcium sulphate or magnesium phosphate scaffolds. Macrophage and vSMC populations were identified that changed spatially and temporally within the scaffold during implantation. NLRP3 inflammasome activation peaked at weeks 2 and 4 and then declined; however, IL-1ß expression was sustained over the course of the experiment. IL-8, a promoter of angiogenesis, was also detected, and together, these responses suggest a role of sterile inflammation. Unexpectedly, the effect was distinct from an injury response as a result of surgical placement and also was not simply a foreign body reaction as a result of placing a rigid bioceramic next to a vein, since, while the materials tested had similar microstructures, only the calcium phosphates tested elicited an angiogenic response. This finding then reveals a potential path towards a new strategy for creating better pro-regenerative biomaterials.

Why Animal Experiments Are Still Indispensable in Bone Research: A Statement by the European Calcified Tissue Society.

Major achievements in bone research have always relied on animal models and in vitro systems derived from patient and animal material. However, the use of animals in research has drawn intense ethical debate and the complete abolition of animal experimentation is demanded by fractions of the population. This phenomenon is enhanced by the reproducibility crisis in science and the advance of in vitro and in silico techniques. 3D culture, organ-on-a-chip, and computer models have improved enormously over the last few years. Nevertheless, the overall complexity of bone tissue cross-talk and the systemic and local regulation of bone physiology can often only be addressed in entire vertebrates. Powerful genetic methods such as conditional mutagenesis, lineage tracing, and modeling of the diseases enhanced the understanding of the entire skeletal system. In this review endorsed by the European Calcified Tissue Society (ECTS), a working group of investigators from Europe and the US provides an overview of the strengths and limitations of experimental animal models, including rodents, fish, and large animals, as well the potential and shortcomings of in vitro and in silico technologies in skeletal research. We propose that the proper combination of the right animal model for a specific hypothesis and state-of-the-art in vitro and/or in silico technology is essential to solving remaining important questions in bone research. This is crucial for executing most efficiently the 3R principles to reduce, refine, and replace animal experimentation, for enhancing our knowledge of skeletal biology, and for the treatment of bone diseases that affect a large part of society. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Versatile Microfluidics for Biofabrication Platforms Enabled by an Agile and Inexpensive Fabrication Pipeline.

Microfluidics have transformed diagnosis and screening in regenerative medicine. Recently, they are showing much promise in biofabrication. However, their adoption is inhibited by costly and drawn-out lithographic processes thus limiting progress. Here, multi-material fibers with complex core-shell geometries with sizes matching those of human arteries and arterioles are fabricated employing versatile microfluidic devices produced using an agile and inexpensive manufacturing pipeline. The pipeline consists of material extrusion additive manufacturing with an innovative continuously varied extrusion (CONVEX) approach to produce microfluidics with complex seamless geometries including, novel variable-width zigzag (V-zigzag) mixers with channel widths ranging from 100-400 µm and hydrodynamic flow-focusing components. The microfluidic systems facilitated rapid mixing of fluids by decelerating the fluids at specific zones to allow for increased diffusion across the interfaces. Better mixing even at high flow rates (100-1000 µL min-1 ) whilst avoiding turbulence led to high cell cytocompatibility (>86%) even when 100 µm nozzles are used. The presented 3D-printed microfluidic system is versatile, simple and efficient, offering a great potential to significantly advance the microfluidic platform in regenerative medicine.

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting.

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 °C and melting temperature of ~90 °C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).