Recreating metabolic interactions of the tumour microenvironment.

Tumours are heterogeneous tissues containing diverse populations of cells and an abundant extracellular matrix (ECM). This tumour microenvironment prompts cancer cells to adapt their metabolism to survive and grow. Besides epigenetic factors, the metabolism of cancer cells is shaped by crosstalk with stromal cells and extracellular components. To date, most experimental models neglect the complexity of the tumour microenvironment and its relevance in regulating the dynamics of the metabolism in cancer. We discuss emerging strategies to model cellular and extracellular aspects of cancer metabolism. We highlight cancer models based on bioengineering, animal, and mathematical approaches to recreate cell-cell and cell-matrix interactions and patient-specific metabolism. Combining these approaches will improve our understanding of cancer metabolism and support the development of metabolism-targeting therapies.

Nano- and Microstructures of Collagen-Nanocellulose Hydrogels as Engineered Extracellular Matrices.

The extracellular matrix (ECM) is the fundamental acellular element of human tissues, providing their mechanical structure while delivering biomechanical and biochemical signals to cells. Three-dimensional (3D) tissue models commonly use hydrogels to recreate the ECM in vitro and support the growth of cells as organoids and spheroids. Collagen-nanocellulose (COL-NC) hydrogels rely on the blending of both polymers to design matrices with tailorable physical properties. Despite the promising application of these biomaterials in 3D tissue models, the architecture and network organization of COL-NC remain unclear. Here, we investigate the structural effects of incorporating NC fibers into COL hydrogels by small-angle neutron scattering (SANS) and ultra-SANS (USANS). The critical hierarchical structure parameters of fiber dimensions, interfiber distance, and coassembled open structures of NC and COL in the absence and presence of cells were determined. We found that NC expanded and increased the homogeneity in the COL network without affecting the inherent fiber properties of both polymers. Cells cultured as spheroids in COL-NC remodeled the hydrogel network without a significant impact on its architecture. Our study reveals the polymer organization of COL-NC hydrogels and demonstrates SANS and USANS as exceptional techniques to reveal nano- and micron-scale details on polymer organization, which leads to a better understanding of the structural properties of hydrogels to engineer novel ECMs.

3D Collagen-Nanocellulose Matrices Model the Tumour Microenvironment of Pancreatic Cancer.

Three-dimensional (3D) cancer models are invaluable tools designed to study tumour biology and new treatments. Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest types of cancer, has been progressively explored with bioengineered 3D approaches by deconstructing elements of its tumour microenvironment. Here, we investigated the suitability of collagen-nanocellulose hydrogels to mimic the extracellular matrix of PDAC and to promote the formation of tumour spheroids and multicellular 3D cultures with stromal cells. Blending of type I collagen fibrils and cellulose nanofibres formed a matrix of controllable stiffness, which resembled the lower profile of pancreatic tumour tissues. Collagen-nanocellulose hydrogels supported the growth of tumour spheroids and multicellular 3D cultures, with increased metabolic activity and matrix stiffness. To validate our 3D cancer model, we tested the individual and combined effects of the anti-cancer compound triptolide and the chemotherapeutics gemcitabine and paclitaxel, resulting in differential cell responses. Our blended 3D matrices with tuneable mechanical properties consistently maintain the growth of PDAC cells and its cellular microenvironment and allow the screening of anti-cancer treatments.

A thermo-responsive collagen-nanocellulose hydrogel for the growth of intestinal organoids.

Three-dimensional (3D) cell culture systems include bioengineered microenvironments that mimic the complexity of human tissues and organs in vitro. Robust biological models, like organoids and spheroids, rely on biomaterials to emulate the biochemical and biomechanical properties found in the extracellular matrix (ECM). Collagen (COL) is the main protein component of the ECM and has been used to generate fibrous matrices for 3D cell culture. Whilst neat COL gels are commonly blended with inert polymers to improve their poor mechanical properties, whether nanocellulose (NC) fibers interact or can develop some synergic bioactive effect to support organoid systems has never been demonstrated. Here, we investigate collagen-nanocellulose (COL-NC) hydrogels as a thermo-responsive matrix for the formation and growth of intestinal organoids. Cellulose nanofibres grafted with fibronectin-like adhesive sites form a porous network with type I collagen, presenting a sol-gel transition and viscoelastic profile similar to those of standard animal-based matrices. Crypts embedded in COL-NC form organoids with evidence of epithelial budding. Cell viability and metabolic activity are preserved as well as the expression of key cell markers. The stiffness of COL-NC hydrogels is shown to be a determinant element for the formation and development organoids. COL-NC hydrogels provide an affordable, performant thermo-responsive and sustainable matrix for organoid growth.

Perspective on Constructing Cellulose-Hydrogel-Based Gut-Like Bioreactors for Growth and Delivery of Multiple-Strain Probiotic Bacteria.

The current perspective presents an outlook on developing gut-like bioreactors with immobilized probiotic bacteria using cellulose hydrogels. The innovative concept of using hydrogels to simulate the human gut environment by generating and maintaining pH and oxygen gradients in the gut-like bioreactors is discussed. Fundamentally, this approach presents novel methods of production as well as delivery of multiple strains of probiotics using bioreactors. The relevant existing synthesis methods of cellulose hydrogels are discussed for producing porous hydrogels. Harvesting methods of multiple strains are discussed in the context of encapsulation of probiotic bacteria immobilized on cellulose hydrogels. Furthermore, we also discuss recent advances in using cellulose hydrogels for encapsulation of probiotic bacteria. This perspective also highlights the mechanism of probiotic protection by cellulose hydrogels. Such novel gut-like hydrogel bioreactors will have the potential to simulate the human gut ecosystem in the laboratory and stimulate new research on gut microbiota.

Cationic Cross-Linked Nanocellulose-Based Matrices for the Growth and Recovery of Intestinal Organoids.

Highly carboxylated nanocellulose fibers can be functionalized with cell adhesive peptides and cationic cross-linked to form matrices for a three-dimensional (3D) cell culture. It is hypothesized that nanocellulose hydrogels cross-linked with divalent cations can provide the required biochemical and mechanical properties for intestinal organoid growth and recovery. Nanocellulose hydrogels are produced by TEMPO- and TEMPO-periodate-mediated oxidation and functionalized with RGD peptides. Mechanical properties are measured by rheology and optical properties quantified by UV-vis spectroscopy. Cellulosic matrices are cross-linked with Ca2+ and Mg2+ and intestinal organoids cultured for 4 days. The organoids are recovered for passaging and RNA extraction. TEMPO-periodate-oxidized nanocellulose fibers form functionalized hydrogels and support the growth of intestinal organoids. The highly transparent cellulosic matrix requires 4 times more Mg2+ than Ca2+ ions to reach the targeted stiffness. Organoids cultured in nanocellulose maintained a major living area for up to 4 days. Cell clusters recovered from magnesium-cross-linked hydrogels can be passaged, and their extracted RNA is intact. Cationic cross-linked nanocellulose hydrogels are promising alternative plant-based matrices for a 3D cell culture systems.

Rapid Gel Card Agglutination Assays for Serological Analysis Following SARS-CoV-2 Infection in Humans.

High-throughput and rapid serology assays to detect the antibody response specific to severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in human blood samples are urgently required to improve our understanding of the effects of COVID-19 across the world. Short-term applications include rapid case identification and contact tracing to limit viral spread, while population screening to determine the extent of viral infection across communities is a longer-term need. Assays developed to address these needs should match the ASSURED criteria. We have identified agglutination tests based on the commonly employed blood typing methods as a viable option. These blood typing tests are employed in hospitals worldwide, are high-throughput, fast (10-30 min), and automated in most cases. Herein, we describe the application of agglutination assays to SARS-CoV-2 serology testing by combining column agglutination testing with peptide-antibody bioconjugates, which facilitate red cell cross-linking only in the presence of plasma containing antibodies against SARS-CoV-2. This simple, rapid, and easily scalable approach has immediate application in SARS-CoV-2 serological testing and is a useful platform for assay development beyond the COVID-19 pandemic.

Engineered Plant-Based Nanocellulose Hydrogel for Small Intestinal Organoid Growth.

Organoids are three-dimensional self-renewing and organizing clusters of cells that recapitulate the behavior and functionality of developed organs. Referred to as "organs in a dish," organoids are invaluable biological models for disease modeling or drug screening. Currently, organoid culture commonly relies on an expensive and undefined tumor-derived reconstituted basal membrane which hinders its application in high-throughput screening, regenerative medicine, and diagnostics. Here, we introduce a novel engineered plant-based nanocellulose hydrogel is introduced as a well-defined and low-cost matrix that supports organoid growth. Gels containing 0.1% nanocellulose fibers (99.9% water) are ionically crosslinked and present mechanical properties similar to the standard animal-based matrix. The regulation of the osmotic pressure is performed by a salt-free strategy, offering conditions for cell survival and proliferation. Cellulose nanofibers are functionalized with fibronectin-derived adhesive sites to provide the required microenvironment for small intestinal organoid growth and budding. Comparative transcriptomic profiling reveals a good correlation with transcriptome-wide gene expression pattern between organoids cultured in both materials, while differences are observed in stem cells-specific marker genes. These hydrogels are tunable and can be combined with laminin-1 and supplemented with insulin-like growth factor (IGF-1) to optimize the culture conditions. Nanocellulose hydrogel emerges as a promising matrix for the growth of organoids.

Photothermal incubation of red blood cells by laser for rapid pre-transfusion blood group typing.

Safe blood transfusion requires compatibility testing of donor and recipient to prevent potentially fatal transfusion reactions. Detection of immunoglobulin G (IgG) antibodies requires incubation at 37 °C, often for up to 15 minutes. Current incubation technology predominantly relies on slow thermal-gradient dependent conduction. Here, we present rapid optical heating via laser, where targeted illumination of a blood-antibody sample in a diagnostic gel card is converted into heat, via photothermal absorption. Our laser-incubator heats the 75 µL blood-antibody sample to 37 °C in under 30 seconds. We show that red blood cells act as photothermal agents under near-infrared laser incubation, triggering rapid antigen-antibody binding. We detect no significant damage to the cells or antibodies for laser incubations of up to fifteen minutes. We demonstrate laser-incubated immunohaematological testing to be both faster and more sensitive than current best practice - with clearly positive results seen from laser incubations of just 40 seconds.

Engineering nanocellulose hydrogels for biomedical applications.

Nanocellulose hydrogels are highly hydrated porous cellulosic soft materials with good mechanical properties. These cellulose-based gels can be produced from bacterial or plant cellulose nanofibrils, which are hydrophilic, renewable, biodegradable and biocompatible. Nanocellulose, whether fibrils (CNF), crystals (CNC) or bacterial (BNC), has a high aspect ratio and surface area, and can be chemically modified with functional groups or by grafting biomolecules. Cellulose functionalization provides enhanced physical and chemical properties and control of biological interactions, tailoring its hydrogels for specific applications. Here, we critically review nanocellulose hydrogels for biomedical applications. Nanocellulose hydrogels have been demonstrated for 3D cell culture, mimicking the extracellular matrix (ECM) properties with low cytotoxicity. For wound dressing and cartilage repair, nanocellulose gels promote cell regeneration while providing the required mechanical properties for tissue engineering scaffolds. The encapsulation of therapeutics within nanocellulose allows the targeted delivery of drugs. Currently, cellulose crosslinking to peptides and proteins enables a new generation of low cost and renewable smart materials used in diagnostics. Last, the organized mesh of fibres contained in hydrogels drives applications in separation of biomolecules and cells. Nanocellulose hydrogels have emerged as a highly engineerable platform for multiple biomedical applications, providing renewable and performant solutions to life sciences.

Nanocellulose Hydrogel for Blood Typing Tests.

The gel test is the most prevalent method for the forward and reverse blood typing tests. It relies on the controlled centrifugation of red blood cells (RBCs) and antibodies through a gel column. This noncontinuous matrix is currently based on microbeads that often lack sensitivity. For the first time, nanocellulose hydrogel is demonstrated as a sustainable and reliable medium for gel-based blood typing diagnostics. Gels with a minimum of 0.3 wt % TEMPO-oxidized cellulose nanofibers (0.92 mmol/g of carboxyl content) separate agglutinated and individual RBCs in the forward test. The addition of glycine is able to balance the osmotic pressure and reduce hemolysis to 5%, while retaining the electrostatic repulsion responsible for the gel network structure and its rheological properties. For the reverse typing, cellulose nanofibers are chemically cross-linked with hexamethylenediamine (HMDA), increasing the gel yield point 8-fold. Sodium chloride is added to achieve the osmolality found in the human plasma and limit cell lysis to 15%, without affecting the gel colloidal stability. Nanocellulose hydrogel constitutes a performant, low cost, and green soft material, providing clear and well-defined results for both blood grouping tests.