A covalently crosslinked bioink for multi-materials drop-on-demand 3D bioprinting of three-dimensional cell cultures

In vitro three-dimensional (3D) cell models have been accepted to better recapitulate aspects of in vivo organ environment than 2D cell culture. Currently, the production of these complex in vitro 3D cell models with multiple cell types and microenvironments remains challenging and prone to human error. Here we report a versatile bioink comprised of a 4-arm PEG based polymer with distal maleimide derivatives as the main ink component and a bis-thiol species as the activator that crosslinks the polymer to form the hydrogel in less than a second. The rapid gelation makes the polymer system compatible with 3D bioprinting. The ink is combined with a drop-on-demand 3D bioprinting platform consisting of eight independently addressable nozzles and high-throughput printing logic for creating complex 3D cell culture models. The combination of multiple nozzles and fast printing logic enables the rapid preparation of many complex 3D structures comprising multiple hydrogel environments in the one structure in a standard 96-well plate format. The platform compatibility for biological applications was validated using pancreatic ductal adenocarcinoma cancer (PDAC) cells with their phenotypic responses controlled by tuning the hydrogel microenvironment

Precise, high-throughput production of multicellular spheroids with a bespoke 3D bioprinter

3D in vitro cancer models are important therapeutic and biological discovery tools, yet formation of multicellular spheroids in a throughput and highly controlled manner to achieve robust and statistically relevant data, remains challenging. Here, we developed an enabling technology consisting of a bespoke drop-on-demand 3D bioprinter capable of high-throughput printing of 96-well plates of spheroids. 3D-multicellular spheroids are embedded inside a tissue-like matrix with precise control over size and cell number. Application of 3D bioprinting for high-throughput drug screening was demonstrated with doxorubicin. Measurements showed that IC 50 values were sensitive to spheroid size, embedding and how spheroids conform to the embedding, revealing parameters shaping biological responses in these models. Our study demonstrates the potential of 3D bioprinting as a robust high-throughput platform to screen biological and therapeutic parameters. Significance Statement In vitro 3D cell cultures serve as more realistic models, compared to 2D cell culture, for understanding diverse biology and for drug discovery. Preparing 3D cell cultures with defined parameters is challenging, with significant failure rates when embedding 3D multicellular spheroids into extracellular mimics. Here, we report a new 3D bioprinter we developed in conjunction with bioinks to allow 3D-multicellular spheroids to be produced in a high-throughput manner. High-throughput production of embedded multicellular spheroids allowed entire drug-dose responses to be performed in 96-well plate format with statistically relevant numbers of data points. We have deconvoluted important parameters in drug responses including the impact of spheroid size and embedding in an extracellular matrix mimic on IC 50 values

Patient-derived xenograft (PDX) models in basic and translational breast cancer research

Patient-derived xenograft (PDX) models of a growing spectrum of cancers are rapidly supplanting long-established traditional cell lines as preferred models for conducting basic and translational preclinical research. In breast cancer, to complement the now curated collection of approximately 45 long-established human breast cancer cell lines, a newly formed consortium of academic laboratories, currently from Europe, Australia, and North America, herein summarizes data on over 500 stably transplantable PDX models representing all three clinical subtypes of breast cancer (ER+, HER2+, and "Triple-negative" (TNBC)). Many of these models are well-characterized with respect to genomic, transcriptomic, and proteomic features, metastatic behavior, and treatment response to a variety of standard-of-care and experimental therapeutics. These stably transplantable PDX lines are generally available for dissemination to laboratories conducting translational research, and contact information for each collection is provided. This review summarizes current experiences related to PDX generation across participating groups, efforts to develop data standards for annotation and dissemination of patient clinical information that does not compromise patient privacy, efforts to develop complementary data standards for annotation of PDX characteristics and biology, and progress toward "credentialing" of PDX models as surrogates to represent individual patients for use in preclinical and co-clinical translational research. In addition, this review highlights important unresolved questions, as well as current limitations, that have hampered more efficient generation of PDX lines and more rapid adoption of PDX use in translational breast cancer research

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Enhanced drug toxicity by conjugation of platinum drugs to polymers with guanidine containing zwitterionic functional groups that mimic cell-penetrating peptides

Inspired by the Ringsdorf model, statistical copolymers with solubility enhancers, platinum drugs and groove binders were compared. In addition, the polymer was furnished with a cell penetrating moiety using a guanidine containing polymer. A block copolymer based on poly(4-vinylbenzyl chloride) and a block carrying zwitterionic monomer prepared from arginine was obtained using RAFT polymerization. Thiol–chloride reaction was then employed to attach thioglycerol (TG) as the water-soluble functional group, 9-aminoacridine (AA) as groove binder to enhance DNA binding and reactive diamino functionality as the bidentate ligand for the conjugation of platinum drugs. The aim of this work was to create a stable bond between the polymer and the drug to answer the question if it is essential to have degradable linkers to generate high drug activity. Three platinated polymers – having only the solubility enhancer, the solubility enhancer and the groove binder and with all three moieties – were compared in regards to their ability to enter the human ovarian carcinoma A2780 cells. Unsurprisingly, the zwitterionic polymer showed the highest uptake, which also coincided with a higher toxicity of the drug. Conjugated to the zwitterionic polymer, the platinum drug showed a higher toxicity than free cisplatin. In summary, even 40 years after the concepts was first established by Ringsdorf, this design still seems to have high validity highlighting that the suitable polymer design can enhance the activity of the drug

Inverse Miniemulsion Periphery RAFT Polymerization: A Convenient Route to Hollow Polymeric Nanoparticles with an Aqueous Core

The recently developed [Chem. Commun. 2012, 48, 11103−11105] inverse miniemulsion periphery RAFT polymerization (IMEPP) approach to prepare hollow polymeric nanoparticles (∼200 nm) with an aqueous core has been explored in detail. The method is based on an amphiphilic macroRAFT agent acting as stabilizer of water droplets in an organic continuous phase while also mediating cross-linking chain growth in a controlled/living manner on the outer periphery of the droplets. The macroRAFT agent comprised a hydrophilic block of poly­(<i>N</i>-(2-hydroxypropyl)­methacrylamide) and a hydrophobic block of either polystyrene or poly­(methyl methacrylate), and the cross-linked shell was formed on polymerization of styrene/divinylbenzene or methyl methacrylate/ethylene glycol dimethacrylate, respectively. The effects of various reaction parameters on the resulting hollow nanoparticles have been systematically investigated, and it has been demonstrated that the shell thickness can be tuned based on initial stoichiometry and monomer conversion. This method is particularly relevant for encapsulation of proteinssuccessful incorporation of proteins (bovine serum albumin) into the miniemulsion did not negatively affect the droplet size and stability

Synthesis of pH-Responsive Nanocapsules via Inverse Miniemulsion Periphery RAFT Polymerization and Post-Polymerization Reaction

We report herein the versatility of inverse miniemulsion periphery RAFT polymerization (IMEPP) and postpolymerization reaction in producing pH-responsive nanocapsules with different functionalities. The robustness of the polymeric nanocapsules was confirmed by their ability to undergo reactions, be dried, and be redispersed in various solvents without any changes in size and core–shell morphology. Nanocapsules bearing carboxylic acid (COOH) functionalities were produced via hydrolysis, while nanocapsules bearing tertiary-amine (N-X₃) functionalities were synthesized via aminolysis. The responsive behavior of the nanocapsules was tested in aqueous solution with pHs ranging from 3 to 12. Nanocapsules with COOH functionalities were found to swell under basic conditions due to the deprotonated carboxylate ions. In contrast, nanocapsule with tertiary amine functionalities underwent swelling in acidic conditions

Secretory pathway antagonism by calicivirus homologues of Norwalk virus nonstructural protein p22 is restricted to noroviruses

Abstract Background Our previous report that the Norwalk virus nonstructural protein p22 is an antagonist of the cellular secretory pathway suggests a new aspect of norovirus/host interaction. To explore conservation of function of this highly divergent calicivirus protein, we examined the effects of p22 homologues from four human and two murine noroviruses, and feline calicivirus on the secretory pathway. Findings All human noroviruses examined induced Golgi disruption and inhibited protein secretion, with the genogroup II.4 Houston virus being the most potent antagonist. Genogroup II.6 viruses have a conserved mutation in the mimic of an Endoplasmic Reticulum export signal (MERES) motif that is highly conserved in human norovirus homologues of p22 and is critical for secretory pathway antagonism, and these viruses had reduced levels of Golgi disruption and inhibition of protein secretion. p22 homologues from both persistent and nonpersistent strains of murine norovirus induced Golgi disruption, but only mildly inhibited cellular protein secretion. Feline calicivirus p30 did not induce Golgi disruption or inhibit cellular protein secretion. Conclusions These differences confirm a norovirus-specific effect on host cell secretory pathway antagonism by homologues of p22, which may affect viral replication and/or cellular pathogenesis.</p

Modular photo-induced RAFT polymerised hydrogels via thiol–ene click chemistry for 3D cell culturing

Cell behaviour changes as a result of the local environment, particularly when transitioning from two dimensional (2D) to three dimensional (3D) environments. It has been acknowledged that there is a need for efficient, tuneable and reproducible methods for making 3D cell cultures to further understand cell behaviour in 3D environments. The development of extracellular matrix (ECM) mimics has gained popularity as a way to create highly tuneable materials that resemble the native environment around cells. The modular nature of synthetic hydrogels means that they have the potential as ECM mimics for 3D cell cultures with tuneable mechanical and chemical properties. Herein, reversible addition fragmentation chain transfer (RAFT) polymerisation was used to synthesise poly(ethylene glycol) methyl ether acrylate (PEGMEA). Hydrogels with tuneable mechanical and cell adhesive properties were synthesised. Norbornene was used as a functional unit for both crosslinking and addition of biomolecules via thiol-ene click chemistry. To obviate the need for UV light for cross-linking of the hydrogel, visible light stimulated eosin-Y was used to induce the thiol-ene reaction. Pancreatic cancer cells (Kras(G12D) and p53(R172H)) were seeded on the hydrogels to confirm that the cytotoxicity of the hydrogels was low. The attachment of CRGDS onto the hydrogel was demonstrated as a means to improve cell adhesion