Automated production of gene-modified chimeric antigen receptor T cells using the Cocoon Platform.

Autologous cell-based therapeutics have gained increasing attention in recent years because of their efficacy at treating diseases with limited therapeutic options. Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical success in hematologic oncology indications, providing critically ill patients with a potentially curative therapy. Although engineered cell therapies such as CAR T cells provide new options for patients with unmet needs, the high cost and complexity of manufacturing may hinder clinical and commercial translation. The Cocoon Platform (Lonza, Basel, Switzerland) addresses many challenges, such as high labor demand, process consistency, contamination risks and scalability, by enabling efficient, functionally closed and automated production, whether at clinical or commercial scale. This platform is customizable and easy to use and requires minimal operator interaction, thereby decreasing process variability. We present two processes that demonstrate the Cocoon Platform's capabilities. We employed different T-cell activation methods-OKT3 and CD3/CD28 Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA)-to generate final cellular products that meet the critical quality attributes of a clinical autologous CAR T-cell product. This study demonstrates a manufacturing solution for addressing challenges with manual methods of production and facilitating the scale-up of autologous cell therapy.

Bioreactor-manufactured cartilage grafts repair acute and chronic osteochondral defects in large animal studies.

OBJECTIVES: Bioreactor-based production systems have the potential to overcome limitations associated with conventional tissue engineering manufacturing methods, facilitating regulatory compliant and cost-effective production of engineered grafts for widespread clinical use. In this work, we established a bioreactor-based manufacturing system for the production of cartilage grafts. MATERIALS & METHODS: All bioprocesses, from cartilage biopsy digestion through the generation of engineered grafts, were performed in our bioreactor-based manufacturing system. All bioreactor technologies and cartilage tissue engineering bioprocesses were transferred to an independent GMP facility, where engineered grafts were manufactured for two large animal studies. RESULTS: The results of these studies demonstrate the safety and feasibility of the bioreactor-based manufacturing approach. Moreover, grafts produced in the manufacturing system were first shown to accelerate the repair of acute osteochondral defects, compared to cell-free scaffold implants. We then demonstrated that grafts produced in the system also facilitated faster repair in a more clinically relevant chronic defect model. Our data also suggested that bioreactor-manufactured grafts may result in a more robust repair in the longer term. CONCLUSION: By demonstrating the safety and efficacy of bioreactor-generated grafts in two large animal models, this work represents a pivotal step towards implementing the bioreactor-based manufacturing system for the production of human cartilage grafts for clinical applications. Read the Editorial for this article on doi:10.1111/cpr.12625.

Influence of particle size and reactive oxygen species on cobalt chrome nanoparticle-mediated genotoxicity.

Patients with cobalt chrome (CoCr) metal-on-metal (MOM) implants may be exposed to a wide size range of metallic nanoparticles as a result of wear. In this study we have characterised the biological responses of human fibroblasts to two types of synthetically derived CoCr particles [(a) from a tribometer (30 nm) and (b) thermal plasma technology (20, 35, and 80 nm)] in vitro, testing their dependence on nanoparticle size or the generation of oxygen free radicals, or both. Metal ions were released from the surface of nanoparticles, particularly from larger (80 nm) particles generated by thermal plasma technology. Exposure of fibroblasts to these nanoparticles triggered rapid (2 h) generation of reactive oxygen species (ROS) that could be eliminated by inhibition of NADPH oxidase, suggesting that it was mediated by phagocytosis of the particles. The exposure also caused a more prolonged, MitoQ sensitive production of ROS (24 h), suggesting involvement of mitochondria. Consequently, we recorded elevated levels of aneuploidy, chromosome clumping, fragmentation of mitochondria and damage to the cytoskeleton particularly to the microtubule network. Exposure to the nanoparticles resulted in misshapen nuclei, disruption of mature lamin B1 and increased nucleoplasmic bridges, which could be prevented by MitoQ. In addition, increased numbers of micronuclei were observed and these were only partly prevented by MitoQ, and the incidence of micronuclei and ion release from the nanoparticles were positively correlated with nanoparticle size, although the cytogenetic changes, modifications in nuclear shape and the amount of ROS were not. These results suggest that cells exhibit diverse mitochondrial ROS-dependent and independent responses to CoCr particles, and that nanoparticle size and the amount of metal ion released are influential.

Nanoparticles can cause DNA damage across a cellular barrier.

The increasing use of nanoparticles in medicine has raised concerns over their ability to gain access to privileged sites in the body. Here, we show that cobalt-chromium nanoparticles (29.5 +/- 6.3 nm in diameter) can damage human fibroblast cells across an intact cellular barrier without having to cross the barrier. The damage is mediated by a novel mechanism involving transmission of purine nucleotides (such as ATP) and intercellular signalling within the barrier through connexin gap junctions or hemichannels and pannexin channels. The outcome, which includes DNA damage without significant cell death, is different from that observed in cells subjected to direct exposure to nanoparticles. Our results suggest the importance of indirect effects when evaluating the safety of nanoparticles. The potential damage to tissues located behind cellular barriers needs to be considered when using nanoparticles for targeting diseased states.

The effect of pre-coating human bone marrow stromal cells with hydroxyapatite/amino acid nanoconjugates on osteogenesis.

Aqueous colloidal suspensions of positively charged, amino acid-functionalized hydroxyapatite (HAp) nanoparticles (HAp/alanine and HAp/arginine) were added to a HBMSC suspension to effect non-specific cell surface deposition due to favourable attractive electrostatic interactions. Subsequent maintenance of these hybrid precursors under in vitro basal (non-osteogenic) culture conditions for up to 21 days, either as a monolayer or as a 3D pellet culture system, resulted in significantly increased levels of markers of osteoblast differentiation in comparison with uncoated cells. In monolayer culture, osteogenic activity could be further enhanced in a dose-dependent manner by surface derivatization of the amino acid-stabilized nanoparticles with the cell surface-specific binding peptide arginine-glycine-aspartic acid (RGD). Significantly, in 3D pellet culture conditions all HAp nanoconjugates promoted osteoblast differentiation, whereas for uncoated cells even soluble osteogenic culture additives were ineffectual. We therefore tested these constructs for in vivo activity by subcutaneous implantation in immunocompromised mice. New osteoid formation was observed in samples recovered after 21 days, comparable to the extensive areas of mineralized extracellular matrix produced in vitro. Overall, these studies outline the potential of biomolecular/hydroxyapatite nanoconjugates to promote osteogenic cell differentiation in vitro and hence provide new models to examine skeletal cell differentiation and function. Moreover, the pre-coating of HBMSCs enables the formation of viable hybrid multicellular 3D constructs with demonstrable activity both in vitro and in vivo.