Athanasios Mantalaris

Bone tissue engineering (BTE) emerged as a practical approach to tackle prosthetic industry limitations. We merge aspects from developmental biology, engineering and medicine with the aim to produce fully functional bone tissue. Mesenchymal stem cells have the capability of self-renewal and specific lineage differentiation. Herein lies their potential for BTE. Among MSCs, human dental pulp stem cells have a higher proliferation rate, shorter doubling times, lower cellular senescence, and enhanced osteogenesis than hBM-SCs under specific conditions. In addition, these cells are readily accessible and can be extracted through a subtle extraction procedure. Thus, they garner fewer moral concerns than most MSCs available and embody a promising cell source for BTE therapies able to replace hBM-MSCs. Interestingly, their study has been limited. Conversely, there is a need for their further study to harness their true value in BTE, with special emphasis in the design of bioprocesses able to produce viable, homogenous bone constructs in a clinical scale. Here, we study the osteogenic differentiation of hDPSCs encapsulated in alginate hydrogels under suspended culture in a novel perfusion bioreactor. The system is compared with traditional 3D static and fed-batch culture methodologies. The novel system performed better, producing higher alkaline phosphatase activity, and more homogeneous, dense and functional bone constructs. Additionally, cell constructs produced by the in-house-designed system were richer in mature osteoblast-like and mineralizing osteocyte-like cells. In conclusion, this study reports the development of a novel bioprocess able to produce hDPSC-based bone-like constructs, providing new insights into hDPSCs’ therapeutic potential and a system able to be transferred from the laboratory bench into medical facilities.

Proteins can perform ideal therapeutic functions. However, their large size and significant surface hydrophilicity and charge prohibit them from reaching intracellular targets. These chemical features also render them poorly encapsulated by nanoparticles used for intracellular delivery. In this work, a novel combination of protein vesicles and hydrophobic ion pairing (HIP) was used to load protein cargo and achieve cytosolic delivery to overcome the limitations of previous protein vesicle properties. Protein vesicles are thermally self-assembling nanoparticles made from elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper and a globular protein fused to a glutamate-rich leucine zipper. To impart stimuli-responsive disassembly, physiological stability, and small size, the ELP sequence was modified to include histidine and tyrosine residues. HIP was used to load and release protein cargo requiring endosomal escape for cytosolic function. HIP vesicles enabled delivery of cytochrome c, a cytosolically active protein, and a significant reduction in viability in both a traditional two-dimensional (2D) human cancer cell line culture and a biomimetic three-dimensional (3D) organoid model of acute myeloid leukemia. By examining the uptake of positively and negatively charged fluorescent protein cargos loaded by HIP, this work revealed the necessity of HIP for cytosolic cargo delivery and how HIP loading influences protein vesicle self-assembly and disassembly using microscopy, small-angle X-ray scattering, and nanoparticle tracking analysis. HIP protein vesicles have the potential to broaden the use of intracellular proteins as therapeutics for various diseases and extend protein vesicles to deliver other biomacromolecules, as the strategy developed here resulted in the first cytosolic protein cargo delivery using protein vesicles.

Stem cells have been widely used to produce artificial bone grafts. Nonetheless, the variability in the degree of stem cell differentiation is an inherent drawback of artificial graft development and requires robust evaluation tools that can certify the quality of stem cell-based products and avoid source-tissue-related and patient-specific variability in outcomes. Omics analyses have been utilised for the evaluation of stem cell attributes in all stages of stem cell biomanufacturing. Herein, metabolomics in combination with machine learning was utilised for the benchmarking of osteogenic differentiation quality in 2D and 3D cultures. Metabolomics analysis was performed with the use of gas chromatography–mass spectrometry (GC-MS). A set of 11 metabolites was used to train an XGboost model which achieved excellent performance in distinguishing between differentiated and undifferentiated umbilical cord blood mesenchymal stem cells (UCB MSCs). The model was benchmarked against samples not present in the training set, being able to efficiently capture osteogenesis in 3D UCB MSC cultures with an area under the curve (AUC) of 82.6%. On the contrary, the model did not capture any differentiation in Wharton’s Jelly MSC samples, which are well-known underperformers in osteogenic differentiation (AUC of 56.2%). Mineralisation was significantly correlated with the levels of fumarate, glycerol, and myo-inositol, the four metabolites found most important for model performance (R² = 0.89, R² = 0.94, and R² = 0.96, and p = 0.016, p = 0.0059, and p = 0.0022, respectively). In conclusion, our results indicate that metabolomics in combination with machine learning can be used for the development of reliable potency assays for the evaluation of Advanced Therapy Medicinal Products.

Background. Chimeric antigen receptor (CAR) T cell therapy, a “living drug” immunotherapy, harnesses the power of T‐cells from a patient (autologous) or healthy donor (allogeneic) to target and kill cancer cells and has shown unprecedented outcomes in patients with relapsed and refractory malignancies. Treatment with CAR‐T cells requires the application of unique skillsets in recognised specialist centres for successful outcomes and requires management by the multidisciplinary team incorporating the specialist pharmacist. Method. A multimodal research strategy was employed for this literature review whereby PubMed, Google Scholar, Embase, Stella Library Search, EMA website, and EBMT website were sources of information. The search was limited from 2020 onwards with key terms referring to CAR‐T cell therapy. Results and Discussion. There are six CAR‐T cell products currently approved by the European Medicines Agency (EMA) and Food and Drug Administration (FDA) which target haematological malignancies with abundant clinical trials underway exploring new and improved CAR designs and antigen targets. As CAR‐T cell therapy is an advanced therapy medicinal product (ATMP), there is need for an extensive regulatory framework underpinning its safety and efficacy. The clinical pharmacist plays an integral role in the provision of safe and effective CAR‐T cell therapy including governance, operational and clinical aspects of treatment. Pharmacists may also be involved through provision of “Qualified Person” (QP) expertise in clinical trials and for release within hospitals under certain circumstances. There is a need for harmonised and accessible guidance on the clinical delivery of ATMPs such as CAR‐T cells, with fully delineated responsibilities of pharmacists involving the oversight and supervision of CAR‐T cell treatment. Conclusion. There is an unmet need to provide suitable and applicable literature for clinical pharmacists who are involved in the delivery of CAR‐T cells. We have provided an overview of T‐cell biology and an explanation of CAR‐T cell design and the biomanufacturing process. We reviewed the complex and multifaceted treatment cycle requiring considerable logistics, and described the involvement of the clinical pharmacist in each part of this cycle from patient selection to postinfusion care. Finally, we look to the challenges and future opportunities that will require the involvement of the clinical pharmacist.

Background Bone tissue engineering emerged as a practical approach to tackle the prosthetic industry limitations. Merging aspects from developmental biology, engineering and medicine with the aim to produce fully-functional bone tissue. Mesenchymal stem cells (MSCs) harbor the capability of self-renewal and specific lineage differentiation. Herein lies their potential for bone tissue engineering. Among MSCs, human dental pulp stem cells (hDPSCs) lodge higher proliferation rate, shorter doubling times, lower cellular senescence, and enhanced osteogenesis than hBM-SCs. In addition, these cells have ease in access and a subtle extraction procedure. Thus, harbouring fewer moral concerns than most MSCs available and embodying a promising cell source for BTE therapies able to replace hBM-MSCs. Interestingly, their study has been limited. Conversely, there is a need for their further study to harness their BTE true value, with special emphasis in the design of bioprocesses able to produce viable, homogenous bone constructs in a clinical scale. Methods Here, we study the in vitro osteogenic differentiation of hDPSCs encapsulated in alginate hydrogels under suspended culture in a novel and scalable perfusion bioreactor, establishing culture conditions; and compare it with three-dimensional (3D) static and fed-batch culture. Results hDPSC-based bone-like constructs produced in the novel system performed above the compared culture strategies, displaying higher alkaline phosphatase activity, more homogeneous, denser and functional bone constructs. In addition, cell constructs produced by the in-house designed system were richer in mature osteoblasts. Conclusion This study reports the development of a novel bioprocess able to produce hDPSC-alginate-based bone-like constructs to be used as bone fillers, while providing new insights into hDPSCs therapeutic potential and a system able to be transferred from the laboratory bench into medical facilities.

Background: As stem cells are considered a promising cell source for tissue engineering, many culture strategies have been extensively studied to generate in vitro stem cell-based tissue constructs. However, most approaches using conventional tissue culture plates are limited by the lack of biological relevance in stem cell microenvironments required for neotissue formation. In this study, a novel perfusion rotating wall vessel (RWV) bioreactor was developed for mass-production of stem cell-based 3D tissue constructs. Methods: An automated RWV bioreactor was fabricated, which is capable of controlling continuous medium perfusion, highly efficient gas exchange with surrounding air, as well as low-intensity pulsed ultrasound (LIPUS) stimulation. Embryonic stem cells encapsulated in alginate/gelatin hydrogel were cultured in the osteogenic medium by using our bioreactor system. Cellular viability, growth kinetics, and osteogenesis/mineralization were thoroughly evaluated, and culture media were profiled at real time. The in vivo efficacy was examined by a rabbit cranial defect model. Results: Our bioreactor successfully maintained the optimal culture environments for stem cell proliferation, osteogenic differentiation, and mineralized tissue formation during the culture period. The mineralized tissue constructs produced by our bioreactor demonstrated higher void filling efficacy in the large bone defects compared to the group implanted with hydrogel beads only. In addition, the LIPUS modules mounted on our bioreactor successfully reached higher mineralization of the tissue constructs compared to the groups without LIPUS stimulation. Conclusion: This study suggests an effective biomanufacturing strategy for mass-production of implantable mineralized tissue constructs from stem cells that could be applicable to future clinical practice.

Cellular microenvironments provide stimuli, including paracrine and autocrine growth factors and physicochemical cues, which support efficient in vivo cell production unmatched by current in vitro biomanufacturing platforms. While three-dimensional (3D) culture systems aim to recapitulate niche architecture and function of the target tissue/organ, they are limited in accessing spatiotemporal information to evaluate and optimize in situ cell/tissue process development. Herein, a mathematical modeling framework is parameterized by single-cell phenotypic imaging and multiplexed biochemical assays to simulate the nonuniform tissue distribution of nutrients/metabolites and growth factors in cell niche environments. This model is applied to a bone marrow mimicry 3D perfusion bioreactor containing dense stromal and hematopoietic tissue with limited red blood cell (RBC) egress. The model characterized an imbalance between endogenous cytokine production and nutrient starvation within the microenvironmental niches and recommended increased cell inoculum density and enhanced medium exchange, guiding the development of a miniaturized prototype bioreactor. The second-generation prototype improved the distribution of nutrients and growth factors and supported a 50-fold increase in RBC production efficiency. This image-informed bioprocess modeling framework leverages spatiotemporal niche information to enhance biochemical factor utilization and improve cell manufacturing in 3D systems. Impact statement: Three-dimensional (3D) culture systems are becoming increasingly important because they recapitulate the architecture and, consequently, physiological function of the target tissue/organ. Design and optimization of these 3D biomanufacturing platforms require evaluation of in situ spatiotemporal information. We have developed an integrated experimental–computational framework that captures the spatiotemporal distribution of cells, nutrients, and cytokines within a marrow biomimicry perfusion bioreactor. The model simulated biochemical factor utilization and guided the design of an improved second-generation bioreactor that achieved 50-fold increase in RBC production with improved cost efficiency. Such a modeling framework provides an essential platform for the optimization of 3D biomanufacturing systems.

Engineering biological processes has become a standard approach to produce various commercially valuable chemicals, therapeutics, and biomaterials. Among these products, bacterial cellulose represents major advances to biomedical and healthcare applications. In comparison to properties of plant cellulose, bacterial cellulose (BC) shows distinctive characteristics such as a high purity, high water retention, and biocompatibility. However, low product yield and extensive cultivation times have been the main challenges in the large-scale production of BC. For decades, studies focused on optimization of cellulose production through modification of culturing strategies and conditions. With an increasing demand for BC, researchers are now exploring to improve BC production and functionality at different categories: genetic, bioprocess, and product levels as well as model driven approaches targeting each of these categories. This comprehensive review discusses the progress in BC platforms categorizing the most recent advancements under different research focuses and provides systematic understanding of the progress in BC biosynthesis. The aim of this review is to present the potential of ‘modern genetic engineering tools’ and ‘model-driven approaches’ on improving the yield of BC, altering the properties, and adding new functionality. We also provide insights for the future perspectives and potential approaches to promote BC use in biomedical applications.

PIP3, a bioactive lipid upstream of the PI3K/AKT/mTOR pathway, decreased protein translated from nanoparticle-delivered mRNA.

Data integration to model-based description of biological systems incorporating gene dynamics improves the performance of microbial systems. Bioprocess performance, typically predicted using empirical Monod-type models, is essential for a sustainable bioeconomy. To replace empirical models, we updated a hybrid gene regulatory network-growth kinetic model, predicting aromatic pollutants degradation and biomass growth in Pseudomonas putida mt-2. We modeled a complex biological system including extensive information to understand the role of the regulatory elements in toluene biodegradation and biomass growth. The updated model exhibited extra complications such as the existence of oscillations and discontinuities. As parameter estimation of complex biological models remains a key challenge, we used the updated model to present a dual-parameter identification approach (the ‘dual approach’) combining two independent methodologies. Approach I handled the complexity by incorporation of demonstrated biological knowledge in the model-development process and combination of global sensitivity analysis and optimisation. Approach II complemented Approach I handling multimodality, ill-conditioning and overfitting through regularisation estimation, global optimisation, and identifiability analysis. To systematically quantify the biological system, we used a vast amount of high-quality time-course data. The dual approach resulted in an accurately calibrated kinetic model (NRMSE: 0.17055) efficiently handling the additional model complexity. We tested model validation using three independent experimental data sets, achieving greater predictive power (NRMSE: 0.18776) than the individual approaches (NRMSE I: 0.25322, II: 0.25227) and increasing model robustness. These results demonstrated data-driven predictive modeling potentially leading to bioprocess’ model-based control and optimisation.