Miniaturization of hiPSC-derived 3D neural cultures in stirred-tank bioreactors for parallelized preclinical assessment of rAAV.

Introduction: Engineered 3D models employing human induced pluripotent stem cell (hiPSC) derivatives have the potential to recapitulate the cell diversity and structure found in the human central nervous system (CNS). Therefore, these complex cellular systems offer promising human models to address the safety and potency of advanced therapy medicinal products (ATMPs), such as gene therapies. Specifically, recombinant adeno-associated viruses (rAAVs) are currently considered highly attractive for CNS gene therapy due to their broad tropism, low toxicity, and moderate immunogenicity. To accelerate the clinical translation of rAAVs, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. The integration of hiPSC-derived CNS models in rAAV development will require, amongst other factors, robust, small-scale, high-throughput culture platforms that can feed the preclinical trials. Methods: Herein, we pioneer the miniaturization and parallelization of a 200 mL stirred-tank bioreactor-based 3D brain cell culture derived from hiPSCs. We demonstrate the applicability of the automated miniaturized Ambr® 15 Cell Culture system for the maintenance of hiPSC-derived neurospheroids (iNSpheroids), composed of neuronal and glial cells. Critical process parameters were optimized, namely, cell density and agitation mode. Results: Under optimized conditions, stable iNSpheroid cultures were attained in the microbioreactors for at least 15 days, with high cell viability and astrocytic and neuronal phenotype maintenance. This culture setup allowed the parallelization of different rAAVs, in different multiplicity of infections (MOIs), to address rAAV-host interactions at a preclinical scale. The iNSpheroids were exposed to rAAV2- and rAAV9-eGFP in the microbioreactors. Transgene expression was detected 14 days post-transduction, revealing different astrocyte/neuron tropism of the two serotypes. Discussion: We advocate that the iNSpheroid cultures in miniaturized bioreactors are reliable and reproducible screening tools for addressing rAAV transduction and tropism, compatible with preclinical demands.

A roadmap towards manufacturing extracellular vesicles for cardiac repair.

For the past two decades researchers have linked extracellular vesicle (EV)-mediated mechanisms to various physiological and pathological processes in the heart, such as immune response regulation, fibrosis, angiogenesis, and the survival and growth of cardiomyocytes. Although use of EVs has gathered momentum in the cardiac field, several obstacles in both upstream and downstream processes during EV manufacture need to be addressed before clinical success can be achieved. Low EV yields obtained in small-scale cultures deter clinical translation, as mass production is a prerequisite to meet therapeutic doses. Moreover, standardizing EV manufacture is critical given the inherent heterogeneity of EVs and the constraints of current isolation techniques. In this review, we discuss the critical steps for the large-scale manufacturing of high-potency EVs for cardiac therapies.

ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming.

Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom-polyclonal antibodies isolated from the plasma of hyperimmunised animals-which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability.

Experimental validation of a rapid staining method and digital cytology for cervical and anal cancer / Validação experimental de um método de coloração rápida e citologia digital para cancro ginecológico e anal

ABSTRACT Cytology is used as detection and screening method of malignant and pre-malignant lesions showing their potential since the original works of Papanicolaou. The cytological smears are usually stained with the Pap staining, although this method is time consuming and requires different reagents. The aim of this study is to assess the quality of an original Blue staining in exfoliative smears comparing it with the standard Papanicolaou staining. The new Blue staining allows staining gynecological cytology with high quality standards at reduced cost and time when compared to the Papanicolaou method.

RESUMO A citologia é utilizada como método de detecção e rastreio de lesões malignas e pré-malignas e mostra seu potencial desde os trabalhos originais de Papanicolaou. Geralmente, os esfregaços citológicos são corados com a coloração de Papanicolaou, apesar desse método exigir muito tempo e vários reagentes. O objetivo deste estudo é avaliar a qualidade de uma coloração original Blue Stain em esfregaços esfoliativos comparando-a com a coloração standard de Papanicolaou. O novo método de coloração Blue Stain permite corar citologias ginecológicas com elevados padrões de qualidade a um custo e tempo reduzidos quando comparado com o método de Papanicolaou.

Improvement of recombinant protein production by an anti-apoptotic protein from hemolymphof Lonomia obliqua

Apoptosis is a major problem in animalcell culture during production of biopharmaceuticals, such as recombinant proteins or viral particles. In the present work baculovirus-insect cell expression system (BEVS/IC) is used as model to produce rotaviruslike-particles, composed by three layers of three different viral proteins (VP2, VP6 and VP7). In this model baculovirus infection also induces host celldeath. Herein a new strategy to enhance cell life span and to increase recombinant rotavirus protein productionof BEVS/IC system was developed. This strategy relies on hemolymph from Lonomia oblique (total extracts or a semi-purified fraction) medium supplementation. The total extract and a purified fraction from hemolymph of Lonomia obliqua were able toprotect Sf-9 cell culture against apoptosis triggered by oxidative stress (using the pro-oxidant agents tert butylhydroperoxide and hydrogen peroxide) and by baculovirus infection. Furthermore, hemolymph enhance final recombinant protein production, as it was observed by the increased amounts of VP6 and VP7, which were measured by the semi-quantitative western blot method. In conclusion, hemolymph medium supplementation can be a promising strategy to improve cell viability and productivity of recombinantprotein in BEVS/IC system.