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A three-dimensional platinum (Pt) microelectrode array embedded on a micromachined silicon (Si) substrate (porosity of 13%, via hole diameter of 40 [[micro]meter]) has been developed. Electrodes are 35-[[micro]meter] wide and 20-[[micro]meter] high, spaced 200 [[micro]meter] apart and arranged in an elliptic geometry. Integrated within a microperfusion chamber, the devices were used for stimulation and recording experiments of hippocampal slice cultures over a period of several days. Index Terms - Electrophysiology, microelectrode array, organotypic cultures, perforated substrate.

Traditional microelectrode arrays (MEAs) are limited to measuring electrophysiological activity in two dimensions, failing to capture the complexity of three-dimensional (3D) tissues such as neural organoids and spheroids. Here, we introduce a flower-shaped MEA (e-Flower) that can envelop submillimeter brain spheroids following actuation by the sole addition of the cell culture medium. Inspired by soft microgrippers, its actuation mechanism leverages the swelling properties of a polyacrylic acid hydrogel grafted to a polyimide substrate hosting the electrical interconnects. Compatible with standard electrophysiology recording systems, the e-Flower does not require additional equipment or solvents and is ready to use with preformed 3D tissues. We designed an e-Flower achieving a curvature as low as 300 micrometers within minutes, a value tunable by the choice of reswelling media and hydrogel cross-linker concentration. Furthermore, we demonstrate the ability of the e-Flower to detect spontaneous neural activity across the spheroid surface, demonstrating its potential for comprehensive neural signal recording.

Engineered 3D neural tissues made of neurons and glial cells derived from human induced pluripotent stem cells (hiPSC) are among the most promising tools in drug discovery and neurotoxicology. They represent a cheaper, faster, and more ethical alternative to in vivo animal testing that will likely close the gap between in vitro animal models and human clinical trials. Micro-Electrode Array (MEA) technology is known to provide an assessment of compound effects on neural 2D cell cultures and acute tissue preparations by real-time, non-invasive, and long-lasting electrophysiological monitoring of spontaneous and evoked neuronal activity. Nevertheless, the use of engineered 3D neural tissues in combination with MEA biochips still involves series of constraints, such as drastically limited diffusion of oxygen and nutrients within tissues mainly due to the lack of vascularization. Therefore, 3D neural tissues are extremely sensitive to experimental conditions and require an adequately designed interface that provides optimal tissue survival conditions. A well-suited technique to overcome this issue is the combination of the Air-Liquid Interface (ALI) tissue culture method with the MEA technology. We have developed a full 3D neural tissue culture process and a data acquisition system composed of high-end electronics and novel MEA biochips based on porous, flexible, thin-film membranes integrating recording electrodes, named as "Strip-MEA," to allow the maintenance of an ALI around the 3D neural tissues. The main motivation of the porous MEA biochips development was the possibility to monitor and to study the electrical activity of 3D neural tissues under different recording configurations, (i) the Strip-MEA can be placed below a tissue, (ii) or by taking advantage of the ALI, be directly placed on top of the tissue, or finally, (iii) it can be embedded into a larger neural tissue generated by the fusion of two (or more) tissues placed on both sides of the Strip-MEA allowing the recording from its inner part. This paper presents the recording and analyses of spontaneous activity from the three positioning configurations of the Strip-MEAs. Obtained results are discussed with the perspective of developing in vitro models of brain diseases and/or impairment of neural network functioning., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Stoppini, Heuschkel, Loussert-Fonta, Gomez Baisac and Roux.)

Traumatic brain injury (TBI) is caused by a wide range of physical events and can induce an even larger spectrum of short- to long-term pathophysiologies. Neuroscientists have relied on animal models to understand the relationship between mechanical damages and functional alterations of neural cells. These in vivo and animal-based in vitro models represent important approaches to mimic traumas on whole brains or organized brain structures but are not fully representative of pathologies occurring after traumas on human brain parenchyma. To overcome these limitations and to establish a more accurate and comprehensive model of human TBI, we engineered an in vitro platform to induce injuries via the controlled projection of a small drop of liquid onto a 3D neural tissue engineered from human iPS cells. With this platform, biological mechanisms involved in neural cellular injury are recorded through electrophysiology measurements, quantification of biomarkers released, and two imaging methods [confocal laser scanning microscope (CLSM) and optical projection tomography (OPT)]. The results showed drastic changes in tissue electrophysiological activities and significant releases of glial and neuronal biomarkers. Tissue imaging allowed us to reconstruct the injured area spatially in 3D after staining it with specific nuclear dyes and to determine TBI resulting in cell death. In future experiments, we seek to monitor the effects of TBI-induced injuries over a prolonged time and at a higher temporal resolution to better understand the subtleties of the biomarker release kinetics and the cell recovery phases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Loussert-Fonta, Stoppini, Neuenschwander, Righini, Prim, Schmidt, Heuschkel, Gomez Baisac, Jovic´, Pfeifer, Extermann and Roux.)

Importance: Convalescent plasma (CP) has been generally unsuccessful in preventing worsening of respiratory failure or death in hospitalized patients with COVID-19 pneumonia., Objective: To evaluate the efficacy of CP plus standard therapy (ST) vs ST alone in preventing worsening respiratory failure or death in patients with COVID-19 pneumonia., Design, Setting, and Participants: This prospective, open-label, randomized clinical trial enrolled (1:1 ratio) hospitalized patients with COVID-19 pneumonia to receive CP plus ST or ST alone between July 15 and December 8, 2020, at 27 clinical sites in Italy. Hospitalized adults with COVID-19 pneumonia and a partial pressure of oxygen-to-fraction of inspired oxygen (Pao2/Fio2) ratio between 350 and 200 mm Hg were eligible., Interventions: Patients in the experimental group received intravenous high-titer CP (≥1:160, by microneutralization test) plus ST. The volume of infused CP was 200 mL given from 1 to a maximum of 3 infusions. Patients in the control group received ST, represented by remdesivir, glucocorticoids, and low-molecular weight heparin, according to the Agenzia Italiana del Farmaco recommendations., Main Outcomes and Measures: The primary outcome was a composite of worsening respiratory failure (Pao2/Fio2 ratio <150 mm Hg) or death within 30 days from randomization., Results: Of the 487 randomized patients (241 to CP plus ST; 246 to ST alone), 312 (64.1%) were men; the median (IQR) age was 64 (54.0-74.0) years. The modified intention-to-treat population included 473 patients. The primary end point occurred in 59 of 231 patients (25.5%) treated with CP and ST and in 67 of 239 patients (28.0%) who received ST (odds ratio, 0.88; 95% CI, 0.59-1.33; P = .54). Adverse events occurred more frequently in the CP group (12 of 241 [5.0%]) compared with the control group (4 of 246 [1.6%]; P = .04)., Conclusions and Relevance: In patients with moderate to severe COVID-19 pneumonia, high-titer anti-SARS-CoV-2 CP did not reduce the progression to severe respiratory failure or death within 30 days., Trial Registration: ClinicalTrials.gov Identifier: NCT04716556.

In this paper we present SpikeOnChip, a custom embedded platform for neuronal activity recording and online analysis. The SpikeOnChip platform was developed in the context of automated drug testing and toxicology assessments on neural tissue made from human induced pluripotent stem cells. The system was developed with the following goals: to be small, autonomous and low power, to handle micro-electrode arrays with up to 256 electrodes, to reduce the amount of data generated from the recording, to be able to do computation during acquisition, and to be customizable. This led to the choice of a Field Programmable Gate Array System-On-Chip platform. This paper focuses on the embedded system for acquisition and processing with key features being the ability to record electrophysiological signals from multiple electrodes, detect biological activity on all channels online for recording, and do frequency domain spectral energy analysis online on all channels during acquisition. Development methodologies are also presented. The platform is finally illustrated in a concrete experiment with bicuculline being administered to grown human neural tissue through microfluidics, resulting in measurable effects in the spike recordings and activity. The presented platform provides a valuable new experimental instrument that can be further extended thanks to the programmable hardware and software.

Objective: Progressive multifocal leukoencephalopathy (PML) is still burdened by high mortality in a subset of patients, such as those affected by hematological malignancies. The aim of this study was to analyze the safety and carry out preliminary evaluation of the efficacy of polyomavirus JC (JCPyV)-specific T cell therapy in a cohort of hematological patients with PML., Methods: Between 2014 and 2019, 9 patients with a diagnosis of "definite PML" according to the 2013 consensus who were showing progressive clinical deterioration received JCPyV-specific T cells. Cell lines were expanded from autologous or allogenic peripheral blood mononuclear cells by stimulation with JCPyV antigen-derived peptides., Results: None of the patients experienced treatment-related adverse events. In the evaluable patients, an increase in the frequency of circulating JCPyV-specific lymphocytes was observed, with a decrease or clearance of JCPyV viral load in cerebrospinal fluid. In responsive patients, transient appearance of punctate areas of contrast enhancement within, or close to, PML lesions was observed, which was interpreted as a sign of immune control and which regressed spontaneously without the need for steroid treatment. Six of 9 patients achieved PML control, with 5 alive and in good clinical condition at their last follow-up., Interpretation: Among other novel treatments, T cell therapy is emerging as a viable treatment option in patients with PML, particularly for those not amenable to restoration of specific immunity. Neurologists should be encouraged to refer PML patients to specialized centers to allow access to this treatment strategy. ANN NEUROL 2021;89:769-779., (© 2021 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Minibrain is a 3D brain in vitro spheroid model, composed of a mixed population of neurons and glial cells, generated from human iPSC derived neural stem cells. Despite the advances in human 3D in vitro models such as aggregates, spheroids and organoids, there is a lack of labeling and imaging methodologies to characterize these models. In this study, we present a step-by-step methodology to generate human minibrain nurseries and novel strategies to subsequently label projection neurons, perform immunohistochemistry and 3D imaging of the minibrains at large multiplexable scales. To visualize projection neurons, we adapt viral transduction and to visualize the organization of cell types we implement immunohistochemistry. To facilitate 3D imaging of minibrains, we present here pipelines and accessories for one step mounting and clearing suitable for confocal microscopy. The pipelines are specifically designed in such a way that the assays can be multiplexed with ease for large-scale screenings using minibrains and other organoid models. Using the pipeline, we present (i) dendrite morphometric properties obtained from 3D neuron morphology reconstructions, (ii) diversity in neuron morphology, and (iii) quantified distribution of progenitors and POU3F2 positive neurons in human minibrains., Competing Interests: SG was employed by HEPIA and is curently employed by company ARMIA Lifesciences PVT Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Govindan, Batti, Osterop, Stoppini and Roux.)

Post-transplant lymphoproliferative disorders (PTLDs) are life-threatening complications of iatrogenic immune impairment after allogeneic hematopoietic stem cell transplantation (HSCT). In the pediatric setting, the majority of PTLDs are related to the Epstein-Barr virus (EBV) infection, and present as B-cell lymphoproliferations. Although considered rare events, PTLDs have been increasingly observed with the widening application of HSCT from alternative sources, including cord blood and HLA-haploidentical stem cell grafts, and the use of novel agents for the prevention and treatment of rejection and graft-vs.-host disease. The higher frequency initially paralleled a poor outcome, due to limited therapeutic options, and scarcity of controlled trials in a rare disease context. In the last 2 decades, insight into the relationship between EBV and the immune system, and advances in early diagnosis, monitoring and treatment have changed the approach to the management of PTLDs after HSCT, and significantly ameliorated the prognosis. In this review, we summarize literature on the impact of combined viro-immunologic assessment on PTLD management, describe the various strategies for PTLD prevention and preemptive/curative treatment, and discuss the potential of novel immune-based therapies in the containment of this malignant complication., (Copyright © 2020 Compagno, Basso, Panigari, Bagnarino, Stoppini, Maiello, Mina, Zelini, Perotti, Baldanti, Zecca and Comoli.)

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.