A Tool for Accurate Stoichiometric Composition of Cryopreservative Media for Fetal and Induced Pluripotent Stem Cell-Derived Human Neural Stem Cells.

Fetal human neural stem cells (fhNSC) are of considerable interest as potential regenerative therapies for neuronal or glial degeneration or destruction resulting from genetic abnormalities, disease, or injury. Realization of this potential requires securing a supply of cells sufficient to meet the needs of transplantation, which are often tens to hundreds of millions of cells per dose. This challenge necessitates the establishment of safe and efficient cell banking protocols. Cryopreservation, involving the slow freezing or vitrification of cells, enables storage of fhNSC for prolonged periods, while maintaining their viability and multipotency required for clinical use. To optimize cryopreservation of fhNSC, attention has become focused on the composition of the medium used to effect cryopreservation by slow freezing/vitrification-i.e., the cryopreservative medium. The cryopreservative medium is typically specified as a dilution of a concentrated cryoprotectant, such as dimethylsulfoxide or glycerol, in cell culture medium that is often combined with serum or another source of necessary growth factors. The present work is devoted to a computational tool for determining the composition of a cryopreservative medium that can be combined with dissociated fhNSC resuspended in a certain volume of culture medium to achieve the criterion of stoichiometric dilution of cryoprotectant favorable to cell viability in the final mixture of cryopreservative medium and cells. © 2021 Wiley Periodicals LLC. Basic Protocol: Culture and passage of fhNSC, counting of enzymatically dissociated fhNSC, and quantitative formulation of cryomedium Alternate Protocol: Procedure when cell medium is not added to the cryomedium.

Generation of Complete Multi-Cell Type Lung Organoids From Human Embryonic and Patient-Specific Induced Pluripotent Stem Cells for Infectious Disease Modeling and Therapeutics Validation.

The normal development of the pulmonary system is critical to transitioning from placental-dependent fetal life to alveolar-dependent newborn life. Human lung development and disease have been difficult to study due to the lack of an in vitro model system containing cells from the large airways and distal alveolus. This article describes a system that allows human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to differentiate and form three-dimensional (3D) structures that emulate the development, cytoarchitecture, and function of the lung ("organoids"), containing epithelial and mesenchymal cell populations, and including the production of surfactant and presence of ciliated cells. The organoids can also be invested with mesoderm derivatives, differentiated from the same human pluripotent stem cells, such as alveolar macrophages and vasculature. Such lung organoids may be used to study the impact of environmental modifiers and perturbagens (toxins, microbial or viral pathogens, alterations in microbiome) or the efficacy and safety of drugs, biologics, and gene transfer. © 2020 Wiley Periodicals LLC. Basic Protocol: hESC/hiPSC dissection, definitive endoderm formation, and lung progenitor cell induction.

A Biomarker for Predicting Responsiveness to Stem Cell Therapy Based on Mechanism-of-Action: Evidence from Cerebral Injury.

To date, no stem cell therapy has been directed to specific recipients-and, conversely, withheld from others-based on a clinical or molecular profile congruent with that cell's therapeutic mechanism-of-action (MOA) for that condition. We address this challenge preclinically with a prototypical scenario: human neural stem cells (hNSCs) against perinatal/neonatal cerebral hypoxic-ischemic injury (HII). We demonstrate that a clinically translatable magnetic resonance imaging (MRI) algorithm, hierarchical region splitting, provides a rigorous, expeditious, prospective, noninvasive "biomarker" for identifying subjects with lesions bearing a molecular profile indicative of responsiveness to hNSCs' neuroprotective MOA. Implanted hNSCs improve lesional, motor, and/or cognitive outcomes only when there is an MRI-measurable penumbra that can be forestalled from evolving into necrotic core; the core never improves. Unlike the core, a penumbra is characterized by a molecular profile associated with salvageability. Hence, only lesions characterized by penumbral > core volumes should be treated with cells, making such measurements arguably a regenerative medicine selection biomarker.

Cyclic olefin polymers: innovative materials for high-density multiwell plates.

Extension of ultra-high-throughput experiment (UHTE) approaches to new assay methodologies is often limited by compromised data quality when samples are miniaturized. Overcoming this challenge requires attending to all components of an automated laboratory system contributing to assay variability. A key but often neglected source is the high-density multiwell platform or microtiter plate. Materials from which plates are fabricated may degrade or otherwise compromise an assay through a variety of sources, including structural weakness, distortion of optical signals, and chemical contamination. Cyclic olefin polymer (COP) resins (CAS Registry Number 26007-43-2, inclusive of polymers and copolymers, sometimes referred to as cyclo-olefin polymers or copolymers) are receiving attention for their structural strength, optical clarity, and biocompatibility. The physical and chemical properties of COP are reviewed for their ramifications on the performance of high-density multiwell plates. Cells known to be difficult to culture in standard plasticware thrive in miniaturized COP wells. In addition, cell-based assays whose data deteriorated when miniaturized in standard plastic reveal a robust recovery of data quality when miniaturized in COP. It is hoped that the material qualities and advantages of COP become better appreciated among the screening and biological communities.

Piezo- and solenoid valve-based liquid dispensing for miniaturized assays.

Miniaturization of biological assays requires dispensing liquids in the submicroliter range of volumes. Accuracy and reproducibility of dispensing this range depend on both the dispenser and the receptacle in which the assay is constructed. Miniaturization technologies developed by Aurora Discovery, Inc. (San Diego, CA) include high-density multiwell plates for assay samples and reagent storage, as well as piezo-based and solenoid valve-based liquid dispensers. Some basic principles of small-volume dispensing by jetting are described to provide context for dispenser design and function. Performance of the latest instruments incorporating these dispensing devices is presented.

Controversies in ASSAY and drug development technologies: a focus on assessing irreproducibility.

Has the impact of irreproducibility on the discovery and development of drugs, as with global warming, metaphorically speaking, crept up on us as we slept? Or is the problem more an issue of heightened awareness? We currently find ourselves in a time when the impact of irreproducibility can easily be amplified by the combinatorial effect of our increasing reliance on advanced technologies and unrealistic expectations of how scientific truths unfold. How and why we got here is a topic that has been written on extensively (1-3) and is probably as complex as any other problem, given the dependence of science today on so many external forces. Through a series of questions, we asked members of our editorial board their opinions on scientific irreproducibility. They chose to answer the same questions from different levels, indicating the depth of the problem and perhaps where they each believe change for the better needs to begin. My thanks to the participants.