Recombinant AAV Production.

The insect cell-baculovirus expression vector (IC-BEV) platform has enabled small research-scale and large commercial-scale production of recombinant proteins and therapeutic biologics including recombinant adeno-associated virus (rAAV)-based gene delivery vectors. The wide use of this platform is comparable with other mammalian cell line-based platforms due to its simplicity, high-yield, comparable quality attributes, and robust bioprocessing features. In this chapter, we describe a rAAV production protocol employing one of the recent modifications of the One-Bac platform that consists of a stable transformed Sf9 cell line carrying AAV Rep2/Cap5 genes that are induced upon infection with a single recombinant baculovirus expression vector harboring the transgene of interest (rAAV genome). The overall protocol consists of essential steps including rBEV working stock preparation, rAAV production, and centrifugation-based clarification of cell culture lysate. The same protocol can also be applied for rAAV vector production using traditional Three-Bac, Two-Bac, and Mono-Bac platforms without requiring significant changes.

Recombinant AAV Purification.

Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.

Functional Baculovirus Particle Quantification via Plaque Assay.

Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.

A Pillar/Perfusion Plate Enhances Cell Growth, Reproducibility, Throughput, and User Friendliness in Dynamic 3D Cell Culture.

Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from a necrotic core due to limited nutrient and oxygen diffusion and waste removal and have a limited in vivo-like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids were loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow velocity was maintained within perfusion wells and the pillar plate was separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in a dynamic 3D cell culture.

Dynamic culture of cerebral organoids using a pillar/perfusion plate for the assessment of developmental neurotoxicity.

Despite the potential toxicity of commercial chemicals to the development of the nervous system (known as developmental neurotoxicity or DNT), conventional in vitro cell models have primarily been employed for the assessment of acute neuronal toxicity. On the other hand, animal models used for the assessment of DNT are not physiologically relevant due to the heterogenic difference between humans and animals. In addition, animal models are low-throughput, time-consuming, expensive, and ethically questionable. Recently, human brain organoids have emerged as a promising alternative to assess the detrimental effects of chemicals on the developing brain. However, conventional organoid culture systems have several technical limitations including low throughput, lack of reproducibility, insufficient maturity of organoids, and the formation of the necrotic core due to limited diffusion of nutrients and oxygen. To address these issues and establish predictive DNT models, cerebral organoids were differentiated in a dynamic condition in a unique pillar/perfusion plate, which were exposed to test compounds to evaluate DNT potential. The pillar/perfusion plate facilitated uniform, dynamic culture of cerebral organoids with improved proliferation and maturity by rapid, bidirectional flow generated on a digital rocker. Day 9 cerebral organoids in the pillar/perfusion plate were exposed to ascorbic acid (DNT negative) and methylmercury (DNT positive) in a dynamic condition for 1 and 3 weeks, and changes in organoid morphology and neural gene expression were measured to determine DNT potential. As expected, ascorbic acid didn't induce any changes in organoid morphology and neural gene expression. However, exposure of day 9 cerebral organoids to methylmercury resulted in significant changes in organoid morphology and neural gene expression. Interestingly, methylmercury did not induce adverse changes in cerebral organoids in a static condition, thus highlighting the importance of dynamic organoid culture in DNT assessment.

Regenerative human liver organoids (HLOs) in a pillar/perfusion plate for hepatotoxicity assays.

Human liver organoids (HLOs) differentiated from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs) can recapitulate structure and function of human fetal liver tissues, thus, considered as a promising tissue model for liver diseases and predictive compound screening. Nonetheless, there are still several technical challenges to adopt HLOs in the drug discovery process, which include relatively long-term cell differentiation with multiple culture media (3 - 4 weeks) leading to batch-to-batch variation, short-term hepatic function after maturation (3 - 5 days), low assay throughput due to Matrigel dissociation and HLO transfer to a microtiter well plate, and insufficient maturity as compared to primary hepatocytes. To address these issues, expandable HLOs (Exp-HLOs) derived from human iPSCs were generated by optimizing differentiation protocols, which were rapidly printed on a 144-pillar plate with sidewalls and slits (144PillarPlate) and dynamically cultured for up to 20 days into differentiated HLOs (Diff-HLOs) in a 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for in situ organoid culture and analysis. Dynamically cultured Diff-HLOs were generated robustly and reproducibly in the pillar/perfusion plate with higher maturity as compared to those in statically cultured HLOs by differentiating Exp-HLOs for 10 days. In addition, Diff-HLOs in the pillar/perfusion plate were tested with acetaminophen and troglitazone for 3 days to assess drug-induced liver injury (DILI) and then incubated in an expansion medium for 10 days to evaluate the recovery of the liver from DILI. The assessment of liver regeneration post injury is critical to understand the mechanism of recovery and determine the threshold drug concentration beyond which there will be a sharp decrease in the liver's regenerative capacity. We envision that bioprinted Diff-HLOs in the pillar/perfusion plate could be used for high-throughput screening (HTS) of hepatotoxic compounds due to short-term differentiation of passage-able Exp-HLOs necessary, stable hepatic function after maturation, high reproducibility, and high throughput with capability of in situ organoid culture, testing, staining, imaging, and analysis.

Chronic dietary iron overload affects hepatic iron metabolism and cognitive behavior in Wistar rats.

BACKGROUND: Iron accumulation in organs affects iron metabolism, leading to deleterious effects on the body. Previously, it was studied that high dietary iron in various forms and concentrations influences iron metabolism, resulting in iron accumulation in the liver and spleen and cognitive impairment. However, the actual mechanism and impact of long-term exposure to high dietary iron remain unknown. As a result, we postulated that iron overload caused by chronic exposure to excessive dietary iron supplementation would play a role in iron dyshomeostasis and inflammation in the liver and brain of Wistar rats. METHODS: Animals were segregated into control, low iron (FAC-Ferric Ammonium Citrate 5000 ppm), and high iron dose group (FAC 20,000 ppm). The outcome of dietary iron overload on Wistar rats was evaluated in terms of body weight, biochemical markers, histological examination of liver and brain tissue, and cognitive-behavioral studies. Also, gene expression of rat brain tissue involving iron transporters Dmt1, TfR1, iron storage protein Fpn1, inflammatory markers Nf-kB, Tnf-α, Il-6, and hepcidin was performed. RESULTS: Our data indicate that excess iron supplementation for 30 weeks leads to decreased body weight, increased serum iron levels, and decreased RBC levels in iron fed Wistar rats. Morris water maze (MWM) studies after 30 weeks showed increased escape latency in the high iron dose group compared with the control group. Histological studies of the high iron dose group showed an iron accumulation in the liver and brain loss of cellular architecture, and cellular degeneration was observed. Excess iron treatment showed upregulation of the Dmt1 gene in iron metabolism and a remarkable increase in the Nf-kB gene in rat brain tissue. CONCLUSION: The results show chronic excess iron supplementation leads to iron accumulation in the liver, leading to inflammation in Wistar rats.

Critical Prandtl Number for Heat Transfer Enhancement in Rotating Convection.

Rotation, which stabilizes flow, can enhance the heat transfer in Rayleigh-Bénard convection (RBC) through Ekman pumping. In this Letter, we present the results of our direct numerical simulations of rotating RBC, providing a comprehensive analysis of this heat transfer enhancement relative to nonrotating RBC in the parameter space of Rayleigh number (Ra), Prandtl number (Pr), and Taylor number (Ta). We show that for a given Ra, there exists a critical Prandtl number (Pr_{cr}) below which no significant heat transfer enhancement occurs at any rotation rate, and an optimal Prandtl number (Pr_{opt}) at which maximum heat transfer enhancement occurs at an optimal rotation rate (Ta_{opt}). Notably, Pr_{cr}, Pr_{opt}, Ta_{opt}, and the maximum heat transfer enhancement all increase with increasing Ra. We also demonstrate a significant heat transfer enhancement up to Ra=2×10^{10} and predict that the enhancement would become even more pronounced at higher Ra, provided Pr is also increased commensurately.

Uniform cerebral organoid culture on a pillar plate by simple and reproducible spheroid transfer from an ultralow attachment well plate.

Human induced pluripotent stem cell (iPSC)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effectivein vitromodel for studying both normal brain development and disorders. However, current brain organoid culture methods face several challenges, including low throughput, high variability in organoid generation, and time-consuming, multiple transfer and encapsulation of cells in hydrogels throughout the culture. These limitations hinder the widespread application of brain organoids including high-throughput assessment of compounds in clinical and industrial lab settings. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies in an ultra-low attachment 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95%-100%. Using this approach, we robustly generated cerebral organoids on the pillar plate and demonstrated an intra-batch coefficient of variation below 9%-19% based on ATP-based cell viability and compound treatment. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowingin situorganoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.

Uniform cerebral organoid culture on a pillar plate by simple and reproducible spheroid transfer from an ultralow attachment well plate.

Human induced pluripotent stem cell (iPSCs)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effective in vitro model for studying both normal brain development and disorders. However, current brain organoid culture methods face several challenges, including low throughput, high variability in organoid generation, and time-consuming, multiple transfer and encapsulation of cells in hydrogels throughout the culture. These limitations hinder the widespread application of brain organoids including high-throughput assessment of compounds in clinical and industrial lab settings. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies (EBs) in an ultra-low attachment (ULA) 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95 - 100%. By differentiating the EBs on the pillar plate, we achieved robust generation of cerebral organoids with a coefficient of variation (CV) below 19%. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowing in situ organoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.

High-throughput assessment of metabolism-mediated neurotoxicity by combining 3D-cultured neural stem cells and liver cell spheroids.

Despite the fact that biotransformation in the liver plays an important role in the augmented toxicity and detoxification of chemicals, relatively little efforts have been made to incorporate biotransformation into in vitro neurotoxicity testing. Conventional in vitro systems for neurotoxicity tests lack the capability of investigating the qualitative and quantitative differences between parent chemicals and their metabolites in the human body. Therefore, there is a need for an in vitro toxicity screening system that can incorporate hepatic biotransformation of chemicals and predict the susceptibility of their metabolites to induce neurotoxicity. To address this need, we adopted 3D cultures of metabolically competent HepaRG cell line with ReNcell VM and established a high-throughput, metabolism-mediated neurotoxicity testing system. Briefly, spheroids of HepaRG cells were generated in an ultralow attachment (ULA) 384-well plate while 3D-cultured ReNcell VM was established on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds were added in the ULA 384-well plate with HepaRG spheroids and coupled with 3D-cultured ReNcell VM on the 384PillarPlate, which allowed us to generate metabolites in situ by HepaRG cells and test them against neural stem cells. We envision that this approach could be potentially adopted in pharmaceutical and chemical industries when high-throughput screening (HTS) is necessary to assess neurotoxicity of compounds and their metabolites.

A Pillar and Perfusion Plate Platform for Robust Human Organoid Culture and Analysis.

Human organoids have the potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo. This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by the lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, these challenges are overcome by engineering "microarray three-dimensional (3D) bioprinting" technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing, and encapsulation techniques are demonstrated on a pillar plate, which is coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels are differentiated into liver and intestine organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

Bolgiano-Obukhov spectrum and mixing efficiency in stably stratified turbulence.

In this paper, using a shell model, we simulate highly turbulent stably stratified flow for weak to moderate stratification at unitary Prandtl number. We investigate the energy spectra and fluxes of velocity and density fields. We observe that for moderate stratification, in the inertial range, the kinetic energy spectrum E_{u}(k) and the potential energy spectrum E_{b}(k) show dual scaling-Bolgiano-Obukhov scaling [E_{u}(k)∼k^{-11/5} and E_{b}(k)∼k^{-7/5}] for kk_{B}. In addition, we find that the mixing efficiency η_{mix} varies as η_{mix}∼Ri for weak stratification, whereas η_{mix}∼Ri^{1/3} for moderate stratification, where Ri is the Richardson number.

Phylogenetic characterization of canine distemper virus from stray dogs in Kathmandu Valley.

Canine distemper is a highly contagious, often fatal disease caused by canine distemper virus (CDV) in domestic dogs and wild carnivores. The virus has caused mass epidemics in both wild and captive carnivores of high conservation value such as tigers, lions and leopards. Hence, understanding and managing CDV outbreaks is particularly important in Nepal, which is home to many species of threatened wild carnivores including tigers, leopards, snow leopards, dholes and wolves, and also contains a large population of stray dogs. Previous studies have suggested that CDV may pose a threat to wild carnivores, but there have not been any studies characterizing the genetic strains of the virus circulating in Nepal's carnivores. We collected invasive and non-invasive biological samples from stray dogs in Kathmandu Valley and genetically characterized the strains of CDV in the dogs to belong to the Asia-5 lineage by using phylogenetic analysis. The same lineage also contained CDV strains sequenced from dogs, civets, red panda and lions in India. Based on our phylogenetic analysis, we think it is likely that CDV is maintained through sylvatic cycle among sympatric carnivores allowing the recurring spillovers and outbreaks. It is crucial to prevent the virus transmission from reservoir hosts to other species, especially threatened populations of large carnivores in Nepal. Hence, we recommend for regular surveillance of CDV targeting wild carnivores in addition to the domestic dogs.

A Pillar and Perfusion Plate Platform for Robust Human Organoid Culture and Analysis.

Human organoids have potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo . This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, we overcome these challenges by engineering "microarray three-dimensional (3D) bioprinting" technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing and encapsulation techniques were demonstrated on a pillar plate, which was coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels were differentiated into liver and intestine organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

A pillar/perfusion plate enhances cell growth, reproducibility, throughput, and user friendliness in dynamic 3D cell culture.

Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from the necrotic core due to limited nutrient and oxygen diffusion and waste removal and have limited in vivo-like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids have been loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow rates were maintained within perfusion wells, and the pillar plate could be separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in dynamic 3D cell culture.

A Study of Neuromyelitis Optica Spectrum Disorders (NMOSD): Disease Pattern Based on Antibody Status.

Introduction: Neuromyelitis optica (NMO) is a central demyelinating disorder, predominantly affecting the optic nerves and spinal cord and autoimmune basis. We aimed to analyze the clinical, laboratory, and imaging features associated with NMO spectrum disorders (NMOSD) according to the aquaporin 4 antibody (AQP4-Ab) serology status. Methods: The inclusion of the patients was based on the Wingerchuk criteria (2006) for NMO, known antibody status and has minimum 1-year follow-up. We analyzed and compared 46 patients with known antibody status. Results: AQP4-Ab positivity was 56.5%. The male to female ratio in the seropositive group was 1:7.7 and 1:1.2 in the seronegative group. The mean age of onset in seropositive patients was 36.8 years (vs 28.8 years in seronegative NMOSD patients). Clinical feature, cerebrospinal fluid (CSF) and magnetic resonance imaging (MRI) features were also different, but data from two subsets did not reach statistical significance. The relapse rate was higher in AQP4 positive NMOSD (84.6% vs 55% in the seronegative group). The recovery rate for AQP4 positive patients was poor (15%). Summary: We found differences in age, gender, and prognosis between the two groups. Antibody status may be a guiding factor in deciding the treatment approach during the first attack of NMOSD.

Establishment of ion channel and ABC transporter assays in 3D-cultured ReNcell VM on a 384-pillar plate for neurotoxicity potential.

Neurotoxicity potential of compounds by inhibition of ion channels and efflux transporters has been studied traditionally using two-dimensionally (2D) cultured cell lines such as CHO and HEK-293 overexpressing the protein of interest. However, these approaches are time consuming and do not recapitulate the activity of ion channels and efflux transporters indigenously expressed in neural stem cells (NSCs) in vivo. To overcome these issues, we established ion channel and transporter assays on a 384-pillar plate with three-dimensionally (3D) cultured ReNcell VM and demonstrated high-throughput measurement of ion channel and transporter activity. RNA sequencing analysis identified major ion channels and efflux transporters expressed in ReNcell VM, followed by validating 3D ReNcell-based ion channel and transporter assays with model compounds. Major ion channel activities were measured by specifically inhibiting potassium channels Kv 7.2 with XE-991 and Kv 4.3 with fluoxetine, and a calcium channel with 2-APB. Activities of major efflux transporters, MDR1, MRP1, and BCRP, were assessed using their respective blockers, verapamil, probenecid, and novobiocin. From this study, we demonstrated that 3D-cultured ReNcell VM on the 384-pillar plate could be a good alternative to rapidly identify environmental chemicals and therapeutic compounds for their role in modulating the activity of ion channels and efflux transporters, potentially leading to neurotoxicity.

TREM2 modulates differential deposition of modified and non-modified Aß species in extracellular plaques and intraneuronal deposits.

Progressive accumulation of Amyloid-ß (Aß) deposits in the brain is a characteristic neuropathological hallmark of Alzheimer's disease (AD). During disease progression, extracellular Aß plaques undergo specific changes in their composition by the sequential deposition of different modified Aß species. Microglia are implicated in the restriction of amyloid deposits and play a major role in internalization and degradation of Aß. Recent studies showed that rare variants of the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) are associated with an increased risk for AD. Post-translational modifications of Aß could modulate the interaction with TREM2, and the uptake by microglia. Here, we demonstrate that genetic deletion of TREM2 or expression of a disease associated TREM2 variant in mice lead to differential accumulation of modified and non-modified Aß species in extracellular plaques and intraneuronal deposits. Human brains with rare TREM2 AD risk variants also showed altered deposition of modified Aß species in the different brain lesions as compared to cases with the common variant of TREM2. These findings indicate that TREM2 plays a critical role in the development and the composition of Aß deposits, not only in extracellular plaques, but also intraneuronally, that both could contribute to the pathogenesis of AD.