Aggregate Cultures Research Articles

Death and survival of gut CD4 T cells following HIV-1 infection ex vivo

Abstract The gastrointestinal (GI) tract is ground zero for the massive and sustained CD4 T cell depletion during acute HIV-1 infection. To date, the molecular mechanisms governing this fundamental pathogenic process remain unclear. HIV-1 infection in the GI tract is associated with chronic inflammation due to a disrupted epithelial barrier that results in microbial translocation. Here, we utilized the Lamina Propria Aggregate Culture model to demonstrate that the profound induction of Granzyme B (GZB) by bacteria in primary gut CD4 T cells ex vivo significantly contributes to HIV-1-mediated CD4 T cell death. Counterintuitively, a substantial fraction of gut GZB+ CD4 T cells harboring high levels of HIV-1 infection survive via a pathway linked to CD120b/TNFR2. Our findings underscore novel mechanisms governing the death and survival of gut CD4 T cells during HIV-1 infection that could inform strategies to counter HIV-1 pathogenesis and persistence in this critical tissue compartment.

Synergistic effects of biological stimuli and flexion induce microcavities promote hypertrophy and inhibit chondrogenesis during in vitro culture of human mesenchymal stem cell aggregates.

Interzone/cavitation are key steps in early stage joint formation that have not been successfully developed in vitro. Further, current models of endochondral ossification, an important step in early bone formation, lack key morphology morphological structures such as microcavities found during development in vivo. This is possibly due to the lack of appropriate strategies for incorporating chemical and mechanical stimuli that are thought to be involved in joint development. We designed a bioreactor system and investigated the synergic effect of chemical stimuli (chondrogenesis-inducing [CIM] and hypertrophy-inducing medium [HIM]) and mechanical stimuli (flexion) on the growth of human mesenchymal stem cells (hMSCs) based linear aggregates under different conditions over 4 weeks of perfusion culture. Computational studies were used to evaluate tissue stress qualitatively. After harvesting, both Safranin-O and hematoxylin & eosin (H&E) staining histology demonstrated microcavity structures and void structures in the region of higher stresses for tissue aggregates cultured only in HIM under flexion. In comparison to either HIM treatment or flexion only, increased glycosaminoglycan (GAG) content in the extracellular matrix (ECM) at this region indicates the morphological change resembles the early stage of joint cavitation; while decreased type II collagen (Col II), and increased type X collagen (Col X) and vascular endothelial growth factor (VEGF) with a clear boundary in the staining section indicates it resembles the early stage of ossification. Further, cell alignment analysis indicated that cells were mostly oriented toward the direction of flexion in high-stress region only in HIM under flexion, resembling cell morphology in both joint cavitation and hypertrophic cartilage in growth plate. Collectively, our results suggest that flexion and HIM inhibit chondrogenesis and promote hypertrophy and development of microcavities that resemble the early stage of joint cavitation and endochondral ossification. We believe the tissue model described in this work can be used to develop in vitro models of joint tissue for applications such as pathophysiology and drug discovery.

Profiling biomanufactured extracellular vesicles of human forebrain spheroids in a Vertical-Wheel Bioreactor.

Extracellular vesicles (EVs) secreted by human brain cells have great potential as cell-free therapies in various diseases, including stroke. However, because of the significant amount of EVs needed in preclinical and clinical trials, EV applicationis still challenging. Vertical-Wheel Bioreactors (VWBRs) have designed features that allow for scaling up the generation of human forebrain spheroid EVs under low shear stress. In this study, EV secretion by human forebrain spheroids derived from induced pluripotent stem cells as 3D aggregates and on Synthemax II microcarriers in VWBRs were investigated with static aggregate culture as a control. The spheroids were characterized by metabolite and transcriptome analysis. The isolated EVs were characterized by nanoparticle tracking analysis, electron microscopy, and Western blot. The EV cargo was analyzed using proteomics and miRNA sequencing. The in vitro functional assays of an oxygen and glucose-deprived stroke model were conducted. Proof of concept in vivo study was performed, too. Human forebrain spheroid differentiated on microcarriers showed a higher growth rate than 3D aggregates. Microcarrier culture had lower glucose consumption per million cells and lower glycolysis gene expression but higher EV biogenesis genes. EVs from the three culture conditions showed no differences in size, but the yields from high to low were microcarrier cultures, dynamic aggregates, and static aggregates. The cargo is enriched with proteins (proteomics) and miRNAs (miRNA-seq), promoting axon guidance, reducing apoptosis, scavenging reactive oxygen species, and regulating immune responses. Human forebrain spheroid EVs demonstrated the ability to improve recovery in an in vitro stroke model and in vivo. Human forebrain spheroid differentiation in VWBR significantly increased the EV yields (up to 240-750 fold) and EV biogenesis compared to static differentiation due to the dynamic microenvironment and metabolism change. The biomanufactured EVs from VWBRs have exosomal characteristics and more therapeutic cargo and are functional in in vitro assays, which paves the way for future in vivo stroke studies.

Robust bioprocess design and evaluation of commercial media for the serial expansion of human induced pluripotent stem cell aggregate cultures in vertical-wheel bioreactors

BackgroundWhile pluripotent stem cell (PSC) therapies move toward clinical and commercial applications at a rapid rate, manufacturing reproducibility and robustness are notable bottlenecks in regulatory approval. Therapeutic applications of PSCs require large cell quantities to be generated under highly robust, well-defined, and economically viable conditions. Small-scale and short-term process optimization, however, is often performed in a linear fashion that does not account for time needed to verify the bioprocess protocols and analysis methods used. Design of a reproducible and robust bioprocess should be dynamic and include a continuous effort to understand how the process will respond over time and to different stresses before transitioning into large-scale production where stresses will be amplified.MethodsThis study utilizes a baseline protocol, developed for the short-term culture of PSC aggregates in Vertical-Wheel® bioreactors, to evaluate key process attributes through long-term (serial passage) suspension culture. This was done to access overall process robustness when performed with various commercially available media and cell lines. Process output variables including growth kinetics, aggregate morphology, harvest efficiency, genomic stability, and functional pluripotency were assessed through short and long-term culture.ResultsThe robust nature of the expansion protocol was demonstrated over a six-day culture period where spherical aggregate formation and expansion were observed with high-fold expansions for all five commercial media tested. Profound differences in cell growth and quality were revealed only through long-term serial expansion and in-vessel dissociation operations. Some commercial media formulations tested demonstrated maintenance of cell growth rates, aggregate morphology, and high harvest recovery efficiencies through three bioreactor serial passages using multiple PSC lines. Exceptional bioprocess robustness was even demonstrated with sustained growth and quality maintenance over 10 serial bioreactor passages. However, some commercial media tested proved less equipped for serial passage cultures in bioreactors as cultures led to cell lysis during dissociation, reduction in growth rates, and a loss of aggregate morphology.ConclusionsThis study demonstrates the importance of systematic selection and testing of bioprocess input variables, with multiple bioprocess output variables through serial passages to create a truly reproducible and robust protocol for clinical and commercial PSC production using scalable bioreactor systems.

Fabrication of a Cancer Cell Aggregate Culture Device That Facilitates Observations of Nutrient and Oxygen Gradients.

Three-dimensional cell culture spheroids are commonly used for drug evaluation studies because they can produce large quantities of homogeneous cell aggregates. As the spheroids grow, nutrients supplied from outer spheroid regions render the inner spheroid areas hypoxic and hyponutrient, which makes them unobservable through confocal microscopy. In this study, we fabricated a cancer cell aggregate culture device that facilitates the observation of nutrient and oxygen gradients. An alginate gel fiber was created in the cell culture chamber to ensure a flow path for supplying the culture medium. A gradient of nutrients and oxygen was generated by positioning the flow channel close to the edge of the chamber. We devised a fabrication method that uses calcium carbonate as a source of Ca2+ for the gelation of sodium alginate, which has a slow reaction rate. We then cultured a spheroid of HCT116 cells, which were derived from human colorectal carcinoma using a fluorescent ubiquitination-based cell cycle indicator. Fluorescence observation suggested the formation of a hypoxic and hyponutrient region within an area approximately 500 µm away from the alginate gel fiber. This indicates the development of a cancer cell aggregate culture device that enables the observation of different nutrition and oxygen states.

Ovarian cell aggregate culture in teleost, marine medaka (Oryzias dancena): basic culture conditions and characterization

Ovarian cell aggregate culture in teleost, marine medaka (<i>Oryzias dancena</i>): basic culture conditions and characterization

Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation

BackgroundHuman pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) has enormous potential for fuelling these cell demands. In this study, the efficient long-term matrix-free suspension culture of hPSC aggregates is shown.Methods and resultsSTBR-controlled, chemical aggregate dissociation and optimized passage duration of 3 or 4 days promotes exponential hPSC proliferation, process efficiency and upscaling by a seed train approach. Intermediate high-density cryopreservation of suspension-derived hPSCs followed by direct STBR inoculation enabled complete omission of matrix-dependent 2D (two-dimensional) culture. Optimized 3D cultivation over 8 passages (32 days) cumulatively yielded ≈4.7 × 1015 cells, while maintaining hPSCs’ pluripotency, differentiation potential and karyotype stability. Gene expression profiling reveals novel insights into the adaption of hPSCs to continuous 3D culture compared to conventional 2D controls.ConclusionsTogether, an entirely matrix-free, highly efficient, flexible and automation-friendly hPSC expansion strategy is demonstrated, facilitating the development of good manufacturing practice-compliant closed-system manufacturing in large scale.

Extracellular vesicle biogenesis of three-dimensional human pluripotent stem cells in a novel Vertical-Wheel bioreactor.

Extracellular vesicles (EVs) secreted by human-induced pluripotent stem cells (hiPSCs) have great potential as cell-free therapies in various diseases, including prevention of blood-brain barrier senescence and stroke. However, there are still challenges in pre-clinical and clinical use of hiPSC-EVs due to the need for large-scale production of a large quantity. Vertical-Wheel bioreactors (VWBRs) have design features that allow the biomanufacturing of hiPSC-EVs using a scalable aggregate or microcarrier-based culture system under low shear stress. EV secretion by undifferentiated hiPSCs expanded as 3-D aggregates and on Synthemax II microcarriers in VWBRs were investigated. Additionally, two types of EV collection media, mTeSR and HBM, were compared. The hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. Protein and microRNA cargo were analysed by proteomics and microRNA-seq, respectively. The in vitro functional assays of microglia stimulation and proliferation were conducted. HiPSCs expanded as 3-D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis and lower autophagy gene expression based on mRNA-seq. The microcarrier cultures had at least 17-23 fold higher EV secretion, and EV collection in mTeSR had 2.7-3.7 fold higher yield than HBM medium. Microcarrier culture with mTeSR EV collection had a smaller EV size than other groups, and the cargo was enriched with proteins (proteomics) and miRNAs (microRNA-seq) reducing apoptosis and promoting cell proliferation (e.g. Wnt-related pathways). hiPSC-EVs demonstrated the ability of stimulating proliferation and M2 polarization of microglia in vitro. HiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR increases yield compared to HBM. The biomanufactured EVs from microcarrier culture in mTeSR have exosomal characteristics and are functional in microglia stimulation, which paves the ways for future in vivo anti-aging study.

Growth prolongation of human induced pluripotent stem cell aggregate in three-dimensional suspension culture system by addition of botulinum hemagglutinin

Human induced pluripotent stem cells (hiPSCs) can be used in regenerative therapy as an irresistible cell source, and so the development of scalable production of hiPSCs for three-dimensional (3D) suspension culture is required. In this study, we established a simple culture strategy for improving hiPSC aggregate growth using botulinum hemagglutinin (HA), which disrupts cell-cell adhesion mediated by E-cadherin. When HA was added to the suspension culture of hiPSC aggregates, E-cadherin-mediated cell-cell adhesion was temporarily disrupted within 24h, but then recovered. Phosphorylated myosin light chain, a contractile force marker, was also recovered at the periphery of hiPSC aggregates. The cell aggregates were suppressed the formation of collagen type I shell-like structures at the periphery by HA and collagen type I was homogenously distributed within the cell aggregates. In addition, these cell aggregates retained the proliferation marker Ki-67 throughout the cell aggregates. The apparent specific growth rate with HA addition was maintained continuously throughout the culture, and the final cell density was 1.7-fold higher than that in the control culture. These cells retained high expression levels of pluripotency markers. These observations indicated that relaxation of cell-cell adhesions by HA addition induced rearrangement of the mechanical tensions generated by actomyosin in hiPSC aggregates and suppression of collagen type I shell-like structure formation. These results suggest that this simple and readily culture strategy is a potentially useful tool for improving the scalable production of hiPSCs for 3D suspension cultures.

Analysis of the effects of bench-scale cell culture platforms and inoculum cell concentrations on PSC aggregate formation and culture.

Introduction: Human pluripotent stem cells (hPSCs) provide many opportunities for application in regenerative medicine due to their ability to differentiate into cells from all three germ layers, proliferate indefinitely, and replace damaged or dysfunctional cells. However, such cell replacement therapies require the economical generation of clinically relevant cell numbers. Whereas culturing hPSCs as a two-dimensional monolayer is widely used and relatively simple to perform, their culture as suspended three-dimensional aggregates may enable more economical production in large-scale stirred tank bioreactors. To be more relevant to this biomanufacturing, bench-scale differentiation studies should be initiated from aggregated hPSC cultures. Methods: We compared five available bench-scale platforms for generating undifferentiated cell aggregates of human embryonic stem cells (hESCs) using AggreWell™ plates, low attachment plates on an orbital shaker, roller bottles, spinner flasks, and vertical-wheel bioreactors (PBS-Minis). Thereafter, we demonstrated the incorporation of an hPSC aggregation step prior to directed differentiation to pancreatic progenitors and endocrine cells. Results and discussion: The AggreWell™ system had the highest aggregation yield. The initial cell concentrations had an impact on the size of aggregates generated when using AggreWell™ plates as well as in roller bottles. However, aggregates made with low attachment plates, spinner flasks and PBS-Minis were similar regardless of the initial cell number. Aggregate morphology was compact and relatively homogenously distributed in all platforms except for the roller bottles. The size of aggregates formed in PBS-Minis was modulated by the agitation rate during the aggregation. In all cell culture platforms, the net growth rate of cells in 3D aggregates was lower (range: -0.01-0.022h-1) than cells growing as a monolayer (range: 0.039-0.045h-1). Overall, this study describes operating ranges that yield high-quality undifferentiated hESC aggregates using several of the most commonly used bench-scale cell culture platforms. In all of these systems, methods were identified to obtain PSC aggregates with greater than 70% viability, and mean diameters between 60 and 260mm. Finally, we showed the capacity of hPSC aggregates formed with PBS-Minis to differentiate into viable pancreatic progenitors and endocrine cell types.

Asteroid impact: the potential of astrocytes to modulate human neural networks within organoids.

Astrocytes are a vital cellular component of the central nervous system that impact neuronal function in both healthy and pathological states. This includes intercellular signals to neurons and non-neuronal cells during development, maturation, and aging that can modulate neural network formation, plasticity, and maintenance. Recently, human pluripotent stem cell-derived neural aggregate cultures, known as neurospheres or organoids, have emerged as improved experimental platforms for basic and pre-clinical neuroscience compared to traditional approaches. Here, we summarize the potential capability of using organoids to further understand the mechanistic role of astrocytes upon neural networks, including the production of extracellular matrix components and reactive signaling cues. Additionally, we discuss the application of organoid models to investigate the astrocyte-dependent aspects of neuropathological diseases and to test astrocyte-inspired technologies. We examine the shortcomings of organoid-based experimental platforms and plausible improvements made possible by cutting-edge neuroengineering technologies. These advancements are expected to enable the development of improved diagnostic strategies and high-throughput translational applications regarding neuroregeneration.

Establishment and Optimization of an Aggregate Culture System of Testicular Cells from Marine Medaka, Oryzias dancena

Although testicular organoids have remarkable potential as testicular models in vitro, there have been few studies about testicular organoids in teleost fish. As a first step to establish a stable culture system for fish testicular organoids, we investigated the efficient conditions for an aggregate culture of dispersed testicular cells from adult marine medaka (Oryzias dancena) by evaluating the effects of culture methods and media composition on an aggregate culture. As the results, we found that culturing dispersed testicular cells in an ultra-low attachment 96 well without Matrigel was most effectively able to induce the formation of testicular cell aggregates among the five different methods tested. Subsequently, through media testing, we confirmed that the modified ESM2 was more optimal for this aggregate culture than the media conventionally used in porcine, human, and rat testicular aggregate cultures. Furthermore, we demonstrated that three supplements in the modified ESM2 including fish serum (FS), basic fibroblast growth factor (bFGF), and embryo extracts (EE) did not influence the number and size of the testicular aggregates formed, but fetal bovine serum and other supplements including β-mercaptoethanol, non-essential amino acids, sodium pyruvate, and sodium selenite were affected significantly. Nevertheless, the removal of three supplements (FS, bFGF, and EE) during culture negatively affected scp3 and sox9a expression levels, indicating their necessity. Finally, we identified that the sperms derived from in vitro cultured testicular aggregates were able to produce offspring after fertilization with naturally matured oocytes. The results from this study will provide fundamental information to develop the techniques for fish testicular organoid culture, which will eventually contribute to the development of reproductive biotechnology for aquaculture and the conservation of endangered fish species.

Fibrin Stiffness Regulates Phenotypic Plasticity of Metastatic Breast Cancer Cells.

The extracellular matrix (ECM)-regulated phenotypic plasticity is crucial for metastatic progression of triple negative breast cancer (TNBC). While ECM faithful cell-based models are available for in situ and invasive tumors, such as cell aggregate cultures in reconstituted basement membrane and in collagenous gels, there are no ECM faithful models for metastatic circulating tumor cells (CTCs). Such models are essential to represent the stage of metastasis where clinical relevance and therapeutic opportunities are significant. Here, CTC-like DU4475 TNBC cells are cultured in mechanically tunable 3D fibrin hydrogels. This is motivated, as in circulation fibrin aids CTC survival by forming a protective coating reducing shear stress and immune cell-mediated cytotoxicity and promotes several stages of late metastatic processes at the interface between circulation and tissue. This work shows that fibrin hydrogels support DU4475 cell growth, resulting in spheroid formation. Furthermore, increasing fibrin stiffness from 57 to 175Pa leads to highly motile, actin and tubulin containing cellular protrusions, which are associated with specific cell morphology and gene expression patterns that markedly differ from basement membrane or suspension cultures. Thus, mechanically tunable fibrin gels reveal specific matrix-based regulation of TNBC cell phenotype and offer scaffolds for CTC-like cells with better mechano-biological properties than liquid.

Macrophage phenotype impacts in vitro equine intrasynovial deep digital flexor tenocyte matrix metalloproteinase gene expression and secretion.

To investigate matrix metalloproteinase (MMP) and their inhibitors tissue inhibitor matrix metalloproteinase (TIMP) gene expression and secretion during equine deep digital flexor tendon (DDFT) tenocyte and macrophage (undifferentiated, proinflammatory, and regulatory) co-culture. Third passage DDF tenocytes and donor-matched macrophages differentiated from peripheral blood CD14+ monocytes from 5 healthy horses ages 9-11 years, euthanized for reasons unrelated to musculoskeletal conditions. Passage 3 DDT tenocyte aggregate cultures were co-cultured with undifferentiated (control), proinflammatory (granulocyte-macrophage colony-stimulating factor; GM-CSF pretreated and lipopolysaccharide + interferon gamma-primed; LPS+IFN-γ) or regulatory (interleukin-4 and interleukin-10-primed; IL-4 + IL-10) macrophages in direct and transwell co-cultures for 72 hours. MMP-1, -2, -3, -9, -13, and TIMP -1, -2 mRNA were measured via real-time Polymerase Chain Reaction (rtPCR). Co-culture media MMP -3, -9, and TIMP -1, -2 concentrations were quantified via ELISA. Direct co-culture of DDF tenocytes with proinflammatory macrophages for 72 hours increased MMP-1, -3, and -13 mRNA levels whereas, MMP-9 mRNA levels decreased. Direct and transwell co-culture with proinflammatory and regulatory macrophages resulted in increased MMP-3 and decreased MMP-9 media concentrations. While direct co-culture with regulatory macrophages significantly increased TIMP-1 mRNA, overall, TIMP mRNA and culture media concentrations were largely unchanged. Cell-to-cell contact between DDF tenocytes and macrophages is not essential to induce MMP gene expression and secretion. Co-culture systems offer a viable in vitro platform to screen and evaluate immunomodulatory properties of therapies aimed at improving equine intrasynovial tendon healing.

Abstract 23 HLA-Homozygous iPSC from Cord Blood for the Generation of Cardiomyocytes for Allogeneic Cell Therapy to the Heart

Abstract Introduction Based on a previous grant on HLA-homozygous (HLAh) iPS-GMP (and ATMP conform) generation from allogeneic licensed cord blood (CB) units, the Düsseldorf CBB could provide the most frequent HLA-types for reprogramming (Liedtke et al. 2020). These HLAh iPSC provide the basis for the generation of cardiomyocytes from iPSC. Patients with acute heart failure or cardiomyopathies have limited options of treatment. iPSC technology allows for de novo generation of functional heart muscle in vitro, enabling the implementation of new therapy concepts for heart regeneration. Objectives The GMP-grade iPSC-lines are the backbone for the granted EU project (HEAL (101056712) on iPSC-cardiomyocyte aggregate manufacturing for allogeneic cell therapy. Besides GMP-compliant up-scaled cell production of cardiomyocytes, the project is designed to overcome many of the scientific, safety and regulatory hurdles necessary to initiate a first-in-man clinical HLAh-based heart regeneration study. Methods After CD34+ selection/short expansion of CD34, cells were reprogrammed by GMP-conform plasmids and stored in a master cell bank with most frequent HLA-haplotypes. All iPSC lines were tested extensively for genetic stability, mycoplasma, virus markers, endotoxins and microorganisms. The differentiation into cardiomyocytes was performed in correspondence to the Wnt modulation, i.e. first activating Wnt pathway and then inhibiting the pathway with the addition of small molecules (Halloin et al. 2019). For the up-scaled production, a 3D-cardiomyocyte aggregate culture in large stirred tank bioreactors (STBR) was applied. Cryopreservation using controlled rate freezing was adapted to tissue freezing. Results Selection of the most frequent homozygous haplotypes facilitated the production of HLAh iPSC lines for differentiation into cardiomyocytes. Efficient progress had been made for the improvement of STBR platform for sufficient production of cardiomyocytes, enabling the differentiation with a purity of up to 95%, measured via cardiac troponin T (cTnT). Discussion Although high yields of cardiomyocytes in a GMP-compliant process is getting more achievable by using STBRs, the safety and regulatory hurdles are still a major problem. For safety testing, toxicology, tumorigenicity assays will be performed in mice and in pig models. This will enable the prediction of arrhythmia-related risks. For a safe distribution of the finally released cell product optimization of cryopreservation of cardiomyocytes aggregates will be performed. Disclaimer: Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency (HADEA). Neither the European Union nor the granting authority can be held responsible for them.

Comparison of cancer stem cell enrichment between spheroids derived from single-cell and multicellular aggregate cultures

Comparison of cancer stem cell enrichment between spheroids derived from single-cell and multicellular aggregate cultures

JAK/STAT3 pathway promotes proliferation of ovarian aggregate-derived stem cells in vitro

BackgroundThe accurate identification and isolation of ovarian stem cells from mammalian ovaries remain a major challenge because of the lack of specific surface markers and suitable in vitro culture systems. Optimized culture conditions for in vitro expansion of ovarian stem cells would allow for identifying requirements of these stem cells for proliferation and differentiation that would pave the way to uncover role of ovarian stem cells in ovarian pathophysiology. Here, we used three-dimensional (3D) aggregate culture system for enrichment of ovarian stem cells and named them aggregate-derived stem cells (ASCs). We hypothesized that mimicking the ovarian microenvironment in vitro by using an aggregate model of the ovary would provide a suitable niche for the isolation of ovarian stem cells from adult mouse and human ovaries and wanted to find out the main cellular pathway governing the proliferation of these stem cells. ResultsWe showed that ovarian aggregates take an example from ovary microenvironment in terms of expression of ovarian markers, hormone secretion and supporting the viability of the cells. We found that aggregates-derived stem cells proliferate in vitro as long-term while remained expression of germline markers. These ovarian stem cells differentiated to oocyte like cells in vitro spontaneously. Transplantation of these stem cells in to chemotherapy mouse ovary could restore ovarian structure. RNA-sequencing analysis revealed that interleukin6 is upregulated pathway in ovarian aggregate-derived stem cells. Our data showed that JAK/Stat3 signaling pathway which is activated downstream of IL6 is critical for ovarian stem cells proliferation. ConclusionsWe developed a platform that is highly reproducible for in vitro propagation of ovarian stem cells. Our study provides a primary insight into cellular pathway governing the proliferation of ovarian stem cells.

Emergent structural and functional properties of hippocampal multi-cellular aggregates.

Hippocampal neural networks are distinctly capable of integrating multi-modal sensory inputs to drive memory formation. Neuroscientific investigations using simplified in vitro models have greatly relied on planar (2D) neuronal cultures made from dissociated tissue. While these models have served as simple, cost-effective, and high-throughput tools for examining various morphological and electrophysiological characteristics of hippocampal networks, 2D cultures fail to reconstitute critical elements of the brain microenvironment that may be necessary for the emergence of sophisticated integrative network properties. To address this, we utilized a forced aggregation technique to generate high-density (>100,000 cells/mm3) multi-cellular three-dimensional aggregates using rodent embryonic hippocampal tissue. We contrasted the emergent structural and functional properties of aggregated (3D) and dissociated (2D) cultures over 28 days in vitro (DIV). Hippocampal aggregates displayed robust axonal fasciculation across large distances and significant neuronal polarization, i.e., spatial segregation of dendrites and axons, at earlier time points compared to dissociated cultures. Moreover, we found that astrocytes in aggregate cultures self-organized into non-overlapping quasi-domains and developed highly stellate morphologies resembling astrocyte structures in vivo. We maintained cultures on multi-electrode arrays (MEAs) to assess spontaneous electrophysiological activity for up to 28 DIV. We found that 3D networks of aggregated cultures developed highly synchronized networks and with high burstiness by 28 DIV. We also demonstrated that dual-aggregate networks became active by 7 DIV, in contrast to single-aggregate networks which became active and developed synchronous bursting activity with repeating motifs by 14 DIV. Taken together, our findings demonstrate that the high-density, multi-cellular, 3D microenvironment of hippocampal aggregates supports the recapitulation of emergent biofidelic morphological and functional properties. Our findings suggest that neural aggregates may be used as segregated, modular building blocks for the development of complex, multi-nodal neural network topologies.

Micronutrient optimization for tissue engineered articular cartilage production of type II collagen.

Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagen in vitro. Inadequate levels of type II collagen are, in part, due to a failure to recapitulate the physiological environment in culture. In this study, we engineered primary rabbit chondrocytes to express a secreted reporter, Gaussia Luciferase, driven by the type II collagen promoter, and applied a Design of Experiments approach to assess chondrogenic differentiation in micronutrient-supplemented medium. Using a Response Surface Model, 240 combinations of micronutrients absent in standard chondrogenic differentiation medium, were screened and assessed for type II collagen promoter-driven Gaussia luciferase expression. While the target of this study was to establish a combination of all micronutrients, alpha-linolenic acid, copper, cobalt, chromium, manganese, molybdenum, vitamins A, E, D and B7 were all found to have a significant effect on type II collagen promoter activity. Five conditions containing all micronutrients predicted to produce the greatest luciferase expression were selected for further study. Validation of these conditions in 3D aggregates identified an optimal condition for type II collagen promoter activity. Engineered cartilage grown in this condition, showed a 170% increase in type II collagen expression (Day 22 Luminescence) and in Young's tensile modulus compared to engineered cartilage in basal media alone.Collagen cross-linking analysis confirmed formation of type II-type II collagen and type II-type IX collagen cross-linked heteropolymeric fibrils, characteristic of mature native cartilage. Combining a Design of Experiments approach and secreted reporter cells in 3D aggregate culture enabled a high-throughput platform that can be used to identify more optimal physiological culture parameters for chondrogenesis.

Large‐Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors (Adv. Healthcare Mater. 24/2022)

Large‐Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors (Adv. Healthcare Mater. 24/2022)