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SUMMARYThis narrative review and meta-analysis summarizes a broad evidence base on the benefits-and also the practicalities, disbenefits, harms and personal, sociocultural and environmental impacts-of masks and masking. Our synthesis of evidence from over 100 published reviews and selected primary studies, including re-analyzing contested meta-analyses of key clinical trials, produced seven key findings. First, there is strong and consistent evidence for airborne transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory pathogens. Second, masks are, if correctly and consistently worn, effective in reducing transmission of respiratory diseases and show a dose-response effect. Third, respirators are significantly more effective than medical or cloth masks. Fourth, mask mandates are, overall, effective in reducing community transmission of respiratory pathogens. Fifth, masks are important sociocultural symbols; non-adherence to masking is sometimes linked to political and ideological beliefs and to widely circulated mis- or disinformation. Sixth, while there is much evidence that masks are not generally harmful to the general population, masking may be relatively contraindicated in individuals with certain medical conditions, who may require exemption. Furthermore, certain groups (notably D/deaf people) are disadvantaged when others are masked. Finally, there are risks to the environment from single-use masks and respirators. We propose an agenda for future research, including improved characterization of the situations in which masking should be recommended or mandated; attention to comfort and acceptability; generalized and disability-focused communication support in settings where masks are worn; and development and testing of novel materials and designs for improved filtration, breathability, and environmental impact.
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COVID-19 , Máscaras , Infecções Respiratórias , SARS-CoV-2 , Humanos , Infecções Respiratórias/prevenção & controle , Infecções Respiratórias/transmissão , COVID-19/prevenção & controle , COVID-19/transmissão , Dispositivos de Proteção Respiratória/normasRESUMO
Sus scrofa domesticus (pig) has served as a superb large mammalian model for biomedical studies because of its comparable physiology and organ size to humans. The derivation of transgene-free porcine induced pluripotent stem cells (PiPSCs) will, therefore, benefit the development of porcine-specific models for regenerative biology and its medical applications. In the past, this effort has been hampered by a lack of understanding of the signaling milieu that stabilizes the porcine pluripotent state in vitro. Here, we report that transgene-free PiPSCs can be efficiently derived from porcine fibroblasts by episomal vectors along with microRNA-302/367 using optimized protocols tailored for this species. PiPSCs can be differentiated into derivatives representing the primary germ layers in vitro and can form teratomas in immunocompromised mice. Furthermore, the transgene-free PiPSCs preserve intrinsic species-specific developmental timing in culture, known as developmental allochrony. This is demonstrated by establishing a porcine in vitro segmentation clock model that, for the first time, displays a specific periodicity at â¼3.7 h, a timescale recapitulating in vivo porcine somitogenesis. We conclude that the transgene-free PiPSCs can serve as a powerful tool for modeling development and disease and developing transplantation strategies. We also anticipate that they will provide insights into conserved and unique features on the regulations of mammalian pluripotency and developmental timing mechanisms.
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Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Animais , Camundongos , Suínos , Reprogramação Celular , Diferenciação Celular , Transgenes , MamíferosRESUMO
Normal developmental progression relies on close interactions between the embryonic and extraembryonic lineages in the pre- and peri-gastrulation stage conceptus. For example, mouse epiblast-derived FGF and NODAL signals are required to maintain a stem-like state in trophoblast cells of the extraembryonic ectoderm, while visceral endoderm signals are pivotal to pattern the anterior region of the epiblast. These developmental stages also coincide with the specification of the first heart precursors. Here, we established a robust differentiation protocol of mouse embryonic stem cells (ESCs) into cardiomyocyte-containing embryoid bodies that we used to test the impact of trophoblast on this key developmental process. Using trophoblast stem cells (TSCs) to produce trophoblast-conditioned medium (TCM), we show that TCM profoundly slows down the cardiomyocyte differentiation dynamics and specifically delays the emergence of cardiac mesoderm progenitors. TCM also strongly promotes the retention of pluripotency transcription factors, thereby sustaining the stem cell state of ESCs. By applying TCM from various mutant TSCs, we further show that those mutations that cause a trophoblast-mediated effect on early heart development in vivo alter the normal cardiomyocyte differentiation trajectory. Our approaches provide a meaningful deconstruction of the intricate crosstalk between the embryonic and the extraembryonic compartments. They demonstrate that trophoblast helps prolong a pluripotent state in embryonic cells and delays early differentiative processes, likely through production of leukemia inhibitory factor (LIF). These data expand our knowledge of the multifaceted signaling interactions among distinct compartments of the early conceptus that ensure normal embryogenesis, insights that will be of significance for the field of synthetic embryo research.
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Male survivors of childhood cancer are at risk of suffering from infertility in adulthood because of gonadotoxic chemotherapies. For adult men, sperm collection and preservation are routine procedures prior to treatment; however, this is not an option for pre-pubertal children. From young boys, a small biopsy may be taken before chemotherapy, and spermatogonia may be propagated in vitro for future transplantation to restore fertility. A robust system that allows for scalable expansion of spermatogonia within a controlled environment is therefore required. Stirred suspension culture has been applied to different types of stem cells but has so far not been explored for spermatogonia. Here, we report that pre-pubertal porcine spermatogonia proliferate more in bioreactor suspension culture, compared with static culture. Interestingly, oxygen tension provides an avenue to modulate spermatogonia status, with culture under 10% oxygen retaining a more undifferentiated state and reducing proliferation in comparison with the conventional approach of culturing under ambient oxygen levels. Spermatogonia grown in bioreactors upregulate the Wnt/ ß-catenin pathway, which, along with enhanced gas and nutrient exchange observed in bioreactor culture, may synergistically account for higher spermatogonia proliferation. Therefore, stirred suspension bioreactors provide novel platforms to culture spermatogonia in a scalable manner and with minimal handling.
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Reatores Biológicos , Técnicas de Cultura de Células/métodos , Proliferação de Células , Espermatogônias/fisiologia , Suspensões , Via de Sinalização Wnt , Animais , Masculino , Espermatogônias/metabolismo , Sus scrofaRESUMO
The retinal pigmented epithelium (RPE) plays a critical role in photoreceptor survival and function. RPE deficits are implicated in a wide range of diseases that result in vision loss, including age-related macular degeneration (AMD) and Stargardt disease, affecting millions worldwide. Subretinal delivery of RPE cells is considered a promising avenue for treatment, and encouraging results from animal trials have supported recent progression into the clinic. However, the limited survival and engraftment of transplanted RPE cells delivered as a suspension continues to be a major challenge. While RPE delivery as epithelial sheets exhibits improved outcomes, this comes at the price of increased complexity at both the production and transplant stages. In order to combine the benefits of both approaches, we have developed size-controlled, scaffold-free RPE microtissues (RPE-µTs) that are suitable for scalable production and delivery via injection. RPE-µTs retain key RPE molecular markers, and interestingly, in comparison to conventional monolayer cultures, they show significant increases in the transcription and secretion of pigment-epithelium-derived factor (PEDF), which is a key trophic factor known to enhance the survival and function of photoreceptors. Furthermore, these microtissues readily spread in vitro on a substrate analogous to Bruch's membrane, suggesting that RPE-µTs may collapse into a sheet upon transplantation. We anticipate that this approach may provide an alternative cell delivery system to improve the survival and integration of RPE transplants, while also retaining the benefits of low complexity in production and delivery.
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Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/transplante , Engenharia Tecidual/métodos , Adesão Celular , Linhagem Celular , Células Cultivadas , Corioide/citologia , Proteínas do Olho/metabolismo , Células-Tronco Embrionárias Humanas , Humanos , Degeneração Macular/terapia , Fatores de Crescimento Neural/metabolismo , Retina/citologia , Retina/metabolismo , Epitélio Pigmentado da Retina/citologia , Serpinas/metabolismoRESUMO
Human male reproductive development has a prolonged prepubertal period characterized by juvenile quiescence of germ cells with immature spermatogonial stem cell (SSC) precursors (gonocytes) present in the testis for an extended period of time. The metabolism of gonocytes is not defined. We demonstrate with mitochondrial ultrastructure studies via TEM and IHC and metabolic flux studies with UHPLC-MS that a distinct metabolic transition occurs during the maturation to SSCs. The mitochondrial ultrastructure of prepubertal human spermatogonia is shared with prepubertal pig spermatogonia. The metabolism of early prepubertal porcine spermatogonia (gonocytes) is characterized by the reliance on OXPHOS fuelled by oxidative decarboxylation of pyruvate. Interestingly, at the same time, a high amount of the consumed pyruvate is also reduced and excreted as lactate. With maturation, prepubertal spermatogonia show a metabolic shift with decreased OXHPOS and upregulation of the anaerobic metabolism-associated uncoupling protein 2 (UCP2). This shift is accompanied with stem cell specific promyelocytic leukemia zinc finger protein (PLZF) protein expression and glial cell-derived neurotropic factor (GDNF) pathway activation. Our results demonstrate that gonocytes differently from mature spermatogonia exhibit unique metabolic demands that must be attained to enable their maintenance and growth in vitro.
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Regulação da Expressão Gênica , Células Germinativas/metabolismo , Estresse Oxidativo , Células-Tronco/metabolismo , Testículo/metabolismo , Animais , Células Germinativas/citologia , Glicólise , Humanos , Masculino , Potencial da Membrana Mitocondrial , Fenótipo , Células-Tronco/citologia , Suínos , Testículo/citologiaRESUMO
RATIONALE: Delivery of continuous positive airway pressure (CPAP) is the standard treatment for obstructive sleep apnoea in children and adults. Treatment adherence is a major challenge, as many patients find the CPAP mask uncomfortable. The study aim was to demonstrate the feasibility of delivered CPAP through customised nasal masks by assessing mask leak and comfort of customised masks compared to commercially available CPAP masks. METHODS: Six healthy adult volunteers participated in a crossover study including commercial masks in three different sizes (petite, small/medium and large) from the same supplier and a customised mask fabricated for each subject using three-dimensional facial scanning and modern additive manufacturing processes. Mask leak and comfort were assessed with varying CPAP levels and mask tightness. Leak was measured in real time using an inline low-resistance Pitot tube flow sensor, and each mask was ranked for comfort by the subjects. RESULTS: Mask leak rates varied directly with CPAP level and inversely with mask tightness. When ranked for comfort, three subjects favoured the customised mask, while three favoured a commercial mask. The petite mask yielded the highest mask leaks and was ranked least comfortable by all subjects. Relative mask leaks and comfort rankings for the other commercial and customised masks varied between individuals. Mask leak was comparable when comparing the customised masks with the highest ranked commercial masks. CONCLUSION: Customised masks successfully delivered target CPAP settings in all six subjects, demonstrating the feasibility of this approach.
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Indirect fundoscopy is challenging for novice learners, as patients are often intolerant of the procedure, impeding development of proficiency. To address this, we developed a canine ocular simulator that we hypothesized would improve student learning compared to live dogs. Six board-certified veterinary ophthalmologists and 19 second-year veterinary students (novices) performed an indirect fundic examination on the model and live dog. Prior to assessment, novices were introduced to the skill with a standardized teaching protocol and practiced (without feedback) with either the model (n = 10) or live dog (n = 9) for 30 minutes. All participants evaluated realism and usefulness of the model using a Likert-type scale. Performance on the live dog and model was evaluated in all participants using time to completion of task, performance of fundic examination using a checklist and global score, identification of objects in the fundus of the model, and evaluation of time spent looking at the fundus of the model using eye tracking. Novices (trained on simulator or live dogs) were compared in fundic examination performance on the live dog and identification of shapes in the model. In general, experts performed the fundic examination faster (p ≤ .0003) and more proficiently than the novices, although there were no differences in eye tracking behavior between groups (p ≥ .06). No differences were detected between training on simulator versus live dog in development of fundoscopy skills in novices (p ≥ .20). These findings suggest that this canine model may be an effective tool to train students to perform fundoscopy.
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Educação em Veterinária , Animais , Competência Clínica , Simulação por Computador , Cães , Retroalimentação , Humanos , EstudantesRESUMO
BACKGROUND: The putative benefit of rhBMP-2 is in the setting of limb reconstruction using structural allografts, whether it be allograft-prosthetic composites, osteoarticular allografts, or intercalary segmental grafts. There are also potential advantages in augmenting osseointegration of uncemented endoprosthetics and in reducing infection. Recombinant human BMP-2 might mitigate nonunion in structural allograft augmented osteosarcoma limb salvage surgery; however, its use is limited because of concerns about the prooncogenic effects of the agent. QUESTIONS/PURPOSES: (1) To assess if BMP-2 signaling influences osteosarcoma cell line growth. (2) To characterize degree of osteosarcoma cell line osteoblastic differentiation in response to BMP-2. (3) To assess if BMP-2 signaling has a consistent effect on local or systemic tumor burden in various orthotopic murine models of osteosarcoma. METHODS: In this study, 143b, SaOS-2 and DLM8-M1 osteosarcoma cell lines were transfected with BMP-2 cDNA controlled by a constitutive promoter (experimental) or an empty vector (control) using a PiggyBac transposon system. Cellular proliferation was assessed using a quantitative MTT colorimetric assay. Osteoblastic differentiation was compared between control and experimental cell lines using quantitative real-time polymerase chain reaction of the osteoblastic markers connective tissue growth factor, Runx-2, Osterix, alkaline phosphatase and osteocalcin. Experimental and control cell lines were injected into the proximal tibia of either NOD-SCID (143b and SaOS-2 xenograft model), or C3H (DLM8-M1 syngeneic model) mice. Local tumor burden was quantitatively assessed using tumor volume caliper measurements and bioluminescence, and qualitatively assessed using post-mortem ex vivo microCT. Lung metastasis was qualitatively assessed by the presence of bioluminescence, and incidence was confirmed using histology. rhBMP-2 soaked absorbable collagen sponges (experimental) and sterile-H2O soaked absorbable collagen sponges (control) were implanted adjacent to 143b proximal tibial cell line injections to compare the effects of exogenous BMP-2 application with endogenous upregulation. RESULTS: Constitutive expression of BMP-2 increased the in vitro proliferation of 143b cells (absorbance values 1.2 ± 0.1 versus 0.89 ± 0.1, mean difference 0.36 [95% CI 0.12 to 0.6]; p = 0.01), but had no effect on SaOS-2 and DLM8-M1 cell proliferation. In response to constitutive BMP-2 expression, 143b cells had no differences in osteoblastic differentiation, while DLM8-M1 cells downregulated the early marker connective tissue growth factor (mean ΔCt 0.2 ± 0.1 versus 0.6 ± 0.1; p = 0.002) and upregulated the early-mid range marker Runx-2 (mean ΔCt -0.8 ± 0.1 versus -1.1 ± 0.1; p = 0.002), and SaOS-2 cells upregulated the mid-range marker Osterix (mean ΔCt -2.1 ± 0.6 versus -3.9 ± 0.6; p = 0.002). Constitutive expression of BMP-2 resulted in greater 143b and DLM8-M1 local tumor volume (143b: 307.2 ± 106.8 mm versus 1316 ± 387.4 mm, mean difference 1009 mm [95% CI 674.5 to 1343]; p < 0.001, DLM8-M1 week four: 0 mm versus 326.1 ± 72.8 mm, mean difference 326.1 mm [95% CI 121.2 to 531]; p = 0.009), but modestly reduced local tumor growth in SaOS-2 (9.5 x 10 ± 8.3x10 photons/s versus 9.3 x 10 ± 1.5 x 10 photons/s, mean difference 8.6 x 10 photons/s [95% CI 5.1 x 10 to 1.2 x 10]; p < 0.001). Application of exogenous rhBMP-2 also increased 143b local tumor volume (495 ± 91.9 mm versus 1335 ± 102.7 mm, mean difference 840.3 mm [95% CI 671.7 to 1009]; p < 0.001). Incidence of lung metastases was not different between experimental or control groups for all experimental conditions. CONCLUSIONS: As demonstrated by others, ectopic BMP-2 signaling has unpredictable effects on local tumor proliferation in murine models of osteosarcoma and does not consistently result in osteosarcoma cell line differentiation. Further investigations into other methods of safe bone and soft tissue healing augmentation and the use of differentiation therapies is warranted. CLINICAL RELEVANCE: Our results indicate that BMP-2 has the potential to stimulate the growth of osteosarcoma cells that are poorly responsive to BMP-2 mediated osteoblastic differentiation. As this differentiation potential is unpredictable in the clinical setting, BMP-2 may promote the growth of microscopic residual tumor burden after resection. Our study provides further support for the recommendation to avoid the use of BMP-2 after limb-salvage surgery in patients with osteosarcoma.
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Proteína Morfogenética Óssea 2/metabolismo , Neoplasias Ósseas/metabolismo , Diferenciação Celular , Proliferação de Células , Osteoblastos/metabolismo , Osteossarcoma/metabolismo , Adolescente , Animais , Proteína Morfogenética Óssea 2/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular , Criança , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos C3H , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Osteoblastos/patologia , Osteossarcoma/genética , Osteossarcoma/patologia , Transdução de Sinais , Carga TumoralRESUMO
Age-related macular degeneration (AMD) is the leading cause of blindness in the industrialized world. AMD is associated with dysfunction and atrophy of the retinal pigment epithelium (RPE), which provides critical support for photoreceptor survival and function. RPE transplantation is a promising avenue towards a potentially curative treatment for early stage AMD patients, with encouraging reports from animal trials supporting recent progression toward clinical treatments. Mature RPE cells have been reported to be superior, but a detailed investigation of the specific changes in the expression pattern of key RPE genes during maturation is lacking. To understand the effect of maturity on RPE, we investigated transcript levels of 19 key RPE genes using ARPE-19 cell line and human embryonic stem cell-derived RPE cultures. Mature RPE cultures upregulated PEDF, IGF-1, CNTF and BDNF-genes that code for trophic factors known to enhance the survival and function of photoreceptors. Moreover, the mRNA levels of these genes are maximized after 42 days of maturation in culture and lost upon dissociation to single cells. Our findings will help to inform future animal and human RPE transplantation efforts.
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Regulação da Expressão Gênica , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/fisiologia , Fator Neurotrófico Derivado do Encéfalo/genética , Técnicas de Cultura de Células , Linhagem Celular , Células Cultivadas , Fator Neurotrófico Ciliar/genética , Proteínas do Olho/genética , Humanos , Fator de Crescimento Insulin-Like I/genética , Fatores de Crescimento Neural/genética , Serpinas/genética , Fatores de Tempo , Regulação para CimaRESUMO
Dystocia is a leading cause of calf mortality, yet there is little available information quantifying the duration and forces applied to assisted deliveries. Objectives of this study were to: (1) develop a method to measure the magnitude and duration of various forces applied to a calf during calving assistance, and (2) quantify the forces applied to beef calves during manual or mechanical calving assistance. Twenty-five primiparous dams requiring calving assistance were enrolled. Calvings were assisted by manual (1 or 2 people pulling) or mechanical (calf extractor) delivery. A set of modified obstetric chains with integrated force measuring devices (Calving Assistance Force Logger; CAF-Log) were applied to the calf for delivery. The CAF-Log system was calibrated using known masses ranging from 25 to 200 kg in increasing increments of 25 kg. Duration of the assisted delivery and force parameters (peak force applied to one leg, peak force applied to both legs, cumulative force, and maximum jerk force) were described and assessed for their associations with method of delivery and ranch. Median duration was 112.6 s (IQR: 88.4-149.7) for manual and 312.6 s (IQR: 221.6-462.3) for mechanical deliveries. Mean peak force applied to one leg was 56.9 kg (SD: 22.9) for manual and 126.8 kg (SD: 48.2) for mechanical deliveries. Mean peak force applied to both legs was 95.4 kg (SD: 34.1) for manual and 188.6 kg (SD: 83.9) for mechanical deliveries. Median cumulative force was 178.3 kg min (IQR: 21.1-38.8) for manual and 380.6 kg min (IQR: 252.1-581.3) for mechanical deliveries. The maximum jerk force for manual deliveries was 36.6 kg/s (IQR: 21.1-38.8) and 77.2 kg/s (IQR: 60.9-97.1) for mechanical deliveries. An interaction occurred between ranch and method of delivery for peak force applied to one leg, peak force applied to both legs, and cumulative force. The CAF-Log system demonstrated that significantly greater forces were applied to mechanically delivered calves compared to manually delivered calves and could be used in future studies to investigate forces applied to a calf during calving assistance and their impacts on cow and calf well-being.
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We have developed an accessible software tool (receptoR) to predict potentially active signaling pathways in one or more cell type(s) of interest from publicly available transcriptome data. As proof-of-concept, we applied it to mouse photoreceptors, yielding the previously untested hypothesis that activin signaling pathways are active in these cells. Expression of the type 2 activin receptor (Acvr2a) was experimentally confirmed by both RT-qPCR and immunochemistry, and activation of this signaling pathway with recombinant activin A significantly enhanced the survival of magnetically sorted photoreceptors in culture. Taken together, we demonstrate that our approach can be easily used to mine publicly available transcriptome data and generate hypotheses around receptor expression that can be used to identify novel signaling pathways in specific cell types of interest. We anticipate that receptoR (available at https://www.ucalgary.ca/ungrinlab/receptoR) will enable more efficient use of limited research resources.
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Mesenchymal stem cells (MSCs) have the capacity to differentiate towards bone, fat, and cartilage lineages. The most widely used culture and differentiation protocols for MSCs are currently limited by their use of serum-containing media and small-scale static culture vessels. Suspension bioreactors have multiple advantages over static culture vessels (e.g., scalability, control, and mechanical forces). This study sought to compare the formation and culture of 3D aggregates of human synovial fluid MSCs within suspension bioreactors and static microwell plates. It also sought to elucidate the benefits of these techniques in terms of productivity, cell number, and ability to generate aggregates containing extracellular matrix deposition. MSCs in serum-free medium were either (1) inoculated as single cells into suspension bioreactors, (2) aggregated using static microwell plates prior to being inoculated in the bioreactor environment, or (3) aggregated using microwell plates and kept in the static environment. Preformed aggregates that were size-controlled at inoculation had a greater tendency to form large, irregular super aggregates after a few days of suspension culture. The single MSCs inoculated into suspension bioreactors formed a more uniform population of smaller aggregates after a definite culture period of 8 days. Both techniques showed initial deposition of extracellular matrix within the aggregates. When the relationship between aggregate size and ECM deposition was investigated in static culture, midsized aggregates (100-300 cells/aggregate) were found to most consistently maximize sGAG and collagen productivity. Thus, this study presents a 3D tissue culture method, which avoids the clinical drawbacks of serum-containing medium that can easily be scaled for tissue culture applications.
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Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.
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Técnicas de Cultura de Células/instrumentação , Organoides/citologia , Testículo/citologia , Animais , Masculino , Especificidade de Órgãos , Espermatogênese , SuínosRESUMO
Induced pluripotent stem cells (iPSCs) are an attractive cell source for regenerative medicine and the development of therapies, as they can proliferate indefinitely under defined conditions and differentiate into any cell type in the body. Large-scale expansion of cells is limited in adherent culture, making it difficult to obtain adequate cell numbers for research. It has been previously shown that stirred suspension bioreactors (SSBs) can be used to culture mouse and human stem cells. Pigs are important preclinical models for stem cell research. Therefore, this study investigated the use of SSBs as an alternative culture method for the expansion of iPSCs. Using an established porcine iPSC (piPSC) line as well as a new cell line derived and characterized in the current study, we report that piPSCs can grow in SSB while maintaining characteristics of pluripotency and karyotypic stability similar to cells grown in traditional two-dimensional static culture. This culture method provides a suitable platform for scale-up of cell culture to provide adequate cell numbers for future research applications involving piPSCs.
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Técnicas de Cultura Celular por Lotes/métodos , Reatores Biológicos/normas , Células-Tronco Pluripotentes Induzidas/fisiologia , Animais , Técnicas de Cultura Celular por Lotes/instrumentação , Linhagem Celular , Proliferação de Células , Células-Tronco Pluripotentes Induzidas/metabolismo , SuínosRESUMO
Three-dimensional (3D) organoids can serve as an in vitro platform to study cell-cell interactions, tissue development, and toxicology. Development of organoids with tissue architecture similar to testis in vivo has remained a challenge. Here, we present a microwell aggregation approach to establish multicellular 3D testicular organoids from pig, mouse, macaque, and human. The organoids consist of germ cells, Sertoli cells, Leydig cells, and peritubular myoid cells forming a distinct seminiferous epithelium and interstitial compartment separated by a basement membrane. Sertoli cells in the organoids express tight junction proteins claudin 11 and occludin. Germ cells in organoids showed an attenuated response to retinoic acid compared to germ cells in 2D culture indicating that the tissue architecture of the organoid modulates response to retinoic acid similar to in vivo. Germ cells maintaining physiological cell-cell interactions in organoids also had lower levels of autophagy indicating lower levels of cellular stress. When organoids were treated with mono(2-ethylhexyl) phthalate (MEHP), levels of germ cell autophagy increased in a dose-dependent manner, indicating the utility of the organoids for toxicity screening. Ablation of primary cilia on testicular somatic cells inhibited the formation of organoids demonstrating an application to screen for factors affecting testicular morphogenesis. Organoids can be generated from cryopreserved testis cells and preserved by vitrification. Taken together, the testicular organoid system recapitulates the 3D organization of the mammalian testis and provides an in vitro platform for studying germ cell function, testicular development, and drug toxicity in a cellular context representative of the testis in vivo.
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Técnicas de Cultura de Células/métodos , Organoides/citologia , Testículo/citologia , Alicerces Teciduais , Animais , Contagem de Células , Técnicas de Cultura de Células/instrumentação , Pré-Escolar , Dietilexilftalato/análogos & derivados , Dietilexilftalato/farmacologia , Humanos , Lactente , Macaca mulatta , Masculino , Camundongos , Organoides/fisiologia , Espermatogênese/efeitos dos fármacos , Espermatogênese/fisiologia , Espermatogônias/citologia , Espermatogônias/efeitos dos fármacos , Espermatogônias/fisiologia , Suínos , Alicerces Teciduais/química , Tretinoína/farmacologiaRESUMO
Mesenchymal stem cells (MSCs) have attracted tremendous research interest due to their ability to repair tissues and reduce inflammation when implanted into a damaged or diseased site. These therapeutic effects have been largely attributed to the collection of biomolecules they secrete (i.e., their secretome). Recent studies have provided evidence that similar effects may be produced by utilizing only the secretome fraction containing extracellular vesicles (EVs). EVs are cell-derived, membrane-bound vesicles that contain various biomolecules. Due to their small size and relative mobility, they provide a stable mechanism to deliver biomolecules (i.e., biological signals) throughout an organism. The use of the MSC secretome, or its components, has advantages over the implantation of the MSCs themselves: (i) signals can be bioengineered and scaled to specific dosages, and (ii) the nonliving nature of the secretome enables it to be efficiently stored and transported. However, since the composition and therapeutic benefit of the secretome can be influenced by cell source, culture conditions, isolation methods, and storage conditions, there is a need for standardization of bioprocessing parameters. This review focuses on key parameters within the MSC culture environment that affect the nature and functionality of the secretome. This information is pertinent to the development of bioprocesses aimed at scaling up the production of secretome-derived products for their use as therapeutics.
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Mammalian cell culture is foundational to biomedical research, and the reproducibility of research findings across the sciences is drawing increasing attention. While many components contribute to reproducibility, the reporting of factors that impact oxygen delivery in the general biomedical literature has the potential for both significant impact, and immediate improvement. The relationship between the oxygen consumption rate of cells and the diffusive delivery of oxygen through the overlying medium layer means parameters such as medium depth and cell type can cause significant differences in oxygenation for cultures nominally maintained under the same conditions. While oxygenation levels are widely understood to significantly impact the phenotype of cultured cells in the abstract, in practise the importance of the above parameters does not appear to be well recognized in the non-specialist research community. On analyzing two hundred articles from high-impact journals we find a large majority missing at least one key piece of information necessary to ensure consistency in replication. We propose that explicitly reporting these values should be a requirement for publication.