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Methods and Protocols Apr 2024The main causes of mortality in horses are the gastrointestinal pathologies associated with septic shock. Stem cells have shown, through systemic injection, a capacity...
The main causes of mortality in horses are the gastrointestinal pathologies associated with septic shock. Stem cells have shown, through systemic injection, a capacity to decrease inflammation and to regenerate injured tissue faster. Nevertheless, to achieve this rapid and total regeneration, systemic injections of 1 to 2 million cells per kilogram of body weight must be considered. Here, we demonstrate for the first time the feasibility and expansion capacity of equine muscle-derived mesenchymal stromal cells (mdMSCs) in a functionally closed, automated, perfusion-based, hollow-fiber bioreactor (HFBR) called the Quantum™ Cell Expansion System (Terumo Blood and Cell Technologies). This feature greatly increases the number of generated cells with a surface area of 1.7 m. The expansion of mdMSCs is very efficient in this bioreactor. The maximum expansion generated twenty times more cells than the initial seeding in nine days. The best returns were observed with an optimal seeding between 10 and 25 million mdMSCs, using the Bull's eye loading method and with a run duration between 7 and 10 days. Moreover, all the generated cells kept their stem properties: the ability to adhere to plastic and to differentiate into chondroblasts, osteoblasts and adipocytes. They also showed the expression of CD-44 and CD-90 markers, with a positive rate above 93%, while CD-45 and MHCII were non-expressed, with a positive rate below 0.5%. By capitalizing on the scalability, automation and 3D culture capabilities of the Quantum™, it is possible to generate large quantities of high-quality equine mdMSCs for gastrointestinal disorders and other clinical applications.
PubMed: 38668139
DOI: 10.3390/mps7020032 -
Biosensors Apr 2024Subtle changes in body temperature affect the outcomes of ill newborns. However, the temperature profile of neonatal brains remains largely unknown. In open-cot care,...
Subtle changes in body temperature affect the outcomes of ill newborns. However, the temperature profile of neonatal brains remains largely unknown. In open-cot care, increased cerebral perfusion is correlated with higher superficial brain temperatures. This study investigated the dependence of brain temperature (relative to rectal temperature) on ambient temperature, body size, cerebral perfusion, and metabolism in infants receiving incubator care. Rectal, scalp, and brain temperatures, superior vena cava flow, and brain oxygenation were assessed using echocardiography, thermo-compensatory temperature monitoring, and near-infrared spectroscopy in 60 newborns. These infants had a mean postconceptional age of 36.9 (2.2) weeks and weighed 2348 (609) g at the time of evaluation. The ambient temperature was maintained at 30.0 (1.0) °C. A higher rectal temperature was associated with greater postconceptional age ( = 0.002), body weight ( < 0.001), and head circumference ( < 0.001). Relative scalp, superficial brain, and deep brain temperatures were associated with smaller head circumference ( < 0.001, = 0.030, and = 0.015, respectively) and superior vena cava flow ( = 0.002, = 0.003, and = 0.003, respectively). In infants receiving incubator care, larger head sizes and increased brain perfusion were associated with lower relative scalp and brain temperatures. When considered alongside previous reports, cerebral perfusion may contribute to maintaining stable cerebral tissue temperature against ambient temperature changes.
Topics: Humans; Infant, Newborn; Brain; Body Temperature; Cerebrovascular Circulation; Female; Male; Body Size; Incubators, Infant; Temperature
PubMed: 38667202
DOI: 10.3390/bios14040209 -
Biotechnology Progress Apr 2024The hollow fiber filter is the primary cell-retention device used in high-density perfusion cell culture and often used in an alternating tangential flow (ATF)...
The hollow fiber filter is the primary cell-retention device used in high-density perfusion cell culture and often used in an alternating tangential flow (ATF) configuration. The limited commercially available diaphragm pumps for ATF prevent utilization of vertical space when scaling beyond 500 L. Stacking hollow fiber filters coupled with viscous cell culture imposes vacuum pressure exceeding facility capabilities. Additionally, the longer filter assembly increases the hold-up volume and exceeds the diaphragm pump's fluid exchange capacity. The conventional tangential flow filtration (TFF) configuration circumvents this issue by exchanging culture from the bioreactor and cell-retention device in a unidirectional recirculation loop; however, the increased filter length when scaled up exacerbates the TFF's inherent issue with product retention from Starling flow. Stacking commercially available 20 cm TFF filters to make up the similar single-module length TFF used for the platform 3 and 50 L perfusion process at 41.5 and 65 cm, respectively, attempts to reduce fouling caused by Starling flow. The permeate of a single-module filter is partitioned into short independent segments through serially stacked filters, each harvested separately. By partitioning the permeate, the sieving coefficient increased for both 3 and 50 L scales. Reduction of Starling flow was confirmed with lower total hydraulic membrane resistance throughout the culture. This work demonstrates a method for increasing sieving coefficient and filter capacity by stacking TFF filters with independent permeate streams.
PubMed: 38655754
DOI: 10.1002/btpr.3472 -
Scientific Reports Apr 2024The advent of micro-physiological systems (MPS) in biomedical research has enabled the introduction of more complex and relevant physiological into in vitro models. The...
The advent of micro-physiological systems (MPS) in biomedical research has enabled the introduction of more complex and relevant physiological into in vitro models. The recreation of complex morphological features in three-dimensional environments can recapitulate otherwise absent dynamic interactions in conventional models. In this study we developed an advanced in vitro Renal Cell Carcinoma (RCC) that mimics the interplay between healthy and malignant renal tissue. Based on the TissUse Humimic platform our model combines healthy renal proximal tubule epithelial cells (RPTEC) and RCC. Co-culturing reconstructed RPTEC tubules with RCC spheroids in a closed micro-perfused circuit resulted in significant phenotypical changes to the tubules. Expression of immune factors revealed that interleukin-8 (IL-8) and tumor necrosis factor-alfa (TNF-α) were upregulated in the non-malignant cells while neutrophil gelatinase-associated lipocalin (NGAL) was downregulated in both RCC and RPTEC. Metabolic analysis showed that RCC prompted a shift in the energy production of RPTEC tubules, inducing glycolysis, in a metabolic adaptation that likely supports RCC growth and immunogenicity. In contrast, RCC maintained stable metabolic activity, emphasizing their resilience to external factors. RNA-seq and biological process analysis of primary RTPTEC tubules demonstrated that the 3D tubular architecture and MPS conditions reverted cells to a predominant oxidative phosphorylate state, a departure from the glycolytic metabolism observed in 2D culture. This dynamic RCC co-culture model, approximates the physiology of healthy renal tubules to that of RCC, providing new insights into tumor-host interactions. Our approach can show that an RCC-MPS can expand the complexity and scope of pathophysiology and biomarker studies in kidney cancer research.
Topics: Humans; Carcinoma, Renal Cell; Kidney Neoplasms; Coculture Techniques; Epithelial Cells; Kidney Tubules, Proximal; Cell Line, Tumor; Lipocalin-2; Spheroids, Cellular
PubMed: 38653823
DOI: 10.1038/s41598-024-60164-w -
Cardiovascular Research Apr 2024In patients with diabetic microvascular complications, decreased perfusion or vascular occlusion, caused by reduced vascular diameter, is a common characteristic that...
BACKGROUND
In patients with diabetic microvascular complications, decreased perfusion or vascular occlusion, caused by reduced vascular diameter, is a common characteristic that will lead to insufficient blood supply. Yet, the regulatory mechanism and effective treatment approach remain elusive.
METHODS AND RESULTS
Our initial findings revealed a notable decrease in the expression of human AQP1 in both diabetic human retina samples (49 healthy vs. 54 diabetic samples) and high-glucose-treated human retinal microvascular endothelial cells. Subsequently, our investigations unveiled a reduction in vascular diameter and compromised perfusion within zebrafish embryos subjected to high glucose treatment. Further analysis indicated a significant downregulation of two aquaporins, aqp1a.1 and aqp8a.1, which are highly enriched in ECs and are notably responsive to hyperglycemic conditions. Intriguingly, the loss of function of aqp1a.1 and/or aqp8a.1 resulted in a reduction of intersegmental vessel diameters, effectively mirroring the phenotype observed in the hyperglycemic zebrafish model.The overexpression of aqp1a.1/aqp8a.1 in zebrafish ECs led to notable enlargement of microvascular diameters. Moreover, the reduced vessel diameters resulting from high-glucose treatment were effectively rescued by the overexpression of these aquaporins. Additionally, both aqp1a.1 and apq8a.1 were localized in the intracellular vacuoles in cultured ECs as well as the ECs of sprouting ISVs, and the loss of Aqps caused the reduction of those vacuoles, which was required for lumenization. Notably, while the loss of AQP1 did not impact EC differentiation from human stem cells, it significantly inhibited vascular formation in differentiated ECs.
CONCLUSION
EC-enriched aquaporins regulate the diameter of blood vessels through an intracellular vacuole-mediated process under hyperglycemic conditions. These findings collectively suggest that aquaporins expressed in ECs hold significant promise as potential targets for gene therapy aimed at addressing vascular perfusion defects associated with diabetes.
PubMed: 38646676
DOI: 10.1093/cvr/cvae085 -
Bioprocess and Biosystems Engineering Jun 2024Since cardiovascular diseases (CVDs) are globally one of the leading causes of death, of which myocardial infarction (MI) can cause irreversible damage and decrease... (Review)
Review
Since cardiovascular diseases (CVDs) are globally one of the leading causes of death, of which myocardial infarction (MI) can cause irreversible damage and decrease survivors' quality of life, novel therapeutics are needed. Current approaches such as organ transplantation do not fully restore cardiac function or are limited. As a valuable strategy, tissue engineering seeks to obtain constructs that resemble myocardial tissue, vessels, and heart valves using cells, biomaterials as scaffolds, biochemical and physical stimuli. The latter can be induced using a bioreactor mimicking the heart's physiological environment. An extensive review of bioreactors providing perfusion, mechanical and electrical stimulation, as well as the combination of them is provided. An analysis of the stimulations' mechanisms and modes that best suit cardiac construct culture is developed. Finally, we provide insights into bioreactor configuration and culture assessment properties that need to be elucidated for its clinical translation.
Topics: Tissue Engineering; Bioreactors; Humans; Electric Stimulation; Animals; Perfusion; Myocardium; Tissue Scaffolds
PubMed: 38643271
DOI: 10.1007/s00449-024-03004-5 -
Tissue Engineering and Regenerative... Jul 2024Various cell culture platforms that could display native environmental cue-mimicking stimuli were developed, and effects of environmental cues on cell behaviors were... (Review)
Review
BACKGROUND
Various cell culture platforms that could display native environmental cue-mimicking stimuli were developed, and effects of environmental cues on cell behaviors were studied with the cell culture platforms. Likewise, various cell culture platforms mimicking native trabecular meshwork (TM) composed of juxtacanalicular, corneoscleral and uveal meshwork located in internal scleral sulcus were used to study effects of environmental cues and/or drug treatments on TM cells and glaucoma development. Glaucoma is a disease that could cause blindness, and cause of glaucoma is not clearly identified yet. It appears that aqueous humor (AH) outflow resistance increased by damages on pathway of AH outflow can elevate intraocular pressure (IOP). These overall possibly contribute to development of glaucoma.
METHODS
For the study of glaucoma, static and dynamic cell culture platforms were developed. Particularly, the dynamic platforms exploiting AH outflow-mimicking perfusion or increased IOP-mimicking increased pressure were used to study how perfusion or increased pressure could affect TM cells. Overall, potential mechanisms of glaucoma development, TM structures and compositions, TM cell culture platform types and researches on TM cells and glaucoma development with the platforms were described in this review.
RESULTS AND CONCLUSION
This will be useful to improve researches on TM cells and develop enhanced therapies targeting glaucoma.
Topics: Trabecular Meshwork; Glaucoma; Humans; Cell Culture Techniques; Intraocular Pressure; Aqueous Humor; Animals
PubMed: 38642251
DOI: 10.1007/s13770-024-00640-6 -
The Journal of Physiology Apr 2024Macrophages (MΦ) play pivotal roles in tissue homeostasis and repair. Their mechanical environment has been identified as a key modulator of various cell functions, and...
Macrophages (MΦ) play pivotal roles in tissue homeostasis and repair. Their mechanical environment has been identified as a key modulator of various cell functions, and MΦ mechanosensitivity is likely to be critical - in particular in a rhythmically contracting organ such as the heart. Cultured MΦ, differentiated in vitro from bone marrow (MΦ), form a popular research model. This study explores the activity of mechanosensitive ion channels (MSC) in murine MΦ and compares it to MSC activity in MΦ enzymatically isolated from cardiac tissue (tissue-resident MΦ; MΦ). We show that MΦ and MΦ have stretch-induced currents, indicating the presence of functional MSC in their plasma membrane. The current profiles in MΦ and in MΦ show characteristics of cation non-selective MSC such as Piezo1 or transient receptor potential channels. While Piezo1 ion channel activity is detectable in the plasma membrane of MΦ using the patch-clamp technique, or by measuring cytosolic calcium concentration upon perfusion with the Piezo1 channel agonist Yoda1, no Piezo1 channel activity was observed in MΦ. The selective transient receptor potential vanilloid 4 (TRPV4) channel agonist GSK1016790A induces calcium entry in MΦ and in MΦ. In MΦ isolated from left-ventricular scar tissue 28 days after cryoablation, stretch-induced current characteristics are not significantly different compared to non-injured control tissue, even though scarred ventricular tissue is expected to be mechanically remodelled and to contain an altered composition of pre-existing cardiac and circulation-recruited MΦ. Our data suggest that the in vitro differentiation protocols used to obtain MΦ generate cells that differ from MΦ recruited from the circulation during tissue repair in vivo. Further investigations are needed to explore MSC identity in lineage-traced MΦ in scar tissue, and to compare mechanosensitivity of circulating monocytes with that of MΦ. KEY POINTS: Bone marrow-derived (MΦ) and tissue resident (MΦ) macrophages have stretch-induced currents, indicating expression of functional mechanosensitive channels (MSC) in their plasma membrane. Stretch-activated current profiles show characteristics of cation non-selective MSC; and mRNA coding for MSC, including Piezo1 and TRPV4, is expressed in murine MΦ and in MΦ. Calcium entry upon pharmacological activation of TRPV4 confirms functionality of the channel in MΦ and in MΦ. Piezo1 ion channel activity is detected in the plasma membrane of MΦ but not in MΦ, suggesting that MΦ may not be a good model to study the mechanotransduction of MΦ. Stretch-induced currents, Piezo1 mRNA expression and response to pharmacological activation are not significantly changed in cardiac MΦ 28 days after cryoinjury compared to sham operated mice.
PubMed: 38642051
DOI: 10.1113/JP284805 -
Journal of the American Heart... May 2024Cellular therapies have been investigated to improve blood flow and prevent amputation in peripheral artery disease with limited efficacy in clinical trials....
BACKGROUND
Cellular therapies have been investigated to improve blood flow and prevent amputation in peripheral artery disease with limited efficacy in clinical trials. Alginate-encapsulated mesenchymal stromal cells (eMSCs) demonstrated improved retention and survival and promoted vascular generation in murine hind limb ischemia through their secretome, but large animal evaluation is necessary for human applicability. We sought to determine the efficacy of eMSCs for peripheral artery disease-induced limb ischemia through assessment in our durable swine hind limb ischemia model.
METHODS AND RESULTS
Autologous bone marrow eMSCs or empty alginate capsules were intramuscularly injected 2 weeks post-hind limb ischemia establishment (N=4/group). Improvements were quantified for 4 weeks through walkway gait analysis, contrast angiography, blood pressures, fluorescent microsphere perfusion, and muscle morphology and histology. Capsules remained intact with mesenchymal stromal cells retained for 4 weeks. Adenosine-induced perfusion deficits and muscle atrophy in ischemic limbs were significantly improved by eMSCs versus empty capsules (mean±SD, 1.07±0.19 versus 0.41±0.16, =0.002 for perfusion ratios and 2.79±0.12 versus 1.90±0.62 g/kg, =0.029 for ischemic muscle mass). Force- and temporal-associated walkway parameters normalized (ratio, 0.63±0.35 at week 3 versus 1.02±0.19 preligation; =0.17), and compensatory footfall patterning was diminished in eMSC-administered swine (12.58±8.46% versus 34.85±15.26%; =0.043). Delivery of eMSCs was associated with trending benefits in collateralization, local neovascularization, and muscle fibrosis. Hypoxia-cultured porcine mesenchymal stromal cells secreted vascular endothelial growth factor and tissue inhibitor of metalloproteinase 2.
CONCLUSIONS
This study demonstrates the promise of the mesenchymal stromal cell secretome at improving peripheral artery disease outcomes and the potential for this novel swine model to serve as a component of the preclinical pipeline for advanced therapies.
Topics: Animals; Mesenchymal Stem Cell Transplantation; Hindlimb; Alginates; Disease Models, Animal; Mesenchymal Stem Cells; Ischemia; Swine; Neovascularization, Physiologic; Peripheral Arterial Disease; Injections, Intramuscular; Regional Blood Flow; Muscle, Skeletal; Translational Research, Biomedical; Cells, Cultured
PubMed: 38639336
DOI: 10.1161/JAHA.123.029880 -
Experimental Neurology Jul 2024Chronic hypoxia in utero causes intrauterine growth restriction (IUGR) of the fetus. IUGR infants are known to be at higher risk for neurodevelopmental disorders, but...
Chronic hypoxia in utero causes intrauterine growth restriction (IUGR) of the fetus. IUGR infants are known to be at higher risk for neurodevelopmental disorders, but the mechanism is unclear. In this study, we analyzed the structure of the cerebral cortex using IUGR model rats generated through a reduced uterine perfusion pressure operation. IUGR rats exhibited thinner cerebral white matter and enlarged lateral ventricles compared with control rats. Expression of neuron cell markers, Satb2, microtubule-associated protein (MAP)-2, α-tubulin, and nestin was reduced in IUGR rats, indicating that neurons were diminished at various developmental stages in IUGR rats, from neural stem cells to mature neurons. However, there was no increase in apoptosis in IUGR rats. Cells positive for Ki67, a marker of cell proliferation, were reduced in neurons and all glial cells of IUGR rats. In primary neuron cultures, axonal elongation was impaired under hypoxic culture conditions mimicking the intrauterine environment of IUGR infants. Thus, in IUGR rats, chronic hypoxia in utero suppresses the proliferation of neurons and glial cells as well as axonal elongation, resulting in cortical thinning and enlarged lateral ventricles. Thrombopoietin (TPO), a platelet growth factor, inhibited the decrease in neuron number and promoted axon elongation in primary neurons under hypoxic conditions. Intraperitoneal administration of TPO to IUGR rats resulted in increases in the number of NeuN-positive cells and the area coverage of Satb2. In conclusion, suppression of neuronal proliferation and axonal outgrowth in IUGR rats resulted in cortical thinning and enlargement of lateral ventricles. TPO administration might be a novel therapeutic strategy for treating brain dysmaturation in IUGR infants.
Topics: Animals; Fetal Growth Retardation; Rats; Neurons; Female; Cell Proliferation; Pregnancy; Thrombopoietin; Neuronal Outgrowth; Neuroprotective Agents; Rats, Sprague-Dawley; Cells, Cultured; Animals, Newborn; Cerebral Cortex
PubMed: 38636773
DOI: 10.1016/j.expneurol.2024.114781