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The Analyst Apr 2024Cellular metabolism has been closely linked to activation state in cells of the immune system, and the oxygen consumption rate (OCR) in particular serves as a valuable...
Cellular metabolism has been closely linked to activation state in cells of the immune system, and the oxygen consumption rate (OCR) in particular serves as a valuable metric for assessing metabolic activity. Several oxygen sensing assays have been reported for cells in standard culture conditions. However, none have provided a spatially resolved, optical measurement of local oxygen consumption in intact tissue samples, making it challenging to understand regional dynamics of consumption. Therefore, here we established a system to monitor the rates of oxygen consumption in tissue slices, using murine lymphoid tissue as a case study. By integrating an optical oxygen sensor into a sealed perfusion chamber and incorporating appropriate correction for photobleaching of the sensor and of tissue autofluorescence, we were able to visualize and quantify rates of oxygen consumption in tissue. This method revealed for the first time that the rate of oxygen consumption in naïve lymphoid tissue was higher in the T cell region compared to the B cell and cortical regions. To validate the method, we measured OCR in the T cell regions of naïve lymph node slices using the optical assay and estimated the consumption rate per cell. The predictions from the optical assay were similar to reported values and were not significantly different from those of the Seahorse metabolic assay, a gold standard method for measuring OCR in cell suspensions. Finally, we used this method to quantify the rate of onset of tissue hypoxia for lymph node slices cultured in a sealed chamber and showed that continuous perfusion was sufficient to maintain oxygenation. In summary, this work establishes a method to monitor oxygen consumption with regional resolution in intact tissue explants, suitable for future use to compare tissue culture conditions and responses to stimulation.
Topics: Animals; Oxygen Consumption; Lymph Nodes; Mice; Mice, Inbred C57BL; Oxygen; T-Lymphocytes
PubMed: 38535830
DOI: 10.1039/d4an00028e -
Annals of Biomedical Engineering Jun 2024We developed the open-source bIUreactor research platform for studying 3D structured tissues. The versatile and modular platform allows a researcher to generate 3D...
We developed the open-source bIUreactor research platform for studying 3D structured tissues. The versatile and modular platform allows a researcher to generate 3D tissues, culture them with oxygenated perfusion, and provide cyclic loading, all in their own lab (in laboratorium) for an all in cost of $8,000 including 3D printer, printing resin, and electronics. We achieved this by applying a design philosophy that leverages 3D printing, open-source software and hardware, and practical techniques to produce the following: 1. perfusible 3D tissues, 2. a bioreactor chamber for tissue culture, 3. a module for applying cyclic compression, 4. a peristaltic pump for providing oxygenated perfusion to 3D tissues, 5. motor control units, and 6. open-source code for running the control units. By making it widely available for researchers to investigate 3D tissue models and easy for them to use, we intend for the bIUreactor to democratize 3D tissue research, therefore increasing the pace and scale of biomedical research discoveries using 3D tissue models.
Topics: Printing, Three-Dimensional; Bioreactors; Humans; Tissue Engineering; Software; Animals
PubMed: 38532173
DOI: 10.1007/s10439-024-03481-5 -
Applied Microbiology and Biotechnology Mar 2024The pharmaceutical industry employs various strategies to improve cell productivity. These strategies include process intensification, culture media improvement, clonal...
The pharmaceutical industry employs various strategies to improve cell productivity. These strategies include process intensification, culture media improvement, clonal selection, media supplementation and genetic engineering of cells. However, improved cell productivity has inherent risk of impacting product quality attributes (PQA). PQAs may affect the products' efficacy via stability, bioavailability, or in vivo bioactivity. Variations in manufacturing process may introduce heterogeneity in the products by altering the type and extent of N-glycosylation, which is a PQA of therapeutic proteins. We investigated the effect of different cell densities representing increasing process intensification in a perfusion cell culture on the production of an IgG1-κ monoclonal antibody from a CHO-K1 cell line. This antibody is glycosylated both on light chain and heavy chain. Our results showed that the contents of glycosylation of IgG1-κ mAb increased in G0F and fucosylated type glycans as a group, whereas sialylated type glycans decreased, for the mAb whole protein. Overall, significant differences were observed in amounts of G0F, G1F, G0, G2FS1, and G2FS2 type glycans across all process intensification levels. G2FS2 and G2 type N-glycans were predominantly quantifiable from light chain rather than heavy chain. It may be concluded that there is a potential impact to product quality attributes of therapeutic proteins during process intensification via perfusion cell culture that needs to be assessed. Since during perfusion cell culture the product is collected throughout the duration of the process, lot allocation needs careful attention to process parameters, as PQAs are affected by the critical process parameters (CPPs). KEY POINTS: • Molecular integrity may suffer with increasing process intensity. • Galactosylated and sialylated N-glycans may decrease. • Perfusion culture appears to maintain protein charge structure.
Topics: Cricetinae; Animals; Antibodies, Monoclonal; Immunoglobulin G; CHO Cells; Cricetulus; Perfusion; Polysaccharides
PubMed: 38530495
DOI: 10.1007/s00253-024-13110-9 -
APL Bioengineering Mar 2024Perfusable microvascular networks offer promising three-dimensional models to study normal and compromised vascular tissues as well as phenomena such as cancer cell...
Perfusable microvascular networks offer promising three-dimensional models to study normal and compromised vascular tissues as well as phenomena such as cancer cell metastasis. Engineering of these microvascular networks generally involves the use of endothelial cells stabilized by fibroblasts to generate robust and stable vasculature. However, fibroblasts are highly heterogenous and may contribute variably to the microvascular structure. Here, we study the effect of normal and cancer-associated lung fibroblasts on the formation and function of perfusable microvascular networks. We examine the influence of cancer-associated fibroblasts on microvascular networks when cultured in direct (juxtacrine) and indirect (paracrine) contacts with endothelial cells, discovering a generative inhibition of microvasculature in juxtacrine co-cultures and a functional inhibition in paracrine co-cultures. Furthermore, we probed the secreted factors differential between cancer-associated fibroblasts and normal human lung fibroblasts, identifying several cytokines putatively influencing the resulting microvasculature morphology and functionality. These findings suggest the potential contribution of cancer-associated fibroblasts in aberrant microvasculature associated with tumors and the plausible application of such platforms in identifying new therapeutic targets and/or agents that can prevent formation of aberrant vascular structures.
PubMed: 38524671
DOI: 10.1063/5.0188238 -
Nature Communications Mar 2024Engineered human cardiac tissues have been utilized for various biomedical applications, including drug testing, disease modeling, and regenerative medicine. However,...
Engineered human cardiac tissues have been utilized for various biomedical applications, including drug testing, disease modeling, and regenerative medicine. However, the applications of cardiac tissues derived from human pluripotent stem cells are often limited due to their immaturity and lack of functionality. Therefore, in this study, we establish a perfusable culture system based on in vivo-like heart microenvironments to improve human cardiac tissue fabrication. The integrated culture platform of a microfluidic chip and a three-dimensional heart extracellular matrix enhances human cardiac tissue development and their structural and functional maturation. These tissues are comprised of cardiovascular lineage cells, including cardiomyocytes and cardiac fibroblasts derived from human induced pluripotent stem cells, as well as vascular endothelial cells. The resultant macroscale human cardiac tissues exhibit improved efficacy in drug testing (small molecules with various levels of arrhythmia risk), disease modeling (Long QT Syndrome and cardiac fibrosis), and regenerative therapy (myocardial infarction treatment). Therefore, our culture system can serve as a highly effective tissue-engineering platform to provide human cardiac tissues for versatile biomedical applications.
Topics: Humans; Endothelial Cells; Induced Pluripotent Stem Cells; Cell Differentiation; Myocytes, Cardiac; Tissue Engineering
PubMed: 38519491
DOI: 10.1038/s41467-024-46928-y -
Best Practice & Research. Clinical... Jun 2024Preeclampsia (PE) is a complex heterogeneous disorder with overlapping clinical phenotypes that complicate diagnosis and management. Although several pathophysiological... (Review)
Review
Preeclampsia (PE) is a complex heterogeneous disorder with overlapping clinical phenotypes that complicate diagnosis and management. Although several pathophysiological mechanisms have been proposed, placental dysfunction due to inadequate remodelling of uterine spiral arteries leading to mal-perfusion and syncytiotrophoblast stress is recognized as the unifying characteristic of early-onset PE. Placental overgrowth and or premature senescence are probably the causes of late-onset PE. The frequency of PE has increased over the last few decades due to population-wide increases in risk factors viz. obesity, diabetes, multifetal pregnancies and pregnancies at an advanced maternal age. Whilst multimodal tools with components comprising risk factors, biomarkers and sonography are used for predicting PE, aspirin is most effective in preventing early-onset PE. The incidence and clinical consequences of PE and eclampsia are influenced by socioeconomic and cultural factors, therefore management strategies should involve multi-sector partnerships to mitigate the adverse outcomes.
Topics: Humans; Female; Pre-Eclampsia; Pregnancy; Eclampsia; Developing Countries; Risk Factors; Maternal Mortality; Aspirin
PubMed: 38513504
DOI: 10.1016/j.bpobgyn.2024.102473 -
Arteriosclerosis, Thrombosis, and... Jun 2024Restoring the capacity of endothelial progenitor cells (EPCs) to promote angiogenesis is the major therapeutic strategy of diabetic peripheral artery disease. The aim of...
BACKGROUND
Restoring the capacity of endothelial progenitor cells (EPCs) to promote angiogenesis is the major therapeutic strategy of diabetic peripheral artery disease. The aim of this study was to investigate the effects of GLP-1 (glucagon-like peptide 1; 32-36)-an end product of GLP-1-on angiogenesis of EPCs and T1DM (type 1 diabetes) mice, as well as its interaction with the classical GLP-1R (GLP-1 receptor) pathway and its effect on mitochondrial metabolism.
METHODS
In in vivo experiments, we conducted streptozocin-induced type 1 diabetic mice as a murine model of unilateral hind limb ischemia to examine the therapeutic potential of GLP-1(32-36) on angiogenesis. We also generated mice to detect whether GLP-1R is required for angiogenic function of GLP-1(32-36). In in vitro experiments, EPCs isolated from the mouse bone marrow and human umbilical cord blood samples were used to detect GLP-1(32-36)-mediated angiogenic capability under high glucose treatment.
RESULTS
We demonstrated that GLP-1(32-36) did not affect insulin secretion but could significantly rescue angiogenic function and blood perfusion in ischemic limb of streptozocin-induced T1DM mice, a function similar to its parental GLP-1. We also found that GLP-1(32-36) promotes angiogenesis in EPCs exposed to high glucose. Specifically, GLP-1(32-36) has a causal role in improving fragile mitochondrial function and metabolism via the GLP-1R-mediated pathway. We further demonstrated that GLP-1(32-36) rescued diabetic ischemic lower limbs by activating the GLP-1R-dependent eNOS (endothelial NO synthase)/cGMP/PKG (protein kinase G) pathway.
CONCLUSIONS
Our study provides a novel mechanism with which GLP-1(32-36) acts in modulating metabolic reprogramming toward glycolytic flux in partnership with GLP-1R for improved angiogenesis in high glucose-exposed EPCs and T1DM murine models. We propose that GLP-1(32-36) could be used as a monotherapy or add-on therapy with existing treatments for peripheral artery disease.
REGISTRATION
URL: www.ebi.ac.uk/metabolights/; Unique identifier: MTBLS9543.
Topics: Animals; Ischemia; Glucagon-Like Peptide-1 Receptor; Neovascularization, Physiologic; Diabetes Mellitus, Experimental; Glycolysis; Glucagon-Like Peptide 1; Mice, Inbred C57BL; Humans; Hindlimb; Mice, Knockout; Male; Signal Transduction; Endothelial Progenitor Cells; Diabetic Angiopathies; Nitric Oxide Synthase Type III; Diabetes Mellitus, Type 1; Cells, Cultured; Angiogenesis Inducing Agents; Peptide Fragments; Mice; Muscle, Skeletal; Disease Models, Animal; Incretins; Angiogenesis
PubMed: 38511325
DOI: 10.1161/ATVBAHA.124.320714 -
Biomedicine & Pharmacotherapy =... May 2024In cancer immunotherapy, chimeric antigen receptors (CARs) targeting specific antigens have become a powerful tool for cell-based therapy. CAR-natural killer (NK) cells...
In cancer immunotherapy, chimeric antigen receptors (CARs) targeting specific antigens have become a powerful tool for cell-based therapy. CAR-natural killer (NK) cells offer selective anticancer lysis with reduced off-tumor toxicity compared to CAR-T cells, which is beneficial in the heterogeneous milieu of solid tumors. In the tumor microenvironment (TME) of glioblastoma (GBM), pericytes not only support tumor growth but also contribute to immune evasion, underscoring their potential as therapeutic targets in GBM treatment. Given this context, our study aimed to target the GBM TME, with a special focus on pericytes expressing CD19, to evaluate the potential effectiveness of CD19 CAR-iNK cells against GBM. We performed CD19 CAR transduction in induced pluripotent stem cell-derived NK (iNK) cells. To determine whether CD19 CAR targets the TME pericytes in GBM, we developed GBM-blood vessel assembloids (GBVA) by fusing GBM spheroids with blood vessel organoids. When co-cultured with GBVA, CD19 CAR-iNK cells migrated towards the pericytes surrounding the GBM. Using a microfluidic chip, we demonstrated CD19 CAR-iNK cells' targeted action and cytotoxic effects in a perfusion-like environment. GBVA xenografts recapitulated the TME including human CD19-positive pericytes, thereby enabling the application of an in vivo model for validating the efficacy of CD19 CAR-iNK cells against GBM. Compared to GBM spheroids, the presence of pericytes significantly enhanced CD19 CAR-iNK cell migration towards GBM and reduced proliferation. These results underline the efficacy of CD19 CAR-iNK cells in targeting pericytes within the GBM TME, suggesting their potential therapeutic value for GBM treatment.
Topics: Pericytes; Humans; Tumor Microenvironment; Glioblastoma; Killer Cells, Natural; Antigens, CD19; Animals; Cell Movement; Receptors, Chimeric Antigen; Induced Pluripotent Stem Cells; Cell Line, Tumor; Immunotherapy, Adoptive; Brain Neoplasms; Mice; Xenograft Model Antitumor Assays
PubMed: 38508081
DOI: 10.1016/j.biopha.2024.116436 -
Investigative Ophthalmology & Visual... Mar 2024Loss-of-function variants in the ANGPTL7 gene are associated with protection from glaucoma and reduced intraocular pressure (IOP). We investigated the role of ANGPTL7 in...
PURPOSE
Loss-of-function variants in the ANGPTL7 gene are associated with protection from glaucoma and reduced intraocular pressure (IOP). We investigated the role of ANGPTL7 in IOP homeostasis and its potential as a target for glaucoma therapeutics.
METHODS
IOP, outflow facility, and outflow tissue morphology of Angptl7 knockout (KO) mice were assessed with and without dexamethasone (Dex). ANGPTL7 was quantified in conditioned media from human trabecular meshwork cells in response to Dex, in effluent from perfused human donor eyes, and in aqueous humor from human patients treated with steroids. Antibodies to ANGPTL7 were generated and tested in three-dimensional (3D) culture of outflow cells and perfused human donor eyes. Rabbits were injected intravitreally with a neutralizing antibody targeting ANGPTL7, and IOP was measured.
RESULTS
IOP was significantly elevated, but outflow facility and outflow tissue morphology were not different between Angptl7 KO mice and littermates. When challenged with Dex, IOP increased in wild-type but not Angptl7 KO mice. In human samples, increased ANGPTL7 was seen in the aqueous humor of patients treated with steroids, regardless of glaucoma status. Using 3D culture, recombinant ANGPTL7 decreased, and ANGPTL7-blocking antibodies increased hydraulic conductivity. Significantly, outflow facility increased in human eyes treated ex vivo with ANGPTL7-blocking antibodies, and IOP decreased for 21 days in rabbits after a single injection of blocking antibodies.
CONCLUSIONS
Using multiple models, we have demonstrated that excess ANGPTL7 increases outflow resistance and IOP and that neutralizing ANGPTL7 has beneficial effects in both naïve and steroid-induced hypertensive eyes, thus motivating the development of ANGPTL7-targeting therapeutics for the treatment of glaucoma.
Topics: Animals; Mice; Humans; Rabbits; Antibodies, Blocking; Glaucoma; Eye; Antibodies, Neutralizing; Mice, Knockout; Steroids; Angiopoietin-like Proteins; Angiopoietin-Like Protein 7
PubMed: 38497513
DOI: 10.1167/iovs.65.3.22 -
Materials Today. Bio Apr 2024In this study, we explore the impact of mechanical stimuli on skin models using an innovative skin-on-a-chip platform, addressing the limitations of conventional...
In this study, we explore the impact of mechanical stimuli on skin models using an innovative skin-on-a-chip platform, addressing the limitations of conventional transwell-cultured skin equivalents. This platform facilitates cyclic mechanical stimulation through compression and stretching, combined with automated media perfusion. Our findings, using bioimaging and bulk RNA sequencing, reveal increased expression of Keratin 10 and Keratin 14, indicating enhanced skin differentiation and mechanical integrity. The increase in desmosomes and tight junctions, observed through Claudin-1 and Desmoplakin 1 & 2 analysis, suggests improved keratinocyte differentiation due to mechanical stimulation. Gene expression analyses reveal a nuanced regulatory response, suggesting a potential connection to the Hippo pathway, indicative of a significant cellular reaction to mechanical stimuli. The results show the important influence of mechanical stimulation on skin model integrity and differentiation, demonstrating the potential of our microfluidic platform in advancing skin biology research and pharmaceutical testing.
PubMed: 38495916
DOI: 10.1016/j.mtbio.2024.101010