Restraint upon Embryonic Metatarsal Ex Vivo Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR Pathway

Mechanical cues play a vital role in limb skeletal development, yet their influence and underpinning mechanisms in the regulation of endochondral ossification (EO) processes are incompletely defined. Furthermore, interactions between endochondral growth and mechanics and the mTOR/NF-ĸB pathways are yet to be explored. An appreciation of how mechanical cues regulate EO would also clearly be beneficial in the context of fracture healing and bone diseases, where these processes are recapitulated. The study herein addresses the hypothesis that the mTOR/NF-ĸB pathways interact with mechanics to control endochondral growth. To test this, murine embryonic metatarsals were incubated ex vivo in a hydrogel, allowing for the effects of quasi-static loading on longitudinal growth to be assessed. The results showed significant restriction of metatarsal growth under quasi-static loading during a 14-day period and concentration-dependent sensitivity to hydrogel-related restriction. This study also showed that hydrogel-treated metatarsals retain their viability and do not present with increased apoptosis. Metatarsals exhibited reversal of the growth-restriction when co-incubated with mTOR compounds, whilst it was found that these compounds showed no effects under basal culture conditions. Transcriptional changes linked to endochondral growth were assessed and downregulation of Col2 and Acan was observed in hydrogel-treated metatarsi at day 7. Furthermore, cell cycle analyses confirmed the presence of chondrocytes exhibiting S-G2/M arrest. These data indicate that quasi-static load provokes chondrocyte cell cycle arrest, which is partly overcome by mTOR, with a less marked interaction for NF-ĸB regulators.

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Cell Cycle Activation of Peripheral Blood Stem and Progenitor Cells Expanded Ex Vivo with SCF, FLT-3 Ligand, TPO, and IL-3 Results in Accelerated Granulocyte Recovery in a Baboon Model of Autologous Transplantation but G0/G1and S/G2/M Graft Cell Content Does Not Correlate with Tranplantability

Stem Cells ◽

10.1634/stemcells.19-5-436 ◽

2001 ◽

Vol 19 (5) ◽

pp. 436-442 ◽

Cited By ~ 7

Author(s):

M. Drouet ◽

F. Herodin ◽

F. Norol ◽

F. Mourcin ◽

J. F. Mayol

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Peripheral Blood ◽

Ex Vivo ◽

Autologous Transplantation ◽

Cell Content ◽

Cell Cycle Activation ◽

Baboon Model ◽

Stem And Progenitor Cells

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Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria

Microbiome ◽

10.1186/s40168-020-00975-x ◽

2021 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Leeann Klassen ◽

Greta Reintjes ◽

Jeffrey P. Tingley ◽

Darryl R. Jones ◽

Jan-Hendrik Hehemann ◽

...

Keyword(s):

Metabolic Pathways ◽

Ex Vivo ◽

Rapid Assessment ◽

Vital Role ◽

Strain Level ◽

Metabolic Phenotype ◽

Specific Cell ◽

Bacterial Cells ◽

Carbohydrate Utilization

AbstractGut microbiomes, such as the microbial community that colonizes the rumen, have vast catabolic potential and play a vital role in host health and nutrition. By expanding our understanding of metabolic pathways in these ecosystems, we will garner foundational information for manipulating microbiome structure and function to influence host physiology. Currently, our knowledge of metabolic pathways relies heavily on inferences derived from metagenomics or culturing bacteria in vitro. However, novel approaches targeting specific cell physiologies can illuminate the functional potential encoded within microbial (meta)genomes to provide accurate assessments of metabolic abilities. Using fluorescently labeled polysaccharides, we visualized carbohydrate metabolism performed by single bacterial cells in a complex rumen sample, enabling a rapid assessment of their metabolic phenotype. Specifically, we identified bovine-adapted strains of Bacteroides thetaiotaomicron that metabolized yeast mannan in the rumen microbiome ex vivo and discerned the mechanistic differences between two distinct carbohydrate foraging behaviors, referred to as “medium grower” and “high grower.” Using comparative whole-genome sequencing, RNA-seq, and carbohydrate-active enzyme fingerprinting, we could elucidate the strain-level variability in carbohydrate utilization systems of the two foraging behaviors to help predict individual strategies of nutrient acquisition. Here, we present a multi-faceted study using complimentary next-generation physiology and “omics” approaches to characterize microbial adaptation to a prebiotic in the rumen ecosystem.

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Phagocytosis of Liposome Particles by Rat Splenic Immature Monocytes Makes Them Transiently and Highly Immunosuppressive In Ex Vivo Culture Conditions

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.110.172510 ◽

2011 ◽

Vol 337 (1) ◽

pp. 42-49 ◽

Cited By ~ 8

Author(s):

Daisuke Takahashi ◽

Hiroshi Azuma ◽

Hiromi Sakai ◽

Keitaro Sou ◽

Daiko Wakita ◽

...

Keyword(s):

Ex Vivo ◽

Culture Conditions ◽

Ex Vivo Culture

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602 STING agonist-based treatment promotes vascular normalization and tertiary lymphoid structure formation in the therapeutic melanoma microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0602 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A637-A637

Author(s):

Manoj Chelvanambi ◽

Ronald Fecek ◽

Jennifer Taylor ◽

Walter Storkus

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Cell ◽

Ex Vivo ◽

In Vitro Assays ◽

Type I ◽

Vascular Normalization ◽

S 100 ◽

Transcriptional Changes

BackgroundThe degree of immune infiltration in tumors, especially CD8+ T cells, greatly impacts patient disease course and response to interventional immunotherapy. Hence, enhancement of TIL prevalence is a preferred clinical endpoint, one that may be achieved via administration of agents that normalize the tumor vasculature (VN) leading to improved immune cell recruitment and/or that induce the development of local tertiary lymphoid structures (TLS) within the tumor microenvironment (TME).MethodsLow-dose STING agonist ADU S-100 (5 μg/mouse) was delivered intratumorally to established s.c. B16.F10 melanomas on days 10, 14 and 17 post-tumor inoculation under an IACUC-approved protocol. Treated and control, untreated tumors were isolated at various time points to assess transcriptional changes associated with VN and TLS formation via qPCR, with corollary immune cell composition changes determined using flow cytometry and immunofluorescence microscopy. In vitro assays were performed on CD11c+ BMDCs treated with 2.5 μg/mL ADU S-100 (vs PBS control) and associated transcriptional changes analyzed via qPCR or profiled using DNA microarrays. For TCRβ-CDR3 analyses, CDR3 was sequenced from gDNA isolated from enzymatically digested tumors and splenocytes.ResultsWe report that activation of STING within the TME leads to slowed melanoma growth in association with increased production of angiostatic factors including Tnfsf15 (Vegi), Cxcl10 and Angpt1, and TLS inducing factors including Ccl19, Ccl21, Lta, Ltb and Tnfsf14 (Light). Therapeutic responses from intratumoral STING activation were characterized by increased vascular normalization (VN), enhanced tumor infiltration by CD8+ T cells and CD11c+ DCs and local TLS neo-genesis, all of which were dependent on host expression of STING. Consistent with a central role for DC in TLS formation, ex vivo ADU S-100-activated mCD11c+ DCs also exhibited upregulated expression of TLS promoting factors including lymphotoxin-α (LTA), IL-36, inflammatory chemokines and type I interferons. TLS formation was associated with the development of a therapeutic TIL TCR repertoire enriched in T cell clonotypes uniquely detected within the tumor but not the peripheral circulation in support or local T cell cross-priming within the TME.ConclusionsOur data support the premise that i.t. delivery of STING agonist promotes a pro-inflammatory TME in support of VN and TLS formation, leading to the local expansion of unique TIL repertoire in association with superior anti-melanoma efficacy.

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Mimicking of Blood Flow Results in a Distinct Functional Phenotype in Human Non-Adherent Classical Monocytes

Biology ◽

10.3390/biology10080748 ◽

2021 ◽

Vol 10 (8) ◽

pp. 748

Author(s):

Elisa Wirthgen ◽

Melanie Hornschuh ◽

Ida Maria Wrobel ◽

Christian Manteuffel ◽

Jan Däbritz

Keyword(s):

Blood Flow ◽

Shear Flow ◽

Ex Vivo ◽

Culture Conditions ◽

Inflammatory Signaling ◽

Gm Csf ◽

Beneficial Effects ◽

Migratory Capacity ◽

Ex Vivo Culture ◽

Static Conditions

Ex vivo culture conditions during the manufacturing process impact the therapeutic effect of cell-based products. Mimicking blood flow during ex vivo culture of monocytes has beneficial effects by preserving their migratory ability. However, the effects of shear flow on the inflammatory response have not been studied so far. Hence, the present study investigates the effects of shear flow on both blood-derived naïve and activated monocytes. The activation of monocytes was experimentally induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), which acts as a pro-survival and growth factor on monocytes with a potential role in inflammation. Monocytes were cultured under dynamic (=shear flow) or static conditions while preventing monocytes' adherence by using cell-repellent surfaces to avoid adhesion-induced differentiation. After cultivation (40 h), cell size, viability, and cytokine secretion were evaluated, and the cells were further applied to functional tests on their migratory capacity, adherence, and metabolic activity. Our results demonstrate that the application of shear flow resulted in a decreased pro-inflammatory signaling concurrent with increased secretion of the anti-inflammatory cytokine IL-10 and increased migratory capacity. These features may improve the efficacy of monocyte-based therapeutic products as both the unwanted inflammatory signaling in blood circulation and the loss of migratory ability will be prevented.

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Temperature Controlled and Monitored Ex Vivo Lung Perfusion System for Research and Training Purposes

Current Directions in Biomedical Engineering ◽

10.1515/cdbme-2019-0074 ◽

2019 ◽

Vol 5 (1) ◽

pp. 293-295

Author(s):

Christina Pongratz ◽

Jens Ziegle ◽

Axel Boese ◽

Michael Friebe ◽

Helena Linge ◽

...

Keyword(s):

Storage Temperature ◽

Ex Vivo ◽

Culture Conditions ◽

Lung Perfusion ◽

Tissue Storage ◽

Ex Vivo Lung Perfusion ◽

Device Development ◽

Preservation Method ◽

Normothermic Perfusion ◽

Short Period

AbstractEx vivo lung perfusion (EVLP) is a preservation method for donor lungs, which keep lungs viable in a physiological environment outside of a body for a short period of time. EVLP is established clinically for lung transplantation. Experimental applications for EVLP are e.g. lung cancer research or medical device development and testing. For preservation, a lung is ventilated artificially in an organ chamber and perfused antegrade through the pulmonary artery. Here we introduce a thermoregulation system for an experimental EVLP system to be used for translational research approaches as well as for training medical staff. To implement physiological culture conditions that are a prerequisite for lung preservation and tissue homeostasis, a thermoregulation is needed to rewarm the explanted lung tissue (storage temperature 4°C). Technically, the EVLP system must be thermally insulated, so loss of caloric is avoided. For monitoring, temperature sensors are integrated within the lung, in the organ chamber and in the afferent perfusate tube, whereby the measured values determine the thermoregulation. Initial tests using thermal packs (cooled to 4-6°C) placed on a heating mat, as a part of the perfusion circuit, showed that the perfusate temperature falls to 34°C, but restores after approximately 60 minutes (36.5°C), whereby the thermal pack is warmed. With this setup longer perfusion times should be obtained rather than without thermoregulation due to normothermic perfusion of the lung.

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EXTH-48. NOVEL COMBINATION THERAPY IN PRECLINICAL GLIOMA MODELS USING THE CELL CYCLE STABILIZING COMPOUND ARGYRIN F AND CHECKPOINT INHIBITION

Neuro-Oncology ◽

10.1093/neuonc/noab196.687 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi174-vi174

Author(s):

Bianca Walter ◽

Denis Canjuga ◽

Simge G Yuez ◽

Michael Ghosh ◽

Przemyslaw Bozko ◽

...

Keyword(s):

Cell Cycle ◽

Ex Vivo ◽

Tumor Infiltrating Lymphocytes ◽

Clonogenic Survival ◽

Autologous Tumor ◽

Cycle Arrest ◽

Resected Tissue ◽

Infiltrating Lymphocytes

Abstract Glioblastoma are incurable aggressive tumors and remain a therapeutic challenge. Glioblastoma frequently harbor alterations in the retinoblastoma pathway with subsequent cell cycle abnormalities. Here, we aimed to investigate the anti-glioma activity of the cell cycle-stabilizing compound Argyrin F and its potential treatment-induced vulnerabilities to exploit possibilities for novel combination therapies. We investigated cell viability, clonogenic survival, cell cycle status and immunoblots of human and murine glioma cells treated with Argyrin F. Moreover, we established an ex vivo glioma model using residual freshly resected tissue from patients, i.e. patient-derived microtumors (PDMs). Additionally, we extracted autologous tumor infiltrating lymphocytes (TILs) to perform co-culturing experiments. We performed mass spectrometry-based immunopeptidomics and used the orthotopic syngeneic SMA560/VM/Dk glioma mouse model. Argyrin F displayed anti-glioma efficacy in glioma cell lines in vitro and in PDM models ex vivo. Moreover, Argyrin F treatment induced cell cycle arrest, reduced clonogenic survival in vitro and prolonged survival in vivo. Argyrin F-treated SMA560 glioma displayed 4.6-fold more glioma-infiltrating CD8+ T cells. We discovered a distinctive treatment-induced immunopeptidome. Combination of Argyrin F plus PD-1 antibody increased cellular toxicity in PDM/TILs co-cultures ex vivo and prolonged overall survival compared with monotherapies in vivo. We conclude that our experimental data suggest a novel combination of Argyrin F plus PD-1 blockade and its clinical translation.

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A new Plasmodium vivax reference sequence with improved assembly of the subtelomeres reveals an abundance of pir genes

Wellcome Open Research ◽

10.12688/wellcomeopenres.9876.1 ◽

2016 ◽

Vol 1 ◽

pp. 4 ◽

Cited By ~ 56

Author(s):

Sarah Auburn ◽

Ulrike Böhme ◽

Sascha Steinbiss ◽

Hidayat Trimarsanto ◽

Jessica Hostetler ◽

...

Keyword(s):

South America ◽

Plasmodium Vivax ◽

Sequence Data ◽

Ex Vivo ◽

Vital Role ◽

Asia Pacific ◽

Reference Sequence ◽

Manual Curation ◽

Illumina Sequence

Plasmodium vivax is now the predominant cause of malaria in the Asia-Pacific, South America and Horn of Africa. Laboratory studies of this species are constrained by the inability to maintain the parasite in continuous ex vivo culture, but genomic approaches provide an alternative and complementary avenue to investigate the parasite’s biology and epidemiology. To date, molecular studies of P. vivax have relied on the Salvador-I reference genome sequence, derived from a monkey-adapted strain from South America. However, the Salvador-I reference remains highly fragmented with over 2500 unassembled scaffolds. Using high-depth Illumina sequence data, we assembled and annotated a new reference sequence, PvP01, sourced directly from a patient from Papua Indonesia. Draft assemblies of isolates from China (PvC01) and Thailand (PvT01) were also prepared for comparative purposes. The quality of the PvP01 assembly is improved greatly over Salvador-I, with fragmentation reduced to 226 scaffolds. Detailed manual curation has ensured highly comprehensive annotation, with functions attributed to 58% core genes in PvP01 versus 38% in Salvador-I. The assemblies of PvP01, PvC01 and PvT01 are larger than that of Salvador-I (28-30 versus 27 Mb), owing to improved assembly of the subtelomeres. An extensive repertoire of over 1200 Plasmodium interspersed repeat (pir) genes were identified in PvP01 compared to 346 in Salvador-I, suggesting a vital role in parasite survival or development. The manually curated PvP01 reference and PvC01 and PvT01 draft assemblies are important new resources to study vivax malaria. PvP01 is maintained at GeneDB and ongoing curation will ensure continual improvements in assembly and annotation quality.

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In-Depth Analysis of the Impact of Different Serum-Free Media on the Production of Clinical Grade Dendritic Cells for Cancer Immunotherapy

Frontiers in Immunology ◽

10.3389/fimmu.2020.593363 ◽

2021 ◽

Vol 11 ◽

Author(s):

João Calmeiro ◽

Luís Mendes ◽

Iola F. Duarte ◽

Catarina Leitão ◽

Adriana R. Tavares ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Cancer Immunotherapy ◽

Ex Vivo ◽

Culture Conditions ◽

Clinical Grade ◽

Serum Free ◽

Depth Analysis ◽

Free Media ◽

The Impact

Dendritic cell (DC)-based antitumor vaccines have proven to be a safe approach, but often fail to generate robust results between trials. Translation to the clinic has been hindered in part by the lack of standard operation procedures for vaccines production, namely the definition of optimal culture conditions during ex-vivo DC differentiation. Here we sought to compare the ability of three clinical grade serum-free media, DendriMACS, AIM-V, and X-VIVO 15, alongside with fetal bovine serum-supplemented Roswell Park Memorial Institute Medium (RPMI), to support the differentiation of monocyte-derived DCs (Mo-DCs). Under these different culture conditions, phenotype, cell metabolomic profiles, response to maturation stimuli, cytokines production, allogenic T cell stimulatory capacity, as well as priming of antigen-specific CD8+ T cells and activation of autologous natural killer (NK) cells were analyzed. Immature Mo-DCs differentiated in AIM-V or X-VIVO 15 presented lower levels of CD1c, CD1a, and higher expression of CD11c, when compared to cells obtained with DendriMACS. Upon stimulation, only AIM-V or X-VIVO 15 DCs acquired a full mature phenotype, which supports their enhanced capacity to polarize T helper cell type 1 subset, to prime antigen-specific CD8+ T cells and to activate NK cells. CD8+ T cells and NK cells resulting from co-culture with AIM-V or X-VIVO 15 DCs also showed superior cytolytic activity. 1H nuclear magnetic resonance-based metabolomic analysis revealed that superior DC immunostimulatory capacities correlate with an enhanced catabolism of amino acids and glucose. Overall, our data highlight the impact of critically defining the culture medium used in the production of DCs for clinical application in cancer immunotherapy. Moreover, the manipulation of metabolic state during differentiation could be envisaged as a strategy to enhance desired cell characteristics.

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