Dataset Information

Spheroid Size Does not Impact Metabolism of the ?-blocker Propranolol in 3D Intestinal Fish Model.

ABSTRACT: Compared to two-dimensional (2D) cell culture, cellular aggregates or spheroids (3D) offer a more appropriate alternative in vitro system where individual cell-cell communication and micro-environment more closely represent the in vivo organ; yet we understand little of the physiological conditions at this scale. The relationship between spheroid size and oxygen microenvironment, an important factor influencing the metabolic capacity of cells, was first established using the fish intestine derived RTgutGC cell line. Subsequently, pharmaceutical metabolism (Propranolol), as determined by high performance liquid chromatography, in this intestinal model was examined as a function of spheroid size. Co-efficient of variation between spheroid size was below 12% using the gyratory platform method, with the least variation observed in the highest cell seeding density. The viable, high oxygen micro-environment of the outer rim of the spheroid, as determined by electron paramagnetic resonance (EPR) oximetry, decreased over time, and the hypoxic zone increased as a function of spheroid size. Despite a trend of higher metabolism in smaller spheroids, the formation of micro-environments (quiescent, hypoxic or anoxic) did not significantly affect metabolism or function of an environmentally relevant pharmaceutical in this spheroid model.

SUBMITTER: Langan LM

PROVIDER: S-EPMC6113889 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Spheroid Size Does not Impact Metabolism of the β-blocker Propranolol in 3D Intestinal Fish Model.

Langan Laura M LM Owen Stewart F SF Trznadel Maciej M Dodd Nicholas J F NJF Jackson Simon K SK Purcell Wendy M WM Jha Awadhesh N AN

Frontiers in pharmacology 20180822

Compared to two-dimensional (2D) cell culture, cellular aggregates or spheroids (3D) offer a more appropriate alternative <i>in vitro</i> system where individual cell-cell communication and micro-environment more closely represent the <i>in vivo</i> organ; yet we understand little of the physiological conditions at this scale. The relationship between spheroid size and oxygen microenvironment, an important factor influencing the metabolic capacity of cells, was first established using the fish i ...[more]

PMID: 30186177

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Project description:Study objectivesPosttraumatic nightmares may exacerbate and perpetuate the daytime symptoms of posttraumatic stress disorder and might represent a therapeutic target. The therapeutic strategy of memory reconsolidation using the β-adrenergic receptor blocker propranolol associated with re-exposure psychotherapy is a promising treatment in patients with posttraumatic stress disorder. Previous studies have established this therapy is effective in reducing overall clinician-assessed posttraumatic stress disorder symptoms, but to date no previous study has specifically focused on posttraumatic nightmares in this therapy. This study provides a preliminary assessment of the evolution of nightmare severity during this therapy protocol compared with the decrease of the other posttraumatic stress disorder symptoms. It evaluates the incidence of side effects and examines the relative effects on posttraumatic nightmares.MethodsPatients were recruited as part of the Paris Mémoire Vive Study. Data were collected using a prospective longitudinal design including 1 baseline visit, 6 therapeutic visits, and 2 follow-up visits. During the 6 therapeutic visits, propranolol was administered orally 60 to 75 minutes prior to the psychotherapeutic session.ResultsOn average, nightmare severity decreased from "severe" to "mild" during the protocol and remained stable 2 months after the last session. Whereas 85% of patients reported nightmares at baseline, only 50% still had them after the protocol. The protocol was generally well tolerated and did not increase nightmare severity for any patient in the study.ConclusionsMemory reconsolidation therapy with propranolol seems promising in reducing nightmare severity, up to and including remission. However, research using a randomized controlled design and assessing maintenance of nightmare extinction is warranted.Clinical trial registrationRegistry: ClinicalTrials.gov; Name: Using Reconsolidation Blockade to Treat Trauma Related Disorders After Paris Attacks: An Effectiveness Study (PARIS-MEM); Identifier: NCT02789982; URL: https://www.clinicaltrials.gov/ct2/show/NCT02789982.CitationMallet C, Chick CF, Maatoug R, Fossati P, Brunet A, Millet B. Memory reconsolidation impairment using the β-adrenergic receptor blocker propranolol reduces nightmare severity in patients with posttraumatic stress disorder: a preliminary study. J Clin Sleep Med. 2022;18(7):1847-1855.

Project description:In a mouse model of oxygen induced retinopathy (OIR), beta adrenergic receptor (BAR) blockade has been shown to recover hypoxia-associated retinal damages. Although the adrenergic signaling is an important regulator of apoptotic and autophagic processes, the role of BARs in retinal cell death remains to be elucidated. The present study was aimed at investigating whether ameliorative effects of BAR blockers may occur through their coordinated action on apoptosis and autophagy. To this aim, retinas from control and OIR mice untreated or treated with propranolol, a non-selective BAR1/2 blocker, were characterized in terms of expression and localization of apoptosis and autophagy markers. The effects of propranolol on autophagy signaling were also evaluated and specific autophagy modulators were used to get functional information on the autophagic effects of BAR antagonism. Finally, propranolol effects on neurodegenerative processes were associated to an electrophysiological investigation of retinal function by recording electroretinogram (ERG). We found that retinas of OIR mice are characterized by increased apoptosis and decreased autophagy, while propranolol reduces apoptosis and stimulates autophagy. In particular, propranolol triggers autophagosome formation in bipolar, amacrine and ganglion cells that are committed to die by apoptosis in response to hypoxia. Also our data argue that propranolol, through the inhibition of the Akt-mammalian target of rapamycin pathway, activates autophagy which decreases retinal cell death. At the functional level, propranolol recovers dysfunctional ERG by recovering the amplitude of a- and b-waves, and oscillatory potentials, thus indicating an efficient restoring of retinal transduction. Overall, our results demonstrate that BAR1/2 are key regulators of retinal apoptosis/autophagy, and that BAR1/2 blockade leads to autophagy-mediated neuroprotection. Reinstating the balance between apoptotic and autophagic machines may therefore be viewed as a future goal in the treatment of retinopathies.

Project description:Background:Three dimensional (3D) cell cultures have been an area of increasing interest and relevance across several research fields including drug discovery, developmental biology and stem cell-based therapies. However, handling 3D structures can be difficult. In particular, the replacement of liquid media and reagents in which liquid is removed using pipettes is difficult to perform as the 3D spheroids can be easily aspirated into the pipette tip. Results:We have developed the 3D-tip, a novel tool that facilitates media change and washing procedures of 3D-spheroid cultures. The 3D-tip contains a mesh with 40-?m pores allowing the aspiration of liquids including media, drugs, buffers and reagents, with the mesh acting as a barrier preventing the spheroids being aspirated into the pipette tip. After aspiration of liquids, the spheroids are gently deposited back into the culture vessel. Our results demonstrate that the 3D-tips offer superior handling of 3D-spheroid cultures in comparison to commonly used methods. We showed that the 3D-tips can easily be used on both fixed and unfixed spheroids and on cancer cell, stem cell and glial cell spheroids.In contrast with the 50/50 media exchange method, the 3D-tips allow a complete media change with minimal loss of spheroids and without damaging their morphology. Our results showed that 86.0% of spheroids remained in the chamber after changing the media using the 3D-tips. In contrast, only 45.0% of spheroids remained using the 50/50 media exchange strategy.In comparison with the centrifugation technique, the 3D-tips preserved spheroids whereas centrifugation led to the loss of spheroids and/or the alteration of the size and shape of the 3D cellular structures. We observed that 87.6 and 84.6% of the fixed and unfixed spheroids remained using the 3D-tip, respectively. In contrast, only 66.3% of the fixed spheroids and 36.4% of the unfixed spheroids were left using the centrifugation method. From a time perspective, the 3D-tips dramatically reduce the time taken for replacing media. Conclusions:This novel pipette tip is suitable for high throughput screening and automation and will revolutionise the techniques used for the production and analysis of 3D spheroids.

Dataset Information

Spheroid Size Does not Impact Metabolism of the ?-blocker Propranolol in 3D Intestinal Fish Model.

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Spheroid Size Does not Impact Metabolism of the β-blocker Propranolol in 3D Intestinal Fish Model.

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