Real-Time Non-Invasive and Direct Determination of Lactate Dehydrogenase Activity in Cerebral Organoids-A New Method to Characterize the Metabolism of Brain Organoids?

Gal Sapir,Rami I Aqeilan,Daniel J Steinberg,Rachel Katz-Brull

doi:10.3390/ph14090878

Gal Sapir, Rami I Aqeilan + Show 2 more

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https://doi.org/10.3390/ph14090878

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Abstract

Organoids are a powerful tool in the quest to understand human diseases. As the developing brain is extremely inaccessible in mammals, cerebral organoids (COs) provide a unique way to investigate neural development and related disorders. The aim of this study was to utilize hyperpolarized 13C NMR to investigate the metabolism of COs in real-time, in a non-destructive manner. The enzymatic activity of lactate dehydrogenase (LDH) was determined by quantifying the rate of [1-13C]lactate production from hyperpolarized [1-13C]pyruvate. Organoid development was assessed by immunofluorescence imaging. Organoid viability was confirmed using 31P NMR spectroscopy. A total of 15 organoids collated into 3 groups with a group total weight of 20–77 mg were used in this study. Two groups were at the age of 10 weeks and one was at the age of 33 weeks. The feasibility of this approach was demonstrated in both age groups, and the LDH activity rate was found to be 1.32 ± 0.75 nmol/s (n = 3 organoid batches). These results suggest that hyperpolarized NMR can be used to characterize the metabolism of brain organoids with a total tissue wet weight of as low as 20 mg (<3 mm3) and a diameter ranging from 3 to 6 mm.

Highlights

In the past decade, the fields of developmental biology and disease modelling were revolutionized by the emergence of organoid cultures [1,2,3]
Human brain organoids are derived from human pluripotent stem cells, which can be induced to form a wide variety of brain regions, such as the forebrain, midbrain, and hindbrain [9,10]
In order to validate the successful generation of cerebral organoids (COs), the organoids were fixated and cryosectioned at weeks 10 and 33 and stained for general neuronal markers and SOX2, which is a nuclear marker for pan-radial glia

Summary

Introduction

The fields of developmental biology and disease modelling were revolutionized by the emergence of organoid cultures [1,2,3]. Organoids are used for studying cancer [4] and various organ development and disease (e.g., kidney [5], liver [6], and pancreas [7]), as well as neural development and diseases [8]. Human brain organoids are derived from human pluripotent stem cells (hPSCs), which can be induced to form a wide variety of brain regions, such as the forebrain, midbrain, and hindbrain [9,10]. Metabolism is a key feature of embryogenesis and cellular disease. Considering the high level of resources needed for organoid preparation, it appears that a metabolic investigation that is non-invasive and non-destructive is especially needed Due to the dynamic nature of organoid development and the fast and regulated changes that occur in them, real-time monitoring of enzyme activities in these research tissues could be beneficial.

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