A high-throughput assay for the measurement of Ca2+-oscillations and insulin release from uniformly sized β-cell spheroids
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eLife Assessment
In this valuable study, Robben et al. describe a 3D beta-cell spheroid platform, a tool allowing high-throughput monitoring of cytoplasmic calcium concentrations and insulin secretion, with calcium signals comparable to those recorded in primary pancreatic islets. The authors demonstrate the method by culturing MIN6 cells in a 3D culture system, and show solid evidence of its utility by recording calcium signals in a high-throughput format and characterizing these calcium signals using pharmacological tools. This highlights the potential utility of the 3D beta-cell spheroids for screening new pharmacological modulators of pancreatic beta-cell function.
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Abstract
Diabetes mellitus is a rapidly growing global health challenge, necessitating the development of more effective anti-diabetic therapies, including drugs that improve insulin release from pancreatic β-cells. Traditional high-throughput screening methods typically rely on 2D β-cell cultures, but such cultures do not mimic the 3D organisation and cell-to-cell communication of β-cells in pancreatic islets of Langerhans. Existing 3D β-cell culture models are hindered by high costs, technical complexity, and limited compatibility with high-throughput screening platforms. In this work, we developed an approach for generating 19 homogeneously shaped pancreatic β-cell spheroids in each well of a 96-well plate, using micropatterned polyethylene glycol (PEG)-based hydrogels and MIN6 insulinoma cells. The uniform shape and positioning of the individual spheroids enabled the simultaneous, real-time imaging of Ca2+ signals in up to 1824 independent spheroids in response to glucose and various test compounds. Using this approach, we show that increasing glucose causes concentration-dependent Ca2+ oscillations in individual spheroids, that these Ca2+ oscillations are sensitive to modulators of ATP-sensitive K+ channels, and that the frequency of Ca2+ oscillations correlate with insulin secretion. Finally, we demonstrate that the neurosteroid pregnenolone sulphate, an agonist of the cation channel TRPM3, increases the frequency of glucose-induced Ca2+ oscillations and enhances insulin release; the TRPM3 antagonist isosakuranetin inhibited these responses. In conclusion, we established a cost-effective and scalable 3D β-cell platform for high-throughput screening of insulin release-modifying compounds, with potential applications in drug development and personalized medicine for the management of diabetes mellitus.
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eLife Assessment
In this valuable study, Robben et al. describe a 3D beta-cell spheroid platform, a tool allowing high-throughput monitoring of cytoplasmic calcium concentrations and insulin secretion, with calcium signals comparable to those recorded in primary pancreatic islets. The authors demonstrate the method by culturing MIN6 cells in a 3D culture system, and show solid evidence of its utility by recording calcium signals in a high-throughput format and characterizing these calcium signals using pharmacological tools. This highlights the potential utility of the 3D beta-cell spheroids for screening new pharmacological modulators of pancreatic beta-cell function.
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Reviewer #1 (Public review):
Summary:
They use cultures of insulinoma MIN6 cells that form spheroids in a micro-patterned PEG-hydrogel to measure Ca2+ oscillations in multiple cells simultaneously.
Strengths:
They demonstrate that insulinoma spheroids are formed in multi-well plates and that Ca2+ imaging can be performed on them.
Weaknesses:
The type of equipment and multi-wells used for the experiments are very specialized to be used as a common tool. Insulinoma cells are tumoral cell lines that divide, unlike primary beta cells. Pancreatic islets are very different from this preparation, as they are highly heterogeneous, whereas these cells all respond equally. It would be good to see the same technique applied to primary cells.
MIN6 cells do not respond to glucose and other secretagogues in the same way as primary cells, and they …
Reviewer #1 (Public review):
Summary:
They use cultures of insulinoma MIN6 cells that form spheroids in a micro-patterned PEG-hydrogel to measure Ca2+ oscillations in multiple cells simultaneously.
Strengths:
They demonstrate that insulinoma spheroids are formed in multi-well plates and that Ca2+ imaging can be performed on them.
Weaknesses:
The type of equipment and multi-wells used for the experiments are very specialized to be used as a common tool. Insulinoma cells are tumoral cell lines that divide, unlike primary beta cells. Pancreatic islets are very different from this preparation, as they are highly heterogeneous, whereas these cells all respond equally. It would be good to see the same technique applied to primary cells.
MIN6 cells do not respond to glucose and other secretagogues in the same way as primary cells, and they cycle, depending on the phase of the cycle to which they are exposed.
The authors should report the number of cells per spheroid and the number of cells that are alive and dead.
I would like to examine the effects of calcium channel blockers on calcium transients, and the use of pregnenolone is already described in the literature, but remains less well known.
MIN6 cells secrete much insulin, because detecting the hormone in ELISAs requires too many primary cells. The authors should discuss the model in greater detail and compare it with primary beta cells. Also, they take 3 mM glucose as the basal concentration, which is low.
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Reviewer #2 (Public review):
Summary:
The study by Robben et al., show 3D beta-cell spheroid platform, a valuable tool allowing high-throughput monitoring of cytoplasmic Ca concentrations and insulin secretion, with Ca signals comparable to those recorded in primary islets. The authors demonstrate a solid method to culturing MIN6 cells in a 3D culture system, recording Ca signals in a high-throughput format and characterizing these Ca signals using pharmacological tools, including TRPM3 channel and K-ATP channel modulators. This highlights the utility of the 3D beta-cell spheroid for screening new ion channel modulators in beta-cells of the pancreas.
Strengths:
- The study shows that the MIN-6-based 3D beta-cell model is better to study Ca-signaling and insulin secretion compared to 2D culture of single MIN-6 cells.
- The method allows …
Reviewer #2 (Public review):
Summary:
The study by Robben et al., show 3D beta-cell spheroid platform, a valuable tool allowing high-throughput monitoring of cytoplasmic Ca concentrations and insulin secretion, with Ca signals comparable to those recorded in primary islets. The authors demonstrate a solid method to culturing MIN6 cells in a 3D culture system, recording Ca signals in a high-throughput format and characterizing these Ca signals using pharmacological tools, including TRPM3 channel and K-ATP channel modulators. This highlights the utility of the 3D beta-cell spheroid for screening new ion channel modulators in beta-cells of the pancreas.
Strengths:
- The study shows that the MIN-6-based 3D beta-cell model is better to study Ca-signaling and insulin secretion compared to 2D culture of single MIN-6 cells.
- The method allows imaging of Ca signaling in many spheroids in parallel followed by collecting medium to measure insulin release and correlate both effects.
- The authors demonstrate that this system is suitable for screening new pharmacological modulators and used as an agonist of the ATP-sensitive potassium channel (diazoxide) and the agonist and antagonist of the TRPM3 channel.
Weaknesses:
- The study is based on only one cell line, the MIN6 insulinoma cells, which may not fully mimic the pancreatic beta-cells within the islet.
- The authors show only spheroids cultured overnight. A long-term culture is missing to assess beta-cell viability long term function.
- The authors tested their platform using only two compounds. Testing a larger compound library is necessary to make a clear conclusion about the suitability of the platform for high-throughput screening.
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Reviewer #3 (Public review):
Summary:
The primary objective of this study is to develop high-throughput screening assays utilizing homogeneous 3D cell cultures that more accurately replicate the intricate architecture and cellular communication found in tissues. The authors have chosen pancreatic islet β-cells as a model system to evaluate agents that modulate insulin release, which is particularly relevant given the increasing prevalence of diabetes mellitus-a significant global health concern. Moreover, the incorporation of human-based 3D spheroids, organoids, or organ-on-chip technologies into drug discovery protocols is essential for enhancing clinical translation, as candidate compounds identified using animal models have often demonstrated limited success in clinical settings.
Strengths:
This study was thoughtfully planned and …
Reviewer #3 (Public review):
Summary:
The primary objective of this study is to develop high-throughput screening assays utilizing homogeneous 3D cell cultures that more accurately replicate the intricate architecture and cellular communication found in tissues. The authors have chosen pancreatic islet β-cells as a model system to evaluate agents that modulate insulin release, which is particularly relevant given the increasing prevalence of diabetes mellitus-a significant global health concern. Moreover, the incorporation of human-based 3D spheroids, organoids, or organ-on-chip technologies into drug discovery protocols is essential for enhancing clinical translation, as candidate compounds identified using animal models have often demonstrated limited success in clinical settings.
Strengths:
This study was thoughtfully planned and skillfully carried out. The use of micropatterned hydrogels to observe 19 spheroids at once is an ingenious aspect, which has been effectively validated with Ca microfluorography. Overall, I found this investigation to be exceptionally well-executed and free from notable flaws, as the results clearly back up the conclusions. Additionally, the developed method achieved the proposed aims, providing a high-throughput format with 3D cultures. I believe this study deserves publication.
Weaknesses:
For an HTS assay, authors should incorporate the Z-factor.
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