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Increased Insulin Secretion from Insulin-Secreting Cells by Construction of Mixed Multicellular Spheroids

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Abstract

Purpose

We previously have shown that multicellular spheroids containing insulin-secreting cells are an effective therapy for diabetic mice. Here we attempted to increase insulin secretion by incorporating other cell types into spheroids.

Materials and Methods

Multicellular spheroids of mouse MIN6 pancreatic β cells were formed in microwells alone and with aortic vascular endothelial MAEC cells or embryo fibroblast NIH3T3 cells. mRNA expression of insulin genes and insulin secretion of MIN6 cells in each spheroid were measured by real-time PCR and an insulin ELIZA kit. Moreover, collagen IV expression in each spheroid was analyzed by western blot.

Results

In all cases, uniformly sized (about 300 μm) multicellular spheroids were obtained. MAEC or NIH3T3 cell incorporation into MIN6 spheroids significantly increased mRNA expression of insulin genes and insulin secretion. In addition, collagen IV expression, which was reported to enhance insulin secretion from pancreatic β cells, also increased in their spheroids.

Conclusions

The formation of mixed multicellular spheroids containing collagen IV-expressing cells can improve the insulin secretion from insulin-secreting MIN6 cells, and mixed multicellular spheroids can be a potent therapeutic option for patients with type I diabetes mellitus.

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Abbreviations

DMEM:

Dulbecco’s Modified Eagle’s medium

ECM:

Extracellular matrix

ELISA:

Enzyme-linked immunosorbent assay

FBS:

Fetal bovine serum

KRB buffer:

Kreb’s Ringer bicarbonate buffer

MAEC:

Mouse aorta endothelial cells

M199:

Medium 199

PAGE:

Polyacrylamide gel electrophoresis

PDMS:

Polydimethylsiloxane

PNIPAAm:

Poly(N-isopropylacrylamide)

PVDF:

Polyvinylidene fluoride

SDS:

Sodium dodecyl sulfate

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ACKNOWLEDGMENTS AND DISCLOSURES

This work was supported in part by a Grant-in-Aid for Exploratory Research (No. 23659283) from Japan Society for the Promotion of Science (JSPS), by a grant from Ritsumeikan Global Innovation Research Organization (R-GIRO), and by an iCeMS Cross-Disciplinary Research Promotion Project.

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Authors and Affiliations

  1. Department of Biopharmaceutics and Drug Metabolism Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan

    Kosuke Kusamori, Makiya Nishikawa, Narumi Mizuno, Tomoko Nishikawa, Akira Masuzawa, Yutaro Tanaka, Yuya Mizukami, Yuki Takahashi & Yoshinobu Takakura

  2. Institute for Innovative NanoBio Drug Discovery and Development Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan

    Makiya Nishikawa, Kazunori Shimizu, Satoshi Konishi & Yoshinobu Takakura

  3. Institute for Integrated Cell-Material Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan

    Makiya Nishikawa & Yuki Takahashi

  4. Ritsumeikan-Global Innovation Research Organization, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan

    Kazunori Shimizu & Satoshi Konishi

  5. Department of Mechanical Engineering, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan

    Satoshi Konishi

Authors
  1. Kosuke Kusamori
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  2. Makiya Nishikawa
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  3. Narumi Mizuno
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  4. Tomoko Nishikawa
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  5. Akira Masuzawa
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  6. Yutaro Tanaka
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  7. Yuya Mizukami
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  8. Kazunori Shimizu
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  11. Yoshinobu Takakura
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Corresponding author

Correspondence to Makiya Nishikawa.

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Kusamori, K., Nishikawa, M., Mizuno, N. et al. Increased Insulin Secretion from Insulin-Secreting Cells by Construction of Mixed Multicellular Spheroids. Pharm Res 33, 247–256 (2016). https://doi.org/10.1007/s11095-015-1783-2

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  • DOI: https://doi.org/10.1007/s11095-015-1783-2

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