Cell Medicine 8(1-2) Abstracts

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Cell Medicine, Vol. 8, pp. 3–7, 2015
2155-1790/15 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517915X689100
Copyright © 2015 Cognizant, LLC.
Printed in the USA. All rights reserved

Review

Cryopreservation of Adipose-Derived Mesenchymal Stem Cells

Chika Miyagi-Shiohira,* Kiyoto Kurima,* Naoya Kobayashi,† Issei Saitoh,‡ Masami Watanabe,§ Yasufumi Noguchi,¶ Masayuki Matsushita,# and Hirofumi Noguchi*

*Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
†Okayama Saidaiji Hospital, Okayama, Japan
‡Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan
§Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
¶Department of Socio-environmental Design, Hiroshima International University, Hiroshima, Japan
#Department of Molecular and Cellular Physiology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan

Mesenchymal stem cells (MSCs) have the potential to differentiate into cells of mesodermal origin such as osteoblasts, adipocytes, myocytes, and chondrocytes. They possess an immunosuppressive effect, which makes them a viable cell population for the cell-based therapy of treatment-resistant immune diseases. Adipose-derived mesenchymal stem cells (ASCs) have been demonstrated to have the ability to acquire the properties of subcutaneous adipose tissue particularly easily, and cryopreservation is currently performed as a routine method for preserving ASCs to safely acquire large numbers of cells. However, many studies have reported that cellular activity after freezing and thawing may be affected by the solutions used for cryopreservation. Dimethyl sulfoxide (DMSO) is commonly used as a cryopreservation medium as it diffuses into the cell through the plasma membrane and protects the cells from the damage caused by freezing. As substitutes for DMSO or animal-derived serum, cell banker series, polyvinylpyrrolidone (PVP), sericin and maltose, and methyl cellulose (MC) have been investigated for their clinical applications. It is critical to develop a reliable cell cryopreservation protocol for regenerative medicine using MSCs.

Key words: Adipose-derived mesenchymal stem cells (ASCs); Cryopreservation; Dimethyl sulfoxide (DMSO); Cell banker series; Polyvinylpyrrolidone (PVP)

Received June 12, 2015; final acceptance July 09, 2015. Online prepub date: August 26, 2015.
Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan. Tel: +81-98-895-3331; Fax: +81-98-895-3331; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 8, pp. 9–23, 2015
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DOI: http://dx.doi.org/10.3727/215517915X689038
Copyright © 2015 Cognizant, LLC.
Printed in the USA. All rights reserved

Choice of Feeders Is Important When First Establishing iPSCs Derived From Primarily Cultured Human Deciduous Tooth Dental Pulp Cells

Issei Saitoh,* Emi Inada,† Yoko Iwase,* Hirofumi Noguchi,‡ Tomoya Murakami,* Miki Soda,* Naoko Kubota,† Hiroko Hasegawa,† Eri Akasaka,† Yuko Matsumoto,† Kyoko Oka,§ Youichi Yamasaki,† Haruaki Hayasaki,* Masahiro Sato¶

*Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Gakkocho-dori, Chuo-ku, Niigata, Japan
†Department of Pediatric Dentistry, Kagoshima University Graduate School of Medical and Dental Sciences, Sakuragaoka, Kagoshima, Japan
‡Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Nishiharatyoaza, Uehara, Okinawa, Japan
§Section of Pediatric Dentistry Department of Oral Growth and Development Fukuoka Dental College, Sawara-ku, Tamura Fukuoka-shi, Fukuoka, Japan
¶Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Sakuragaoka, Kagoshima, Japan

Feeder cells are generally required to maintain embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs). Mouse embryonic fibroblasts (MEFs) isolated from fetuses and STO mouse stromal cell line are the most widely used feeder cells. The aim of this study was to determine which cells are suitable for establishing iPSCs from human deciduous tooth dental pulp cells (HDDPCs). Primary cultures of HDDPCs were cotransfected with three plasmids containing human OCT3/4, SOX2/KLF4, or LMYC/LIN28 and pmaxGFP by using a novel electroporation method, and then cultured in an ESC qualified medium for 15 days. Emerging colonies were reseeded onto mitomycin C-treated MEFs or STO cells. The colonies were serially passaged for up to 26 passages. During this period, colony morphology was assessed to determine whether cells exhibited ESC-like morphology and alkaline phosphatase activity to evaluate the state of cellular reprogramming. HDDPCs maintained on MEFs were successfully reprogrammed into iPSCs, whereas those maintained on STO cells were not. Once established, the iPSCs were maintained on STO cells without loss of pluripotency. Our results indicate that MEFs are better feeder cells than STO cells for establishing iPSCs. Feeder choice is a key factor enabling efficient generation of iPSCs.

Key words: Deciduous tooth; Dental pulp; Feeder cell; Induced pluripotent stem cells (iPSCs); Mouse embryonic fibroblasts (MEFs); STO cells

Received June 12, 2015; final acceptance July 20, 2015. Online prepub date: August 26, 2015.
Address correspondence to Issei Saitoh, D.D.S., Ph.D., Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, 2-5274 Gakkocho-dori, Chuo-ku, Niigata 951-8514, Japan. Tel: +81-25-227-2911; Fax: +81-25-227-2911; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 8, pp. 25–29, 2015
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DOI: http://dx.doi.org/10.3727/215517915X689047
Copyright © 2015 Cognizant, LLC.
Printed in the USA. All rights reserved

Review

Islet Culture/Preservation Before Islet Transplantation

Hirofumi Noguchi,* Chika Miyagi-Shiohira,* Kiyoto Kurima,* Naoya Kobayashi,† Issei Saitoh,‡ Masami Watanabe,§ Yasufumi Noguchi,¶ and Masayuki Matsushita#

*Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
†Okayama Saidaiji Hospital, Okayama, Japan
‡Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan
§Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
¶Department of Socio-environmental Design, Hiroshima International University, Hiroshima, Japan
#Department of Molecular and Cellular Physiology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan

Although islet culture prior to transplantation provides flexibility for the evaluation of isolated islets and the pretreatment of patients, it is well known that isolated islets deteriorate rapidly in culture. Human serum albumin (HSA) is used for medium supplementation instead of fetal bovine serum (FBS), which is typically used for islet culture research, to avoid the introduction of xenogeneic materials. However, FBS contains several factors that are beneficial to islet viability and which also neutralize the endogenous pancreatic enzymes or exogenous enzymes left over from the isolation process. Several groups have reported the comparison of cultures at 22°C and 37°C. Recent studies have demonstrated the superiority of 4°C preservation to 22°C and 37°C cultures. We herein review the current research on islet culture/preservation for clinical islet transplantation.

Key words: Islet transplantation; Culture condition; Preservation solution; University of Wisconsin (UW) solution; Low temperature

Received June 12, 2015; final acceptance July 01, 2015. Online prepub date: August 26, 2015.
Address correspondence to Hirofumi Noguchi, M.D., Ph.D., Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan. Tel: +81-98-895-3331; Fax: +81-98-895-3331; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 8, pp. 31–38, 2015
2155-1790/15 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517915X689083
Copyright © 2015 Cognizant, LLC.
Printed in the USA. All rights reserved

Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors

Hiroyuki Hanayama,* Kazuo Ohashi,† Rie Utoh,‡ Hirofumi Shimizu,* Kazuya Ise,* Fuminori Sakurai,§ Hiroyuki Mizuguchi,§ Hiroyuki Tsuchiya,† Teruo Okano,‡ and Mitsukazu Gotoh*

*Department of Regenerative Surgery, Fukushima Medical University, Hikarigaoka, Fukushima, Japan
†iPS Cell-based Projects on Cell Transplantation and Cell Dynamics, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan
‡Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Shinjuku, Tokyo, Japan
§Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan

To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells.

Key words: Dispersed islet cell; Fiber-modified adenoviral vector; Islet transplantation; Tissue engineering; Gene therapy

Received June 12, 2015; final acceptance July 17, 2015. Online prepub date: August 26, 2015.
Address correspondence to Kazuo Ohashi, M.D., Ph.D., iPS Cell-based Projects on Cell Transplantation and Cell Dynamics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel: +81-6-6879-4091; Fax: +81-6-6879-8154; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 8, pp. 39–46, 2015
2155-1790/15 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517915X689029
Copyright © 2015 Cognizant, LLC.
Printed in the USA. All rights reserved

Human Laminin Isotype Coating for Creating Islet Cell Sheets

Shingo Yamashita,*† Kazuo Ohashi,*†‡ Rie Utoh,* Teruo Okano,* and Masakazu Yamamoto†

*Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Shinjuku-ku, Tokyo, Japan
†Department of Surgery, Institute of Gastroenterology, Tokyo Women’s Medical University, Shinjuku-ku, Tokyo, Japan
‡iPS Cell-based Projects on Cell Transplantation and Cell Dynamics, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan

Our experimental approach toward the development of new islet-based treatment for diabetes mellitus has been the creation of a monolayered islet cell construct (islet cell sheet), followed by its transplantation into a subcutaneous pocket. Previous studies describe rat laminin-5 (chain composition: α3, β3, γ2) as a suitable extracellular matrix (ECM) for surfaces comprised of a coated temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). To progress toward the clinical application of this approach, the present study attempted to identify an optimal human ECM as a coating material on PIPAAm surfaces, which allowed islet cells to attach on the surfaces and subsequently to be harvested as a monolithic cell sheet. Dispersed rat islet cells were seeded onto PIPAAm dishes coated with various human laminin isotypes: human laminin (HL)-211, HL-332, HL-411, HL-511, and HL-placenta. Plating efficiency at day 1, the confluency at day 3, and glucose-stimulated insulin secretion test at day 3 were performed. The highest value of plating efficiency was found in the HL-332-PIPAAm group (83.1 ± 0.7%). The HL-332-PIPAAm group also showed the highest cellular confluency (98.6 ± 0.5%). Islet cells cultured on the HL-332-PIPAAm surfaces showed a positive response in the glucose-stimulated insulin secretion test. By reducing culture temperature from 37°C to 20°C in the HL-332-PIPAAm group, cells were able to be harvested as a monolithic islet sheet. The present study showed that HL-332 was an optimal human-derived ECM on a PIPAAm coating for preparing islet cell sheets.

Key words: Cell sheet; Pancreatic islets; Laminin; Extracellular matrix (ECM); Dispersed islet cells; Temperature-responsive polymer

Received June 12, 2015; final acceptance July 23, 2015. Online prepub date: August 21, 2015.
Address correspondence to Kazuo Ohashi, M.D., Ph.D., iPS Cell-based Projects on Cell Transplantation and Cell Dynamics, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan. Tel: +81-06-6879-4091; Fax: +81-06-6879-8154; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 8, pp. 47–56, 2015
2155-1790/15 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517915X689056
Copyright © 2015 Cognizant, LLC.
Printed in the USA. All rights reserved

Spheroid Formation and Evaluation of Hepatic Cells in a Three-Dimensional Culture Device

Yoshitaka Miyamoto,*† Masashi Ikeuchi,†‡ Hirofumi Noguchi,§ Tohru Yagi,¶ and Shuji Hayashi*

*Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, Tsurumai-cho, Showa-ku, Nagoya, Japan
†Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan
‡PRESTO, Japan Science and Technology (JST), Saitama, Japan
§Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
¶School of Information Science and Engineering, Tokyo Institute of Technology, Tokyo, Japan

In drug discovery, it is very important to evaluate liver cells within an organism. Compared to 2D culture methods, the development of 3D culture techniques for liver cells has been successful in maintaining long-term liver functionality with the formation of a hepatic-specific structure. The key to performing drug testing is the establishment of a stable in vitro evaluation system. In this article, we report a Tapered Stencil for Cluster Culture (TASCL) device developed to create liver spheroids in vitro. The TASCL device will be applied as a toxicity evaluation system for drug discovery. The TASCL device was created with an overall size of 10 mm × 10 mm, containing 400 microwells with a top aperture (500 μm × 500 μm) and a bottom aperture (300 μm diameter circular) per microwell. We evaluated the formation, recovery, and size of HepG2 spheroids in the TASCL device. The formation and recovery were both nearly 100%, and the size of the HepG2 spheroids increased with an increase in the initial cell seeding density. There were no significant differences in the sizes of the spheroids among the microwells. In addition, the HepG2 spheroids obtained using the TASCL device were alive and produced albumin. The morphology of the HepG2 spheroids was investigated using FE-SEM. The spheroids in the microwells exhibited perfectly spherical aggregation. In this report, by adjusting the size of the microwells of the TASCL device, uniform HepG2 spheroids were created, and the device facilitated more precise measurements of the liver function per HepG2 spheroid. Our TASCL device will be useful for application as a toxicity evaluation system for drug testing.

Key words: Human hepatocellular carcinoma (HepG2) cells; Spheroid; Three-dimensional (3D) culture; Tapered stencil for cluster culture (TASCL); Medical evaluation

Received June 12, 2015; final acceptance July 13, 2015. Online prepub date: August 26, 2015.
Address correspondence to Yoshitaka Miyamoto, Ph.D., Department of Advanced Medicine in Biotechnology and Robotics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Tel: +81-52-719-1873; Fax: +81-52-719-1977; E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it  or This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Cell Medicine, Vol. 8, pp. 57–62, 2015
2155-1790/15 $90.00 + .00
DOI: http://dx.doi.org/10.3727/215517915X689074
Copyright © 2015 Cognizant, LLC.
Printed in the USA. All rights reserved

Fluorescence Quenching of CdSe/ZnS Quantum Dots by Using Black Hole Quencher Molecules Intermediated With Peptide for Biosensing Application


Sreenadh Sasidharan Pillai,* Hiroshi Yukawa,† Daisuke Onoshima,‡ Vasudevanpillai Biju,§ and Yoshinobu Baba*†‡§

*Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-Ku, Nagoya, Japan
†FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Furo-cho, Chikusa-Ku, Nagoya, Japan
‡Institute of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-Ku, Nagoya, Japan
§Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Hayashi-Cho, Takamatsu, Kagawa, Japan

Quantum dots (QDs) have recently been investigated as fluorescent probes for detecting a very small number of biomolecules and live cells; however, the establishment of molecular imaging technology with on–off control of QD fluorescence remains to be established. Here we have achieved the fluorescence off state of QDs with the conjugation of black hole quencher (BHQ) molecules intermediated with peptide by using streptavidin-QDs585 and biotin-pep-BHQ-1. The fluorescence of streptavidin-QDs585 was decreased by the addition of biotin-pep-BHQ-1 in a dose-dependent manner. It has been suggested that the decrease in QDs585 fluorescence occurred through a Förster resonance energy transfer (FRET) mechanism from the analysis of fluorescence intensity and lifetime of streptavidin-QDs585 and QDs585-pep-BHQ-1. QDs585 fluorescence could be quenched by more than 60% efficiency in this system. The sequence of intermediate peptide (pep) was GPLGVRGK, which can be cleaved by matrix metalloproteinases (MMPs) produced by cancer cells. QDs585-pep-BHQ-1 is thus expected to detect the MMP production by the recovery of QDs585 fluorescence as a new bioanalytical agent for molecular imaging.

Key words: Quantum dots (QDs); Black hole quencher (BHQ); Fluorescence resonance energy transfer; Matrix metalloproteinase

Received June 12, 2015; final acceptance August 10, 2015. Online prepub date: August 26, 2015.
Address correspondence to Sreenadh Sasidharan Pillai, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-Ku, Nagoya 464-8603, Japan. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it  or Hiroshi Yukawa, FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Furo-cho, Chikusa-Ku, Nagoya 464-8603, Japan. E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it