References

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[1] J. Lee, A. Ametani, A. Enomoto, Y. Sato, H. Motosima, F. Ike, S Kaminogawa, Screening for the immunopotentiating activity of the immune response by Bifidobacterium adolescentis M101-4, Biosci. Biotech. Ciochem. 57 (1993) 21272132.

[2] A. Hosono, J. Lee, A. Ametani, M. Natsume, M. Hirayama, T. Adachi, S. Kaminogawa, Comparison of the Immunopotentiating Activity with Structural Characteristics among Water-soluble Polysaccharides Isolated from the Genus Bifidobacterium, Bioscience Microflora. 17 (1998) 97-104.

[3] A. Hosono, J. Lee, A. Ametani, M. Natsume, M. Hirayama, T. Adachi, S. Kaminogawa, Characterization of a water-soluble polysaccharide fraction with immunopotentiating activity from Bifidobacterium adolescentis M101-4, Biosci Biotechnol Biochem. 61 (1997) 312-316.

[4] A. Hosono, A. Ozawa, R. Kato, Y. Ohnishi, Y. Nakanishi, T. Kimura, R. Nakamura, Dietary fructooligosaccharides induce immunoregulation of intestinal IgA secretion by murine Peyer's patch cells, Biosci Biotechnol Biochem. 67 (2003) 758-764.

SUPPLEMENTATION OF SERICIN IS EFFECTIVE IN MAINTENANCE OF ISLET SURVIVAL AND FUNCTION UNDER SERUM-FREE CULTURE

Akiko Ogawa1, Satoshi Terada1, Masao Miki1, Toshihisa Kimura2, Akio Yamaguchi2, Masahiro Sasaki3, and Hideyuki Yamada3

1Department of Applied Chemistry and Biotechnology, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan; Department of Surgery, Faculty of Medicine, University of Fukui, 23-3 Shimoaizuki, Matsuoka-cho, Yoshida-gun, Fukui 910-1193, Japan; 3Technology Department, Seiren Co., Ltd., 1-10-1 Keya, Fukui 918-8560, Japan

Abstract: Diabetes is a chronic syndrome due to an insufficiency of insulin production or to insulin tolerance of the target tissue. Islet of Langerhans is the only tissue that produces insulin, the hormone reducing blood glucose level. Therefore islet transplantation is an extremely promising treatment for severe diabetes. Practical islet transplantations have been performed worldwide, including in Japan. However, isolated islets must be transplanted within several hours because their functions and viability reduce as time passes and a suitable islet-culture method for clinical treatment has not been established. Islets are usually cultured in FBS-supplemented medium but the use of FBS has several problems such as batch-to-batch variations and infection from several different pathogens. Therefore the development of a serum-free medium for islets culture is eagerly sought. In the present study, we examined the possibility of sericin as a supplement for islet culture. Rat islets were cultured in RPMI mediums containing sericin, FBS, or no supplement, and the concentration of rat insulin in their supernatants were then measured by ELISA. In the absence of a supplement, half the islets felt apart during culture, while 75% of islets cultured with sericin remained morphologically intact in the presence of sericin. Islets cultured with sericin maintained steady insulin production, while those cultured without supplement rapidly ceased insulin production.

Key words: diabetes, insulin, cell therapy, islet, sericin, serum-free culture

1. INTRODUCTION

More than 16 million people in Japan suffer from diabetes or incipient diabetes. There are some relevant treatments, such as diet therapy and exercise training, but the most effective treatment is insulin injection. However its effect is transient and so patients need to inject insulin daily; thus this treatment has the risk that patients will suffer hypoglycemia, a cause of coma bringing on a critical state if infected with a surfeit of insulin. Thus treatments for diabetes that are both permanent and safe are desired.

Pancreas transplantation or islet transplantation is the only effective treatment for complete recovery. While there is a strong risk of infection during pancreas transplantation surgery, islets can be easily transplanted by infusions [1, 2]. Therefore it is anticipated that islet transplantation will become an effective treatment for diabetes. Already, seven islet transplantations have been performed in Japan, and medical insurance is now applied to islet transplantation in Canada. In the US, Medicare, the tax supported system that provides medical care for old people, is about to be made applicable. Even so, some problems remain before islet transplantation will be established as a general treatment. The major problem is to how build an islet supply system [3, 4].

Pancreas islets are widely cultured in mediums containing fetal bovine serum (FBS). FBS, however, is unfavorable for islet culture because of the risk of virus infection and its batch-to-batch variations. To achieve a safe, high-quality islet culture system, alternative medium supplements have been urgently sought.

This study aimed to establish an effective FBS-independent islet culture for cell therapy. Previously, we reported that sericin improved the proliferation of various cells including mouse hybrydoma [5]. In this work, we assess the usefulness of sericin as a novel supplement for serum-free islet culture.

2. MATERIALS AND METHODS

2.1 Rat islets isolation and culture

Rat islets were prepared by collagenase (Nitta Gelatin, Osaka, Japan) digestion, as described previously [6], from male Lewis rats (8-10 weeks old; Charles River Japan, Kanagawa). The obtained islets were cultured in RPMI medium supplemented with 10% FBS for 2 - 10 days before the experiment proceeded.

2.2 Conditioned medium culture

Morphologically intact rat islets were selected under a stereoscopic microscope and transferred by a micropipette with a 20-^1 culture supernatant into each well of a 96-well round-bottom plate (Sumitomo Bakelite, Tokyo) containing 100 of a fresh 10% FBS-supplemented RPMI medium. To study the effect of sericin on islet survival and insulin production, rat islets were cultured in this conditioned media, no-supplemented, sericin-supplemented, or FBS-supplemented RPMI medium. The medium was exchanged on day 3 according to the following procedure; single rat islets with 20-^1 culture supernatant were collected using a micropipette under a stereoscopic microscope. They were then rinsed with 1 ml of fresh medium with each supplement to take off the culture supernatant before they were transferred to each of the wells that contained 100-^1 fresh medium in a new 96-well round-bottom plate (Figure 1). The culture was ended when disintegration of islets was observed under a stereoscopic microscope.

2.3 INSULIN ANALYSIS

Insulin abundance in the culture supernatants was then determined by ELISA (Sibayagi, Gunma).

3. RESULTS AND DISCUSSION

All rat islets remained assembled until the first medium exchange (Figure 2). After that, four (33%) of the islets in the absence of supplement collapsed, while only two (17%) of them in the presence of sericin and one (8%) of those in the presence of FBS did. On day 6, 11 islets in the presence of FBS remained morphologically intact, while only half of them in the basal medium did. In the presence of sericin, 9/12 (75%) of the islets remained in a morphologically intact state.

Culture in 120

96-well-round bottom plate

Counting the number of morphologically intact islets

Medium exchange

— Insulin analysis Islet in 20 | I medium

Islet in 20 |1 medium

Culture in 120 |l

At day 6

Counting the number of morphologically intact islets

Insulin analysis

Figure 1. Flow chart of islet serum-free culture.

96-well-round bottom plate

Rince in 1 ml

* 24-well plate

Fresh medium 100 |il

Culture period (d)

Figure 2. Survival of morphologically intact islets. Each islet was separately harvested into each well of a 96-well round plate. The islets were cultured in the presence of 10% FBS (closed circles), 0.01% sericin (open triangles), or in the absence of any supplement (open circles).

Culture period (d)

Figure 2. Survival of morphologically intact islets. Each islet was separately harvested into each well of a 96-well round plate. The islets were cultured in the presence of 10% FBS (closed circles), 0.01% sericin (open triangles), or in the absence of any supplement (open circles).

Insulin production of rat islets was measured by ELISA and the results are shown in figure 3. Islets in the presence of FBS produced abundant insulin and this high production was retained over the culture. Islets in the presence of sericin also maintained insulin production, though the amount of insulin was lower than in the presence of FBS. Islets cultured in the absence of any supplement produced only a small amount of insulin during the first 3 days (Figure 3 a) and insulin production then ceased.

Most islets cultured in the presence of FBS maintained intact morphology and high insulin production during the culture, whereas only half of the islets cultured in the absence of FBS retained morphology and the insulin production in the culture with the absence of any supplement dramatically decreased. In the presence of sericin, islets successfully maintained morphology and insulin production, but the effects of sericin were inferior to those of FBS. The effects of sericin could be improved by raising the sericin concentration, because the culture of insulinoma produced more insulin in a higher concentration of sericin (data not shown). In conclusion, sericin is an effective medium supplement for islet serum-free culture.

A 8000

B 8000

No Sericin FBS

Figure 3. Islet insulin release. Cultured supernatants were collected on days 3 (A) and 6 (B). Insulin concentrations were determined by ELISA. Each column indicates the insulin concentration of each culture's supernatant. Closed, hatched, and open bars indicate islets cultured in the medium supplemented with 10% FBS, 0.01% sericin, and no supplement, respectively.

& 6000

6000

4000

4000

2000

2000

Sericin

4. REFERENCES

1. Alejandro, R., Lehmann, R., Ricordi, C., Kenyon, N. S., Angélico, M. C., Burke, G., Esquenazi, V., Nery, J., Betancourt, A. E., Kong, S. S., Miller, J., and Mintz, D. H.(1997) Long-term function (6 years) of islet allografts in type 1 diabetes, Diabetes, 46, 1983-1989.

2. Shapiro, A. M., Lakey, J. R., Ryan, E. A., Korbutt, G. S., Toth, E., Warnock, G. L., Kneteman, N. M., and Rajotte, R. V. (2000) Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocoticoid-free immunosuppressive regimen, N. Engl. J. Med., 343, 230-238.

3. Zwillich, T. (2000) Islet transplants not yet ready for prime time, Science, 289, 531533.

4. Burridge, P. W., Shapiro, A. M. J., Ryan, E. A., and Lakey, J. R. T. (2002) Future trends in clinical islet transplantation, Transplant. Proc., 34, 3347-3348.

5. Terada, S., Nishimura, T., Sasaki, M., Yamada, H., and Miki, M.(2002) Sericin, a protein derived from silkworms, accelerates the proliferation of several mammalian cell lines including a hybridoma, Cytotechnology, 40, 3-12.

6. Gotoh, M., Maki, T., Kiyoizumi, T., Satomi, S., and Monaco, A. P. (1985) An improved method for isolation of mouse pancreatic islets, Transplantation, 40, 437438.

SUPPRESSION OF TWO-STAGE CELL TRANSFORMATION BY ELECTROLYZED REDUCED WATER CONTAINING PLATINUM NANOPARTICLES

Ryuhei Nishikawa1, Kiichiro Teruya1'2, Yoshinori Katakura12, Kazumichi

Otsubo , Shinkatsu Morisawa , Qianghua Xu , and Sanetaka Shirahata ,

1Graduate School of Systems Life sciences, Kyushu University; 2Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; 3Nihon Trim Co.Ltd., 1-8-34 Oyodonaka, Kita-ku, Osaka 5310076, Japan; 4College of Life Sciences, Zhejiang University, No. 268 Kaixuan Road, Hangzhou 310029, Zhejiang, P.R. China

Abstract: According to the two-stage cell transformation theory, cancer cells first receive initiation, which is mainly caused by DNA damage and then promotion, which enhance transformation. Since murine Balb/c 3T3 cells lose contact inhibition by cell transformation, the cells have been widely used for transformation experiments. Electrolyzed reduced water (ERW) is a health beneficial alkaline drinking water containing high concentration of dissolved hydrogen, and can scavenge intracellular reactive oxygen species (ROS). ERW contains a small amount of platinum (Pt) nano-particles as atomic hydrogen donor and ROS-scavenger. Therefore, ERW supplemented with Pt nanoparticles (ERW/Pt) can be considered as a model of strong ERW. Here, we report that ERW/Pt can prevent transformation of Balb/c 3T3 cells. ERW was prepared by electrolysis of 0.002 M NaOH. Balb/c 3T3 cells were treated with 3-methyl cholantrene (MCA) as an initiation compound, followed by the treatment with phorbol-12-myristate-13-acetate (PMA) as a promotion compound. The cell transformation induced by MCA/PMA was strongly suppressed by ERW/Pt treatment, especially at the stage of promotion. Analysis of intracellular ROS level showed that ERW/Pt could decrease excess intracellular ROS induced by PMA. These results suggested that ERW/Pt suppressed cell transformation at promotion stage by its ROS scavenging effect.

Key words: active hydrogen; Balb/c 3T3 cells; electrolyzed reduced water; platinum nanoparticles; reactive oxygen species; two-stage cell transformation

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Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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