| 1.Research Institution | Tokyo Women's Medical University | |
| 2.Research Area | Integrated Fields | |
| 3.Research Field | Tissue Engineering | |
| 4.Term of Project | FY1996〜FY2000 | |
| 5.Project Number | 96I00201 | |
| 6.Title of Project | Biomaterials for Tissue Engineering |
| Name | Institution,Department | Title of Position |
| Teruo OKANO | Tokyo Women's Medical University, College of Medicine | Professor |
8.Core Members
| Names | Institution,Department | Title of Position |
| Kazunori KATAOKA | University of Tokyo, Graduate School of Engineering | Professor |
| Takao AOYAGI | Tokyo Women's Medical University, College of Medicine | Assistant Professor |
| Akihiko KIKUCHI | Tokyo Women's Medical University, College of Medicine | Res. Assistant Professor |
9.Cooperating Researchers
| Names | Institution,Department | Title of Position |
| Toshihiko HAYASHI | University of Tokyo, Graduate School of Arts and Sciences | Professor |
| Shin-ichi KOSAKA | National Institute of Neuroscience, Dept. of Neurochem. | Chairman |
| Tetsuya TERASAKI | Tohoku University, Graduate School of Pharmaceu. Sci. | Professor |
10.Summary of Research Results
|
In this fiscal year, novel thermoresponsive cell culture surfaces were developed to accelerate cell detachment. We immobilized thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) onto porous PET membranes with 0.4μm-pore instead of conventional polystyrene cell culture dishes (TCPS). Cells attached and grown on the modified porous membrane were detached much faster than from the PIPAAm-modified TCPS. This is probably due to a rapid hydration of surface-grafted PIPAAm molecules with water molecules penetrating from the bottom surface through pores. Thus, configuration of base materials did have effect on the accelerated cell detachment. We further developed novel thermoresponsive dishes with poly(IPAAm-co-carboxy-isopropylacrylamide(CIPAAm)). As CIPAAm contains ionizable carboxyl groups, surface hydrophilicity could increase upon decreasing temperature. In fact, cell detachment from modified surfaces is faster than PIPAAm-modified TCPS surfaces. Those two approaches are important for protecting cell sheets from possible functional deterioration arising from longer exposure at lower temperature. With the aid of mask patterns, PIPAAm-patterned surfaces were made. Using patterned surfaces, cells were proliferated on the whole surface area at 37℃, while only cells on PIPAAm domains detach at low temperature. Applying this procedure, new type of cells can be seeded on PIPAAm domains at second heating at 37℃ and thus succeeded the co-culture of different cell types. Examining various sets of cell types, hepatocytes co-cultured with endothelial cells have higher functionality as judged by the albumin production. By this approach, it is possible to produce tissue elements of more complicated tissues those mimic organs in the living systems, and to utilize basic cellular functional analyses. We extended our researches on tissue engineering using cultured cell sheet manipulation (cell sheet engineering). By utilizing PIPAAm-modified culture dishes, confluent culture of cells could manipulate to new surfaces with minimum invasion. With this procedure, confluent culture of endothelial cells (endothelial cell sheet) was stratified onto hepatocyte cultures and examined hepatocyte viability and functionality. Stratified hepatocytes have longer survival rate for more than 40 days compared with hepatocytes survived only 10 days without any other cells. When cells are mixed and seeded on the culture dishes, it is difficult and eventually impossible to co-culture those cells. Thus our system is the first case to show stable co-culture systems with the aid of cell sheet engineering. This technique is applied for various cell types, we succeeded to produce cultured skin layers with good maintenance of cell-cell connection and extracellular matrix underneath of cell sheet. This recovered skin layer has higher functionality than those utilized in skin transplantation clinically in terms of protection from infection. Recently the obtained cultured skin sheets are applied clinically in our hospital with success. Cultured cardiac myocytes are also obtained with autonomous pulsation. Electrical stimulation was transmitted from one sheet to the other for double-layered cell sheets. Thus functionally connected stratified cardiac myocyte sheets are obtained. Those sheets will be applied to treat the patient with heart failure. In conclusion, we believe that our newly developed cell sheet engineering using PIPAAm-modified dishes might be novel approach in tissue engineering for therapeutic use. |
11.Key Words
(1)Poly(N-isopropylacrylamide)、(2)Thermoresponsive Cell Culture Dishes、(3)Cellular Sheet Engineering
(4)Patterned Co-Culture、(5)Stratification、(6)Liver Lobule-like Structure
(7)Myocardiac Cell Sheet、(8)Transplantation of Cultured Skin Sheet
12.References
| Author | Title of Article | |||
| T. Shibanuma, T. Okano et al. | Thermosensitive phase-separation behavior of poly(acrylic acid)-graft-poly(N, N-demethylacrylamide)aqueous solution | |||
| Journal | Volume | Year | Pages Concerned | |
| Macromolecules | 33 | 2000 | 444-450 | |
| Author | Title of Article | |||
| O. H. Kwon, T. Okano et al. | Rapid Cell Sheet Detachement from PIPAAm-Grafted Porous Cell Culture Membranes | |||
| Journal | Volume | Year | Pages Concerned | |
| J. Control. Rel | 64 | 2000 | 143-153 | |
| Author | Title of Article | |||
| J. E. Chung, M. Yokoyama and T. Okano | Inner Core Segment Design for Drug Delivery Control of Thermo-Responsive Polymeric Micells | |||
| Journal | Volume | Year | Pages Concerned | |
| J. Control. Rel. | 62 | 2000 | 115-127 | |
| Author | Title of Article | |||
| K. Uchida, T. Okano et al. | Temparature-dependentmodulation of blood platelet movement and morphology on poly(N-isopropylacrylamide)-grafted surfaces | |||
| Journal | Volume | Year | Pages Concerned | |
| Biomaterials | 21 | 2000 | 923-929 | |
| Author | Title of Article | |||
| M. Yamato, T. Okano et al. | Release of absorbed fibrnectin from temperature-responsive culture surfaces requires cellular activity | |||
| Journal | Volume | Year | Pages Concerned | |
| Biomaterials | 21(10) | 2000 | 981-986 | |
| Author | Title of Article | |||
| K. Uchida, T. Okano et al. | Temperature-sensitive poly(N-isopropylacrylamide)-grafted surfaces modulate blood platelet interactions | |||
| Journal | Volume | Year | Pages Concerned | |
| Macromol. Rapid Commun. | 21 | 2000 | 169-173 | |
| Author | Title of Article | |||
| K. Yamamoto. T. Okano et al. | Temperature-responsive chromatographic separation of Bisphenol with water as a sole mobile phase | |||
| Journal | Volume | Year | Pages Concerned | |
| Enviromental Sciences | 7(1) | 2000 | 47-56 | |
| Author | Title of Article | |||
| K. Uchida, T. Okano et al. | Two different types of nonthrombogenic surfaces:PEG suppresses platelet adhesion ATP-independently but HEMA-St block copolymer requires ATP consumption of platelets to prevent adhesion | |||
| Journal | Volume | Year | Pages Concerned | |
| J. Biomed. Mater. Res. | 50 | 2000 | 585-590 | |
| Author | Title of Article | |||
| A. Kushida, T. Okano et al. | Temperature-responsive culture dishes allow non-enzymatic harvest of differentiated Madin-Darby canine kidney (MDCK) cell sheets | |||
| Journal | Volume | Year | Pages Concerned | |
| J. Biomed. Master. Res. | 51 | 2000 | 216-223 | |
| Author | Title of Article | |||
| M. Kurisawa, M. Yokoyama and T. Okano | Transfection efficiency increases by incorporating hydrophobic monomer units into polymeric gene carriers | |||
| Journal | Volume | Year | Pages Concerned | |
| J. Control. Rel. | 68 | 2000 | 1-8 | |
| Author | Title of Article | |||
| K. Yamamoto, T. Okano et al. | Preparation of packing materials for temperature-responsive chromatography. Modification of silica surface with poly(N-isopropylacrylamide) | |||
| Journal | Volume | Year | Pages Concerned | |
| Chromatography | 21(3) | 2000 | 209-215 | |
| Author | Title of Article | |||
| M. Hirose, T. Okano et al. | Creation of designed shape cell sheets that are nonivasively harvested and moved onto another surface | |||
| Journal | Volume | Year | Pages Concerned | |
| Biomacromolecules | 1 | 2000 | 377-381 | |
| Author | Title of Article | |||
| H. Kanazawa, T. Okano et al. | Temperature-responsive chromatographic separation of amino acid phenylthionydantoins using aqueous media as the mobile phase | |||
| Journal | Volume | Year | Pages Concerned | |
| Analytical Chemistry | 72(24) | 2000 | 5961-5966 | |
| Author | Title of Article | |||
| M. Ebara, T. Aoyagi, K. Sakai and T. Okano | Introducing reactive carboxyl side chains retains phase transition temperature sensitivity in N-isopropylacrylamide copolymer gels | |||
| Journal | Volume | Year | Pages Concerned | |
| Macromolecules | 33 | 2000 | 8312-8316 | |
| Author | Title of Article | |||
| M. Kurisawa, M. Yokoyama and T. Okano | Gene experssion control by temperature with thermo-responsive polymeric gene carriers | |||
| Journal | Volume | Year | Pages Concerned | |
| J. Contorol. Rel. | 69 | 2000 | 127-137 | |
| Author | Title of Article | |||
| O. H. Kwon, A. Kikuchi, M. Yamato and T. Okano | Acceleration of cell sheet reccovery for tissue engineering | |||
| Journal | Volume | Year | Pages Concerned | |
| Transaction of the Materials Research Society of Japan | 25(4) | 2000 | 899-902 | |
| Author | Title of Article | |||
| A. Kushida, T. Okano et al. | Non-enzymatic harvest of renal epithelial cell sheets using temperature-responsive culture dishes | |||
| Journal | Volume | Year | Pages Concerned | |
| Transaction of the Materials Research Society of Japan | 25(4) | 2000 | 903-906 | |
| Author | Title of Article | |||
| M. Hirose, T. Okano et al. | Temperature-Responsive Surface for Novel Co-Culture Systems of Hepatocytes with Endothelial Cells: 2-D Patterned and Double Layered Co-Cultures | |||
| Journal | Volume | Year | Pages Concerned | |
| Yonsei Medical Journal. | 41.(6) | 2000 | 803-813 | |
| Author | Title of Article | |||
| A. Kushida, M. Yamato, A. Kikuchi and T. Okano | Two-dimensional manipulation of differentiated Madin-Darby canine kidney(MDCK) cell sheets : the noninvasived harvest from temperature-responsive culture dishes and transfer to other surfaces | |||
| Journal | Volume | Year | Pages Concerned | |
| J. Biomed. Mater. Res | 54 | 2001 | 37-46 | |
| Author | Title of Article | |||
| M. Yamato, T. Okano | Novel Patterned cell co-culture utilizing thermally responsive grafted polymer surfaces | |||
| Journal | Volume | Year | Pages Concerned | |
| J. Biomed. Mater. Res. | 55 | 2001 | 137-140 | |
| Author | Title of Article | |||
| T. Shimizu and T. Okano | Biomaterials for Tissue Engineering | |||
| Journal | Volume | Year | Pages Concerned | |
| Tissue Engineering 6 | 58(12) | 2000 | 851-854 | |
| Author | Title of Article | |||
| H. Kanazawa, Y. Matsushima and T. Okano | Temperature-responsive chromatography | |||
| Journal | Volume | Year | Pages Concerned | |
| Advances in chromatography | 41 | 2000 | 311-336 | |
| Author | Title of Article | |||
| K. Emoto, M. Iijima, Y. Nagasaki, K. Kataoka, | Functionality of polymeric micelle hydrogel with organized three dimensional architecture on surfaces | |||
| Journal | Volume | Year | Pages Concerned | |
| J. Amer. Chem. Soc | 122(11) | 2000 | 2653-2654 | |
| Author | Title of Article | |||
| H. Otsuka, Y. Nagasaki, K. Kataoka | Dynamic wettability study on the functionalized PEGylated layer on a polylactide surface constructed by the coating of aldehyde-ended poly(ethylene glycol)(PEG)/polylactide (PLA) block copolymer | |||
| Journal | Volume | Year | Pages Concerned | |
| Science and Technology of Advanced Materials | 1(1) | 2000 | 21-29 | |
| Author | Title of Article | |||
| H. Otsuka, Y. Nagasaki, K. Kataoka | Surface characterization of functionalized polylactide (PLA) through the coating with heterobifunctional poly(ethylene glycol)(PEG)/PLA block copolymers | |||
| Journal | Volume | Year | Pages Concerned | |
| Biomacromolecules | 1(1) | 2000 | 39-48 | |
| Author | Title of Article | |||
| K. Emoto, Y. Nagasaki, K. Kataoka | Core-shell structured hydrogel thin layer on surfaces by lamination of poly(ethylene glycol)-b-poly(D, L-lactide) micelle and polyallylamine | |||
| Journal | Volume | Year | Pages Concerned | |
| Langmuir | 16(13) | 2000 | 5738-5742 | |
| Author | Title of Article | |||
| H. Otsuka, K. Kataoka et al. | Novel approaches for the construction of cell specific surfaces using heterobifunctional poly(ethylene glycol)(PEG) | |||
| Journal | Volume | Year | Pages Concerned | |
| J. Photopolym. Sci. Tech. | 13(1) | 2000 | 25-28 | |
| Author | Title of Article | |||
| K. Emoto, K. Kataoka et al. | Preparation of non-fouling surface through the coating with core-polymerized block copolymer micelles having aldehyde-ended PEG shell | |||
| Journal | Volume | Year | Pages Concerned | |
| Colloids and Surfaces B: Biointerface | 18(3-4) | 2000 | 337-346 | |
| Author | Title of Article | |||
| Y. Akiyama, H. Otsuka, Y. Nagasaki, M. Kato, K. Kataoka | Selective synthesis of heterobifuncitional poly(ethylene glycol) derivatives containing both mercapto and acetal terminals | |||
| Journal | Volume | Year | Pages Concerned | |
| Bioconjugate Chemistry | 11(6) | 2000 | 947-950 | |
| Author | Title of Article | |||
| H. Otsuka, Y. Nagasaki, Y. Sakurai, T. Okano, K. Kataoka | Functionalization of polylactide (PLA) surface using end-functionalized block copolymer of a-acetal-poly(ethylene glycol)(PEG)/PLA | |||
| Journal | Volume | Year | Pages Concerned | |
| Trans. Mater. Res. Soc. Jpn | 25(4) | 2000 | 895-898 | |
| Author | Title of Article | |||
| E. Uchimura, H. Otsuka, T. Okano, Y. Sakurai, K. Kataoka | Totally synthetic polymer with lectin-like function:Induction of killer cells by the copolymer of 3-acrylamidophenylboronic acd with N, N-dimethylacrylamide | |||
| Journal | Volume | Year | Pages Concerned | |
| Biotechnology and Bioengineering | 72(3) | 2001 | 307-314 | |
| Author | Title of Article | |||
| H. Otsuka, Y. Nagasaki, K. Kataoka | Self-assembly of poly(ethylene glycol) (PEG) based block copolymers for biomedical applications | |||
| Journal | Volume | Year | Pages Concerned | |
| Current Opinion in Colloid & Interface Science | 6 | 2001 | 3-10 | |
| Author | Title of Article | |||
| Motohiro Hirose, Toshihiko Hayashi et al. | Implications for Biological Functions of Type IV Collagen Gels | |||
| Journal | Volume | Year | Pages Concerned | |
| Transactions of the Materials Research Society of Japan | 25[4] | 2000 | 907-910 | |
| Author | Title of Article | |||
| Nobuko AKUTSU, Toshihiko HAYASHI et al. | Repression of fibroblast Migration into the Collagen Gel Incubated with Glucose Is Due to Glycation-mediated Cross-linking of Collagen | |||
| Journal | Volume | Year | Pages Concerned | |
| CONNECTIVE TISSUE | 32 | 2000 | 267-272 | |
| Author | Title of Article | |||
| T. Kato, T. Hayashi et al. | Distribution and Isoform Characterization of Type XII Collagen in Bovine Cornea | |||
| Journal | Volume | Year | Pages Concerned | |
| Opththalmic Research | 32 | 2000 | 215-221 | |
| Author | Title of Article | |||
| Mizuno, K., Hayashi, T et al. | The fibril structure of type V collagen triple-helical domain. | |||
| Journal | Volume | Year | Pages Concerned | |
| Micron. | 32 | 2000 | 317-323 | |
| Author | Title of Article | |||
| Keiko SAKAI and Toshihiko HAYASHI | Prevention of Fibrosarcoma HT 1080 Cell Detachment by Multimeric Fibronectin Formed in vitro | |||
| Journal | Volume | Year | Pages Concerned | |
| CONNECTIVE TISSUE | 32 | 2000 | 27-32 | |
| Author | Title of Article | |||
| Masami Harimoto, Motohiro Hirose, Toshihiko Hayashi, and Yoriko Atomi | Maintenance of a Round Cell Shape and Physiological Responsiveness to Insulin of Mature Adipocytes by Culturing in Type I Collagen Gel | |||
| Journal | Volume | Year | Pages Concerned | |
| CONNECTIVE TISSUE | 32 | 2000 | 11-16 | |
| Author | Title of Article | |||
| T. Kitazawa, K. Hosoya, T. Takahashi, Y. Sugiyama, T. Terasaki | In vivo and in vitro evidence of a carrier-mediated efflux transport system for estrone-3-sulfate across the blood-cerebrospinal fluid barrier. | |||
| Journal | Volume | Year | Pages Concerned | |
| J. Pharm. Pharmacol | 52 | 2000 | 281-288 | |
| Author | Title of Article | |||
| H. Asaba, K. Hosoya, H. Takanaga, S. Ohtsuki, E. Tamura and T. Terasaki | Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydro-epiandrosterone sulfate, via organic anion transporting polypeptide 2. | |||
| Journal | Volume | Year | Pages Concerned | |
| J. Neurochem | 75 | 2000 | 1907-1916 | |
| Author | Title of Article | |||
| K. Hosoya, H. Asaba, T. Terasaki | Brain-to-blood efflux transport of estrone-3-sulfate at the blood-brain barrier in rats. | |||
| Journal | Volume | Year | Pages Concerned | |
| Life Sci | 67 | 2000 | 2669-2711 | |
| Author | Title of Article | |||
| K. Hosoya, K. Tetsuka, K. Nagase, M. Tomi, S. Saeki, , A. Kikuchi, T. Okano and T. Terasaki | Conditionally immortalized brain capillary endothelial cell lines established from a transgenic mouse harboring temperature-sensitive Simian Virus 40 large T-antigen gene. | |||
| Journal | Volume | Year | Pages Concerned | |
| AAPS Pharmsci | 2 | 2000 | article27 | |
| Author | Title of Article | |||
| T. Terasaki and K. Hosoya | Conditionally immortalized cell lines as a new in vitro model for the study of barrier functions. | |||
| Journal | Volume | Year | Pages Concerned | |
| Biol. Pharm. Bull | 24 | 2000 | 111-118 | |
| Author | Title of Article | |||
| K. Hosoya, T. Takashima, K. Tetsuka, T. Nagura, S. Ohtsuki, T. Terasaki | mRNA expression and transport characterization of conditionally immortalized rat brain capillary endothelial cell lines; A new in vitro BBB model for drug targeting. | |||
| Journal | Volume | Year | Pages Concerned | |
| J. Drug Target | in press | 2001 | ||
| Author | Title of Article | |||
| T. Kitazawa, K. Hosoya, M. Watanabe, T. Takashima. S. Ohtsuki, T. Terasaki | Characterization of the amino acid transport of new immortalized choroid plexus epithelial cell lines: A novel in vitro system for investigating transport functions at the blood-cerebrospinal fluid barrier. | |||
| Journal | Volume | Year | Pages Concerned | |
| Pharm. Res | in press | 2001 | ||
| Author | Title of Article | |||
| Hisako Ohgawara | Strategies for immunoisolation in islet transplantation : challenges for the twenty-first century. | |||
| Journal | Volume | Year | Pages Concerned | |
| J Hepatobiliary Pancreat Surg. | 7 | 2000 | 374-379 | |
| Author | Title of Article | |||
| Shin'oka T, et al. | Current Status of Tissue Engineering for Cardiovascular Structure | |||
| Journal | Volume | Year | Pages Concerned | |
| Journal of Artificial Organ | 3 | 2000 | 102-106 | |
| Author | Title of Book | ||
| T. Okano, A. Kikuchi and M. Yamato | Intelligent hydrogel and new biomedical application | ||
| Publisher | Year | Pages | |
| Han Rim Won Publishing Co. | 2000 | ||
| Author | Title of Book | ||
| M. Yamato, T. Okano et al. | Tissue Engineering for Therapeutic Use 5 | ||
| Publisher | Year | Pages | |
| Elsevier Science | 2000 | ||
| Author | Title of Book | ||
| H. Otsuka, Y. Nagasaki, K. Kataoka | Novel approaches for the construction of functionalized poly(ethylene glycol) (PEG) layer on surfaces using heterobifuncitonal PEG/polylactide (PLA) block copolymers and their micelles, Polymers from Renewable Resources: Polyesters of Biomedical and Environmental Importance, ACS Symposium Series | ||
| Publisher | Year | Pages | |
| Eds. C. Scholz, R. A. Gross, American Chemical Society, Washington, D. C. | 2000 | ||
| Author | Title of Book | ||
| K. Emoto, M. Iijima, Y. Nagasaki, K. Kataoka | Surface modification with polymeric micelles from poly(ethylene gylcol)-poly(D, L-lactide) block copolymers for the creation of high performance biomaterials, | ||
| Publisher | Year | Pages | |
| Biomaterials and Drug Delivery toward New Millenium, Eds., K. D. Park, I. C. Kwon, N. Yui, S. Y. Jeong, K. Park, Hanrimwon, Seoul, Korea | 2000 | ||
| Author | Title of Book | ||
| T. Terasaki, S. Ohtsuki, H. Takanaga, K. Tetsuka, A. Kikuchi, T. Okano and K. Hosoya | Brain Barrier Function: Its Analysis and reconstitution, in Tissue Engineering for Therapeutic Use 4 | ||
| Publisher | Year | Pages | |
| Elsevier Science B. V. | 2000 | ||