Summary of Research Project Results Under the JSPS FY2000
"Research for the future Program"



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

7.Projetct Leader
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

[Reference Articles]
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

[Reference Books]
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  


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