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1 Tissue Engineering

2 Tissue Engineering Recovery of Lost Body Parts or their functions Develop Biocompatible Artificial Tissues or Organs (Artificial Bone, Artificial Skin, Artificial Vessel, Artificial Hearing) Necessary to understand the characteristics of the cell and tissue and to control their growth Human ear grown on mouse Robert Langer and Jay Vacanti, MIT, 1995

3 Objective

5 Tow Methods for Artificial Organ Development

6 Xenotransplantation ( 이종이식 ) Implantation The Lancet, 354 (1001):S32-S34, 1999

7 Autotransplantation ( 자가이식 )

8 Purposes of Cellular Engineering To understand cellular dynamics To control cellular behavior To make cellular networks To develop artificial organs, tissue To enhance biocompatibility of implant material

9 Tissue Dynamics 1. Cell replication-an increase in cell number 2. Cell differentiation-changes in gene expression and the acquisition of a particular function 3. Cell motility-the motion of a cell into a particular niche or location 4. Cell apoptosis (programmed death)- the controlled death of a cell 5. Cell adhesion-the physical binding of a cell to its immediate environment, which may be a neighboring cell, extracellular matrix, or an artificial surface.

10 세포공학의현황과전망 : 세포치료 에있어서줄기세포의역할과활용

11 목적 난치병치료-현재는? 줄기세포치료기술의현재와미래 줄기세포의경제성 무엇이중요한가?

12 세포치료 세포치료 세포치료란환자의손상된장기나조직을치료하기위하여외부에서세포를주입하거나세포의성장및분화를촉진하여치료대상이되는부위의재활및재생을유도하는치료방법. 최초의세포치료 1912 년에갑상선항진환자에갑상선세포를이식한것 1971 년노벨상을수상한골수이식수술도훌륭한세포치료의한예

13 줄기세포 양서류의경우다리가절단된후에줄기세포의역할에의해서다리가재생되는사실이이미보고되어있음.

14 줄기세포 줄기세포의종류 배아줄기세포 : 전능 (Pluripotent) 세포, 거의모든세포로분화가능 성체줄기세포 : 다능 (Multipotent) 세포, 분화능에있어서일정한계 줄기세포획득방법 태아줄기세포 : 유산된태아 성체줄기세포 : 골수, 제대혈, 조직 배아줄기세포 : 수정란, 체세포핵치환

15 성체줄기세포 신체의거의모든부위에서발견 골수, 제대혈, 뇌, 간, 지방, 장, 표피, 망막, 췌장등 자신의세포이므로면역거부반응문제최소화 분화능에있어서는일정한계

16 배아줄기세포 1998년톰슨에의해인간포배 (blastocyte) 에서최초로배아줄기세포를분리배양 수정란사용으로인간생명파괴의윤리적문제야기 거의모든세포로분화가능한전능세포

17 성체줄기세포와배아줄기세포비교 분화능력기형종수명윤리문제극복과제 배아줄기세포 거의모든세포 발생가능성큼 제한없음, 많은수의증식가능윤리적부담 면역학적거부반응특정세포로분화 성체줄기세포 일정한세포 발생가능성작음 일정시간배양후증식능력상실비교적크지않음 줄기세포의획득및증식, 성질유지

18 체세포핵치환줄기세포주 인간난자에환자의체세포의핵을이식하여환자자신의유전정보를가진세포주확립 면역거부반응이없고, 환자자신의맞춤세포주

19 임상적적용의예

20 골수줄기세포를이용한심혈관재생 Three aspects of modification in stem cell therapy Ex vivo expansion 골수 Mobilization Homing 허혈심근

21 골수줄기세포심혈관내에직접주입 Baseline 6months follow up 1 year follow up 심근경색환자의심장동작회복을확인.

22 척수손상 척수손상으로 19년간하반신마비환자의척수에제대혈줄기세포를주입 일부감각이회복되는것이보고됨 안정성과효과에대한추가연구가필요

23 청각손상 2003년하버드의대의연구결과배아줄기세포를유모세포로분화시키는데에성공함으로써줄기세포로청각손상회복의전망

24 줄기세포이용한세포치료의문제점 줄기세포의대량증식 원하는세포로의분화 면역학적거부반응 주입시목적기관위치 이식후환자에서의기능유지 기형종 ( 암세포등 ) 으로발전

25 세계각국의줄기세포연구현황 미국 1998 년인간줄기세포배양세계최초성공 2002 년인간줄기세포를혈관조직으로분화성공 줄기세포분화분야에서선두적 배아줄기세포연구에비교적소극적이었으나, 캘리포니아주에서 Proposition 71 통과 (2005) 로매년 3 조투입 영국 1997 년세계최초로포유류 ( 돌리양 ) 복제성공 2004 년인간복제배아연구승인 세계최초로세계줄기세포은행설립 줄기세포를국가중점연구개발과제에포함 일본 복제배아보다임상에활용할연구에주력 2004 년줄기세포로혈관분화성공 복제배아를줄기세포나신경세포로분화해만든새로운약의효과시험, 복제배아에특정한유전자를삽입혹은제거해질 병의발생원리규명 등두연구분야에대해한해 100 억원투입

26 선진국과의기술격차 전반적기술은선진국의 60~70% 수준 배아줄기세포획득이나배양기술은세계적 지속적인투자가필요한생명공학분야의특성상모든분야에서선진국을따라잡기는힘들것 특히세포분화와동물실험연구에서협력연구가필요 - 과학기술부 -

27 세포치료요법과관련기술에관한시장규모 단위 : 억달러 기 술 2005 년 2010 년 2015 년 줄기세포 제대혈 조직공학 Blood transfusion products 세포기반유전자요법 Encapsulated cell therapy 세포기반암백신 이종이식 요소기술 ( 세포주, 세포배양액, 세포운반체 ) 총 계 자료 : A Jain PharmaBiotech Report 2005

28 산업전망 세포치료수요증가 2002 년세포이식건수는세계적으로대략 50,000 건으로추정되며 2007 년에는 159,000 건으로약 3 배정도증가할전망 현재줄기세포가차지하고있는비중은전체세포치료시장의 7.5% 인 20 억달러에불과하나향후 10 년간 18.5% 의고성장예측 기업환경 막대한연구개발비용이필요한관련사업의특성상취약한재무구조와수익모델을확보하지못한벤처기업들의인수와합병이이루어질것으로기대. 결국은글로벌제약기업의주도아래에서이루어질것. 줄기세포를이용한세포치료이외에신약후보물질테스트에시장이새롭게떠오를것임 ( 연간 10 억달러시장형성기대 )

29 기업현황 국제기업현황 세계적으로 85개의줄기세포관련세포치료회사가존재 Aastrom, Stem-cell, Osiris Therapeutics, Lexion Therapeuticals 등의바이오벤처기업의상업화임상이활발 Baxter, Novartis, Johnson & Johnson, Smith and Nephew, Boston Scientific, Medtronic, Wyeth, Schering, Becton Dickinson, Stryker, Genzyme과같은다국적제약기업및의료기기회사의참여확대 연구가상용화되어투자를회수하기위해서는장기간이소요되나열악한재정환경을가진기업들은감당하기어려워관련기업들의폐업 (Advanced Tissue Sciences, Artecel, StemRon 등 ), 자산매각 (Nexell Therapeurtics는 Baxter에자산매각 ), 합병 (Diacrin와 GenVec 과의합병 ), 인수 (Neurotech의 StemCell 인수 ) 등이급증 국내기업현황 줄기세포주를제외한줄기세포분화및기전연구및기술확보매우취약하며관련된지적재산권확보도미약 취약분야에대한공동연구부족 : 세포치료제기술특허동향분석자료에따르면한국특허에서국제공동연구에의한특허점유율은 2.6% 에불과 ( 특허청, 05. 5) 분자발생조절, 마커및항체개발, 세포신호전달, 생분자, 생물정보학등의전문인력부족 줄기세포은행, 제대혈네트워크, 전임상및임상시험등의인프라미흡 국내줄기세포기업의자본구조는매우취약

30 Where are we? 줄기세포의 1,2,3 차산업 Clinical Therapy Differentiation Homing 제 3 차산업 Mass Production of SC 제 2 차산업 제 1 차산업 SJK2005

31 CAN WE MAKE MONEY? 치료의불특허성 그러나상업적기술을이용한치료에는기술료부과가능 줄기세포주도특허가능 20년동안만보장 : 상업화에걸리는시간 + 그동안유사기술이나올가능성 장비등에특허가능

32 Be Wise Biotech Economist group Biotech Legal Advice group Biotech Ethics monitoring group 경제적가치평가 산업모델분석 산업지원 법적제도적장치마련 사회적합의도출 민간사회단체, 종료단체 SJK 2005

33 A Cell Motility Study Guidance of Cellular Growth

34 Tan J. et. al., Proc Natl Acad Sci. (2003) The tractions can be calculated from the recorded deflection of the posts Ability to control the compliance of the substrate geometrically instead of chemically No need for recording the unstressed state of the substrate, because the posts were manufactured with sufficient precision

35 Building Methods of Cellular Network Chemical cue Non-biological Biological proteins Topographical cue Cliffs, grooves, spikes, tubes, mesh and random roughness

36 Research Goal Patterning neuronal network with the small number of cells Previous studies on patterned neuronal network have focused on high density of cells and the collective characteristics such as firing rates from lots of cells. In order to investigate the interaction between neurons in the network, patterning the small number of neurons is necessary. The electrical connectivity should be maintained. What are the challenges? Patterning polylysine on the electrode with very small dimension Maintaining small number of neurons for long time. Analysis of the patterned network in the respect of a single cell.

37 Why Microelectrode Array? Culture neurons for the long term Measuring neuronal responses simultaneously over a long period of time Precise Control of the environmental conditions around the neurons Direct visualization Hippocampal neurons at 5 days after culture

38 Microelectrode Electrode 200 μm Teflon Ring 8 4 array Each electrode size : 10 10µm 2 Interelectrode Spacing : 200 µm

Reason to use patterned networks Patterned

cell density: 200 cells/ mm2 Unpatterned

cells/ mm2 In order to get action potential

39 Reason to use patterned networks Patterned Random Patterned neuronal culture Plating cell density: 200 cells/ mm2 Unpatterned neuronal culture Plating cell density: 800 cells/ mm2 In order to get action potential from randomly cultured neurons, much more neurons should be plated than patterned neurons. Patterning neurons enhances activities of the network.

etch(3um) and PR reflow(10um) The supportive structure is needed for preventing the

40 Fabrication of stamp master Method 1 1) 1 st Photo Lithography 2) Silicon RIE-dry etching 3) 2 nd Photo Lithography 4) Photo Resist Reflow 2 μm 10 μm Shallow Si etch(3um) and PR reflow(10um) The supportive structure is needed for preventing the fine stamp pattern from collapsing especially for the very fine pattern like several microns.

41 Microstamping Procedure 1) Sonication in 50% ethanol for 10 min 2) PDMS stamp molding with stampmater 4) Stamping for 1 hr (50 g/cm 2 ) Plasma treated surface 3) Poly-L-lysine (diluted in BBS) Inking for 2 hr Line width = 2 μm Line width = 4 μm Line width = 6 μm

42 Aligner for Chemical Stamping Rotational manipulator Pressure Gauge X, Y, Z-axis manipulator Pressure monitor Vacuum clamp

43 Stamped Surface Stamped pattern on the microelectrode array (FITC-labeled Poly-L-lysine) Hippocampal neurons at 5 days after culture

Neuronal Culture conditions Hippocampal neuron culture

embryos Cultured in serum-free B27/neurobasal medium

Cell density 100 cells/ mm2, 200 cells/ mm2, 400 cells/

44 Neuronal Culture conditions Hippocampal neuron culture Collected from 18-day gestation Sprague-Dawley rat embryos Cultured in serum-free B27/neurobasal medium (25mM L-glutamine) For culturing neurons only Plating Cell density 100 cells/ mm2, 200 cells/ mm2, 400 cells/ mm2 100 cells/ mm2 200 cells/ mm2 400 cells/ mm2 4 hours after plating cells

45 Immunocytochemical descriptions of cells in networks Synaptophysin Nuclei MAP-2 Overlapped image Ch-20

46 SEM image

47 Evoked Action Potential Stimulation electrode Istim = 100 μa, 50ms Ch 22 Ch 20 Istim = 200 μa, 50ms Ch22 Ch20

48 Pharmacological Study The spontaneous activities showed the dose-dependency to DNQX(6,7- dinitroquinoxaline-2,3-dione), AMPA receptor antagonist. Before the treatment of DNQX 1μM DNQX 2μM DNQX After washing out DNQX

49 Topological Method for the Guidance of Cellular Growth

Forming regular sinusoidal grooves SRG (Surface Relief Grating) Control of depth

50 Surface Relief Grating Technique Mirror Ar Laser 488nm Photo polymer He-Ne laser Detector <Optical SETUP> <SEM Image> Phase Interference by Lloyd s mirror setup Forming regular sinusoidal grooves SRG (Surface Relief Grating) Control of depth and width Depth : Time of Beam Irradiation Width : Incident angle of Beam <AFM Image>

51 Photo-Responsive Polymer Molecular Migration Polarization <Surface Relief Grating> Photo-Responsive Azo-dye benzene copolymer C 12 H 10 N 2 Cis-trans isomerization Aligned parallel to axis of beam polarization Molecular Migration by modulation of polarization

without grating> Cultured neurons on polymer with

52 Reaction of Neuron to Grating 50 μm <Hippocampal neuronal cells On polymer with Grating > <On polymer without grating> Cultured neurons on polymer with Grating Extending neurites perpendicular to groove direction

53 Contact Guidance of Neuron 50 μm Random growth on smooth surface Perpendicular growth on the grooved surface Turning neurites on grating

Quantification of Cell Elongation On Surface

Angle Lengths of Neurites Vs Angle Neurons

55 Quantification of Cell Elongation On Surface Relief Grating On Smooth Surface Frequencies of Neurites Orientation Frequencies of Neurites Orientation Lengths of Neurites Vs Angle Lengths of Neurites Vs Angle Neurons show the best alignment on 1.4um wide, 450nm deep grooves

09È«¼®¿µ5~152s

09È«¼®¿µ5~152s Korean Journal of Remote Sensing, Vol.23, No.2, 2007, pp.45~52 Measurement of Backscattering Coefficients of Rice Canopy Using a Ground Polarimetric Scatterometer System Suk-Young Hong*, Jin-Young Hong**,