저자는 본 연구에서 사용되는 돼지의 혈청을 제공하는 이미징 티타늄 섹션과 쌍둥이 바이오 제품에 대한 자신의 귀중한 조언을 Leica Microsystems에 감사하고 싶습니다. 우리는 또한 RC1HL099863 - 01 &quot;장치를 지원 순환계의 biocompatibility을 향상시키기 위해 Autologous EPC 내막&quot;부여를 통해 그들의 지원에 대한 NIH 감사드립니다. 또한, 우리는 알렉 산드라 잔츤의 국립 과학 재단 (National Science Foundation) 대학원 연구 원정대의 지원에 감사하고 있습니다. 우리는 또한 수술의 여러 측면 지원 및 연구 동물의 취급에 대한 조지 빠른, 마이크 로우와 Ianthia 파커 감사 드리고. 스티븐 오웬 우리 시딩 장치의 가공 각지에서 귀중한되고 티타늄 튜브를 절단했다.

<ol>
	<li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+bleeding+and+clotting+in+the+cardiac+surgery+patient:+from+vascular+endothelium+to+circulatory+assist+device+surface.">Pathophysiology of bleeding and clotting in the cardiac surgery patient: from vascular endothelium to circulatory assist device surface.</a> Circulation. 122, 2068-2077 (2010).</li><li>Achneck, H. E., Sileshi, B., Lawson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+biology+of+bleeding+and+clotting+in+the+surgical+patient.">Review of the biology of bleeding and clotting in the surgical patient.</a> Vascular. 16, 6-13 (2008).</li><li>Arvidsson, S., Askendal, A., Tengvall, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+plasma+contact+activation+on+silicon,+titanium+and+aluminium.">Blood plasma contact activation on silicon, titanium and aluminium.</a> Biomaterials. 28, 1346-1354 (2007).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biocompatibility+of+titanium+cardiovascular+devices+seeded+with+autologous+blood-derived+endothelial+progenitor+cells:+EPC-seeded+antithrombotic+Ti+Implants.">The biocompatibility of titanium cardiovascular devices seeded with autologous blood-derived endothelial progenitor cells: EPC-seeded antithrombotic Ti Implants.</a> Biomaterials. 32, 10-18 (2011).</li><li>Kang, C., Bonneau, M., Brouland, J. P., Bal dit Sollier, C., Drouet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+pig+models+of+venous+thrombosis+mimicking+human+disease.">In vivo pig models of venous thrombosis mimicking human disease.</a> Thromb Haemost. 89, 256-263 (2003).</li><li>Ueberrueck, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+ovine+and+porcine+animal+models+for+biocompatibility+testing+of+vascular+prostheses.">Comparison of the ovine and porcine animal models for biocompatibility testing of vascular prostheses.</a> J Surg Res. 124, 305-311 (2005).</li><li>Velik-Salchner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+values+for+thrombelastography+(ROTEM)+and+selected+coagulation+parameters+in+porcine+blood.">Normal values for thrombelastography (ROTEM) and selected coagulation parameters in porcine blood.</a> Thromb Res. 117, 597-602 (2006).</li><li>Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American Heart Association Scientific Sessions, Abstract Oral Sessions, Medical Aspects End Stage Heart Failure: Transplantation and Device Therapies. , (2010).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+titanium+ventricular+assist+device+surfaces+after+gold/palladium+coating+for+scanning+electron+microscopy.">Regenerating titanium ventricular assist device surfaces after gold/palladium coating for scanning electron microscopy.</a> Microsc Res Tech. 73, 71-76 (2010).</li><li>Ford, J. W., Welling, T. H., Stanley, J. C., Messina, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKH26+and+125I-PKH95:+characterization+and+efficacy+as+labels+for+in+vitro+and+in+vivo+endothelial+cell+localization+and+tracking.">PKH26 and 125I-PKH95: characterization and efficacy as labels for in vitro and in vivo endothelial cell localization and tracking.</a> J Surg Res. 62, 23-28 (1996).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기에 제시 세포 시딩 티 튜브의 방법은 신속하고 균일하게 endothelialize으로 의사와 과학자들을 수 있도록 혈액을 연락 implantable 장치의 표면. 우리가 분리하고 주변의 혈액 샘플에서 EPCs를 확장하고 있기 때문에, 더 큰 침략 절차는 이러한 세포를 수확하기 위해 필요하지 않습니다. 또한, EPCs는 autologous 않으므로 셀 씨앗 주입 어떤 면역 반응의 위험이 제거됩니다. 그 프로토콜에 보여준 원칙은 티 튜브에 대한뿐만 아니라 심혈관 의학에서 널리 많은 다른 biomaterials, 활용하실 수 있습니다. 이 프로토콜의 중요한 단계는 우리가 발견한 것처럼, 티 튜브의 세심한 청소하는 오염 물질 타협 셀 준수의 자기편 필름. 또한, 느린 회전 속도가 (튜브 직경에 반비례) 시딩 과정에서 필수적이며, 그러한 것을 EPCs이 서서히 정착하고 티 튜브가 움직이는으로 표면을 준수합니다. 이식 직전 시딩 우리의 방법은 허무 전직의 생체내 문화 시간을 피할 수, 세포가 개별적으로 준수하고 나중에 즉시 흐름의 다시 설립 다음 시트로 embolization의 가능성을 피하기 위해, 생체내에 합류 시트를 형성하고 있습니다. 우리의 이전 연구 EPCs가 합류 레이어 성장되면, 그들은 또한 내피 시트의 모든 가능한 sloughing을 최소화들이 단단히 준수 엑스트라 세포 매트릭스를 만들 것을 보여줍니다. embolic 분리의 가능성을 완전히 배제할 수 없다지만, 이것은 전체 장치 표면의 혈전증보다 가지각색의 낮은 위험이 치료가되지 않도록 설계되는 문제 같습니다. 낮은 전단에 주입 우리의 접근 방식은, 하부 대정 맥의 prothrombotic 환경 혈액 호환성 및 장치 5,6의 혈전증을 연구에 대한 가장 신뢰할 수있는 큰 동물 모델 중 하나를 사용합니다. 모든 동물의 보호와 실험은 실험실 동물의 관리 및 사용을위한 건강 지침의 국립 연구소와 듀크 대학은 기관 애니멀 케어 및 사용위원회의 승인 이후에 따라 실시합니다. 성공적으로 biomaterials 및 장치를 테스트하기 위해이 주입 방법을 이용하기 위해서는 인내 IVC 세그먼트를 해골로 만들다 및 장치 주변에는 출혈이 &#39;잘못된 루멘&#39;에서 유래없는 이러한 장치 삽입을위한 준비의 모든 정맥 분기 선박을 ligate하는 것이 중요합니다. 또 다른 중요한 단계는 내부 튜브 표면에 세포가 veinotomy의 폐쇄하는 동안 습기가 남아 reperfusion이 시작되기 전에 이러한 장치 루멘에 DPBS의 추가입니다. 장치가 입력된 위치에서 찾을 수없는 경우, 그것은 IVC에서 &#39;업스트림&#39;마이 그 레이션 할 수 있습니다. PVC 관의 3mm 섹션 튜브가 단단히 현재의 위치에 고정되도록 - 이것은 정맥 벽 통해 2를 통해 봉합 (4 - O Prolene)를 배치하여 예방할 수 있습니다. 연구자가 별도로 특허 튜브 explantation 후 그림 8에 표시된 찬란 사전 라벨 세포를 찾는 데 어려움이 있으면 그것은 세포가 일관된 시트에서 벗겨 가지고 가능성이 높습니다. 이것은 튜브와 함께 정맥을 고정 후, 3 티 튜브 섹션의 주변 혈관 및 창피 - 조립 매우 부드러운 절개로 막을 수 있습니다. 우리의 기술은 EPC - 시딩을 통해 심장 혈관 장치 혈전증을 방지하기위한 증명 개념을 제공합니다. 이 기술은 환자 자신의 내피 전구 세포로 늘어선 &#39;biogenic&#39;임플란트의 개발에 사용할 수 있습니다. 우리 돼지의 동물 모델의 타당성 연구는 임상 실천에이 &#39;맞춤형 의약품&#39;의 번역위한 첫 번째 단계를 제공합니다.

<table border="1"><tbody><tr><td> 시약 </td><td> 회사명 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> Acepromazine </td><td> Boehringer Ingelheim - </td><td> BIC670025 </td><td> NAC # 10280002 </td></tr><tr><td> Alconox 전원 정밀 클리너 </td><td> Alconox </td><td> 1104 </td><td></td></tr><tr><td> 발포어 외과 견인기 </td><td> 애들러 </td><td> N / A </td><td></td></tr><tr><td> Baytril </td><td> 바이엘 </td><td> APVMA 46028/0705 </td><td></td></tr><tr><td> 버터플라이 니들 (19G) </td><td> Terumo </td><td> SV19CLK </td><td></td></tr><tr><td> Chlorhexidine </td><td> 3M </td><td> 9200 </td><td></td></tr><tr><td> 클램프 (45도) </td><td> Aesculap </td><td> FC339T </td><td></td></tr><tr><td> DPBS (-/-) </td><td> Gibco </td><td> 14190-144 </td><td></td></tr><tr><td> DPBS (+/+) </td><td> Gibco </td><td> 14040-133 </td><td></td></tr><tr><td> DuraPrep </td><td> 3M </td><td> 8635 </td><td></td></tr><tr><td> EBM - 2 중간 </td><td> Lonza </td><td> CC - 3156 </td><td> 모두 혈청 자유롭고 완전하게 성장 매체에 대한 자료 </td></tr><tr><td> EGM - 2 SingleQuots </td><td> Lonza </td><td> CC - 3162 </td><td> 모두 혈청 자유롭고 완전하게 성장 매체에 EBM - 2와 함께 사용 </td></tr><tr><td> Electrocautery 도구 </td><td> Valleylab </td><td> SurgII - 20 </td><td></td></tr><tr><td> 사포 </td><td> 3M </td><td> 60-0700-0425-8 </td><td> 240 그릿 </td></tr><tr><td> Euthasol의 안락사 솔루션 </td><td> Virback 동물 보건 </td><td></td><td> 아나다 # : 200-071 </td></tr><tr><td> 펜타 닐 패치 </td><td> Actavis </td><td></td><td> NDC # 67767-120-18 </td></tr><tr><td> Flunixin </td><td> 셔링 - 플로 </td><td></td><td> NAC # : 10470183 </td></tr><tr><td> 폴리 카테터 (16F) </td><td> 음유 시인 </td><td> 730116 </td><td></td></tr><tr><td> HBSS </td><td> 시그마 </td><td> H8264 - 500ML </td><td></td></tr><tr><td> 히트 건 </td><td> 밀워키 </td><td> 8988-20 </td><td></td></tr><tr><td> 헤파린 </td><td></td><td></td><td> NDC # : 25021 </td></tr><tr><td> Histopaque - 1077 </td><td> 시그마 </td><td> H8889 - 500ML </td><td></td></tr><tr><td> 삽관법 튜브 </td><td> Mallinckrodt </td><td> 86,113 </td><td></td></tr><tr><td> Isoflurane </td><td> MWI, 메리디언 </td><td></td><td> NDC # 13985-030 </td></tr><tr><td> IV 카테터 (18G) </td><td> 백톤 디킨슨 - </td><td> 381547 </td><td></td></tr><tr><td> 케타민을 </td><td> 포르 다지 </td><td></td><td> NDC # 0856-2013 </td></tr><tr><td> 레벨 </td><td> 스완슨 </td><td> LLA001 </td><td></td></tr><tr><td> Luer - 잠금 팁 모자 </td><td> CML 공급 </td><td> 909-001 </td><td></td></tr><tr><td> Marcaine </td><td> Hospira </td><td></td><td> NDC # : 0409-1560-10 </td></tr><tr><td> Metzenbaum 가위 </td><td> 애들러 </td><td> N / A </td><td></td></tr><tr><td> 마이크로 introducer (5 층) </td><td> 갤트 </td><td> KIT 002-01 </td><td></td></tr><tr><td> 모기 집게 </td><td> 애들러 </td><td> N / A </td><td></td></tr><tr><td> 모터 </td><td> Herbach 및 Rademan </td><td> H1 - 08 </td><td></td></tr><tr><td> Oxymorphone </td><td> 엔도 실험실 </td><td></td><td> NDC : 63481-624-10 </td></tr><tr><td> PKH26 다이 키트 </td><td> 시그마 </td><td> PKH26GL - 1KT </td><td></td></tr><tr><td> 돼지의 혈청 </td><td> 쌍둥이 바이오 제품 </td><td> 100-115 </td><td> 전체 성장 매체 2 % 농도 </td></tr><tr><td> 포츠 가위 </td><td> 애들러 </td><td> N / A </td><td></td></tr><tr><td> 정확한 비스타 스킨 스테플러 </td><td> 3M </td><td> 3998 </td><td></td></tr><tr><td> PVC 관 </td><td> McMaster-Carr/Insultab </td><td> 7132K117 </td><td> ID 15.88 mm를 확장 복구 ID 7.95 mm </td></tr><tr><td> 직각 중간 크기 </td><td> 애들러 </td><td> N / A </td><td></td></tr><tr><td> 메스 블레이드 (# 10-15) </td><td> 음유 시인 </td><td> 373910 </td><td></td></tr><tr><td> 실리콘 튜브 </td><td> 맥매스터 - 카 </td><td> 51735K26 </td><td> 16.64 mm OD, 9.52 mm ID를 </td></tr><tr><td> 주사기 (5 CC) </td><td> 백톤 - 디킨슨 (BD) </td><td> 309603 </td><td></td></tr><tr><td> Tegaderm </td><td> 3M </td><td> 90,001 </td><td></td></tr><tr><td> 세 방향 꼭지 </td><td> 켄들 </td><td> 170060 </td><td></td></tr><tr><td> 티 튜브 </td><td> Tico 티타늄 </td><td> N / A </td><td> 로 ½ &quot;OD, 0.065&quot;벽, 0.370 &quot;ID, 0.1737 파운드 / 피트 지정 </td></tr><tr><td> 트립신 </td><td> Lonza </td><td> CC - 5012 </td><td></td></tr><tr><td> 트립신 중화 솔루션 (TNS) </td><td> Lonza </td><td> CC - 5002 </td><td> 0.03 % </td></tr><tr><td> Vetropolycin </td><td> Pharmaderm 동물 보건 </td><td></td><td> NAC # : 12920110 </td></tr><tr><td> Vicryl 봉합사 (3-0) </td><td> Ethicon </td><td> J808T </td><td></td></tr><tr><td> 워터 목욕 Sonicator </td><td> 브랜슨 </td><td> B200 </td><td></td></tr></tbody></table>

autologous endothelial progenitor cell-seeding technology and biocompatibility testing for cardiovascular devices in large animal model

Implantable 심혈관 장치 thromboemboli 형성 하나, 둘, 셋의 위험을 제기 인공 재료 (예 : 티타늄 (TI), polytetrafluoroethylene 확장)에서 제조됩니다. 우리는 autologous 혈액 파생 인간이나 돼지의 내피 전구 세포 (EPCs) 4 티 튜브의 내부 표면을 줄 수있는 방법을 개발했습니다. 돼지는 하부 대정 맥 (IVC)에 돼지의 EPCs의 합류 레이어를 포함 티 튜브를 이온 주입함으로써, 우리는 대형 동물 모델의 prothrombotic 환경에서 셀 씨앗 표면의 개선 biocompatibility를 테스트하고 수정되지 않은 노출된 금속 표면에 비해 5,6,7 (그림 1). 이 방법은 주입의 분 이내에 장치를 endothelialize 및 생체내에서 antithrombotic 함수를 테스트하는 데 사용할 수 있습니다. 주변 혈액 50kg 요크셔 돼지와 EPCs 4,8를 분리 교양의 mononuclear 세포 분율로부터 얻은 것입니다. 티 튜브 (9.4 mm ID는) 3 4.5 cm 세로 섹션으로 미리 잘라 열 수축 튜브로 재조 립했다. 시딩 장치 티 튜브의 느린 회전을 허용하는, 건설되었습니다. 우리는 돼지에 개복술을 수행하고 대장과 방광을 externalized. 샤프하고 무딘 절개는 근위 오른쪽 신장 동맥 말초의 분기점에서 IVC를 해골로 만들다하는 데 사용되었다. 티 튜브는 다음 균일한 셀 - 코팅 9 10 달성 피 X 30 분에서 EPC 정지 및 회전 autologous 찬란 - 라벨로 가득 차 있었다. 100 USP / kg 헤파린의 관리 후, I​​VC 및 요추 정맥의 양쪽이 고정되어 있었다. 4cm의 veinotomy이 수행되고 장치가 삽입 인산염 버퍼 호수로 가득했습니다. veinotomy가 4-0 Prolene 실행 봉합과 폐쇄되면서 하나의 클램프는 데 공기 IVC로 제거되었습니다. 프로 시저의 끝에, 근막이 0 - PDS (polydioxanone 봉합)와 approximated 되었음 피하 공간 2-0 Vicryl로 닫히고 피부 폐쇄 잘라. 3 이후 - 21 일, 돼지가 euthanized 있었다 엉 - 블록을 explanted 및 고정 장치. 티 튜브는 분해 및 내부 표면은 형광 현미경으로 몇 군데 있었다. 우리는 EPC - 씨앗을 품고 튜브 특허 남은 반면, 베어 메탈 티 튜브가 완전히 occluded 것으로 나타났습니다. 또한, 우리는 내부 혈액 접촉 표면에 EPCs의 합류 레이어를 증명할 수 있었다. 결론, 우리의 기술은 장치의 혈전증을 방지하기 위해 autologous EPCs와 함께 주입 몇 분 이내에 티 튜브를 endothelialize하는 데 사용할 수 있습니다. 우리의 외과 방법은 최소한의 혈액 손실 및 EPC - 씨앗 표면 장애와 같은 수정된 장치의 개선 biocompatibility 테스트를 허용합니다.

Watch this Scientific Journal Video about Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model at JoVE.com

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

혈액 - 문의 autologous 세포 및 시험 biocompatibility과 biomaterials 것은 시딩의 티타늄에 대한 방법을 설명합니다. 이 방법은 돼지의 venae cavae에 주입 수술의 분 이내에 씨앗 내피 전구 세포와 티타늄 튜브를 사용합니다. 이 기술은 다른 많은 implantable 생명 의학 장치에 적용할 수 있습니다.

Autologous 내피 전구 세포 - 심는 기술 및 대형 동물 모델에서 순환기 장치에 대한 Biocompatibility 테스트

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

autologous-endothelial-progenitor-cell-seeding-technology

Research

JoVE Journal

Bioengineering

20.3K 조회수.  Duke University. 혈액 - 문의 autologous 세포 및 시험 biocompatibility과 biomaterials 것은 시딩의 티타늄에 대한 방법을 설명합니다. 이 방법은 돼지의 venae cavae에 주입 수술의 분 이내에 씨앗 내피 전구 세포와 티타늄 튜브를 사용합니다. 이 기술은 다른 많은 implantable 생명 의학 장치에 적용할 수 있습니다.

비디오: Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

태반의 맞은 편에 생체이 물과 나노 물질의 전송 속도의 결정을 사용하여<em&gt; 생체</em&gt; 인간 태반 관류 모델

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

연구

생체공학

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

전기 세포 기판 임피던스 내피 증식, 장벽 기능의 정량화에 대한 감지 및 운동성

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

독성 흡입 화학 시험에 대한 시험 관내 세포 배양 모델에서

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Microtissue 안정화를위한 미세 유체 장치에서 레이어별로 층 콜라겐 증착

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention.

내피 세포 캡처 및 유지에 대한 강자성 베어 메탈 스텐트

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

셀 시드를 위한 사용자 지정 Agarose 우물 및 자기 조립 금형 3D 인쇄를 사용 하 여 조직 반지 제작

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

스테셀레이트 비계에 단계별 세포 파종하여 발아 혈관을 연구합니다.

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

생물학적 조직의 자극을 위한 전기 및 자기장 장치

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing novel therapeutics for PH requires a clinically relevant large animal model of increased pulmonary vascular resistance and RVF. This manuscript discusses the latest development of the previously published ovine PH-RVF model that utilizes left pulmonary artery (PA) ligation and main PA occlusion. This model of PH-RVF is a versatile platform to control not only the disease severity but also the RV&#39;s phenotypic response.
Adult sheep (60-80 kg) underwent left PA (LPA) ligation, placement of main PA cuff, and insertion of RV pressure monitor. PA cuff and RV pressure monitor were connected to subcutaneous ports. Subjects underwent progressive PA banding twice per week for 9 weeks with sequential measures of RV pressure, PA cuff pressures, and mixed venous blood gas (SvO2). At the initiation and endpoint of this model, ventricular function and dimensions were assessed using echocardiography. In a representative group of 12 animal subjects, RV mean and systolic pressure increased from 28 &#177; 5 and 57 &#177; 7 mmHg at week 1, respectively, to 44 &#177; 7 and 93 &#177; 18 mmHg (mean &#177; standard deviation) by week 9. Echocardiography demonstrated characteristic findings of PH-RVF, notably RV dilation, increased wall thickness, and septal bowing. The longitudinal trend of SvO2 and PA cuff pressure demonstrates that the rate of PA banding can be titrated to elicit varying RV phenotypes. A faster PA banding strategy led to a precipitous decline in SvO2 &#60; 65%, indicating RV decompensation, whereas a slower, more paced strategy led to the maintenance of physiologic SvO2 at 70%-80%. One animal that experienced the accelerated strategy developed several liters of pleural effusion and ascites by week 9. This chronic PH-RVF model provides a valuable tool for studying molecular mechanisms, developing diagnostic biomarkers, and enabling therapeutic innovation to manage RV adaptation and maladaptation from PH.

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

폐 고혈압 과 우심 심실 부전을 위한 대형 동물 모델: 왼쪽 폐 동맥 리깅 및 양에 있는 진보적인 주요 폐 동맥 밴딩

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.