We thank the laboratory of Dr. Stephen Moss for providing embryonic rat brain tissues. M.D.T. designed the original protocol. This work was funded by National Institutes of Health P41 Tissue Engineering Resource Center Grant EB002520. K.C. was supported with Postdoctoral Fellowship from German Research Foundation (DFG) (CH 1400/2-1).

뇌 조직 분리 프로토콜은 터프 츠 대학 기관 동물 관리 및 사용위원회의 승인과 관리 및 사용 실험 동물의 (기관 동물 관리 및 사용위원회 B2011-45)에 대한 건강 가이드의 국립 연구소에 부합했다. 1. 실크 비계 준비 <ol><li> 누에 나방의 고치에서 실크 용액의 조제는 이전에 19, 20 설명한대로. <ol><li> 가위를 사용하여 8 개의 동일한 조각으로 각각의 누에 고치를 잘라. 조각난 고치 5 g을 약 11 고치를 사용합니다. (15 분) </li><li> 0.02 M 나 2 CO 3 용액 2 L를 준비하고 핫 플레이트를 사용하여 끓여야 가져. (15 분) </li><li> 조각난 고치의 5g의 무게를 30 분 동안 나 2 CO 3 용액을 끓인다. 주걱으로 끓는 실크에게 분에 한 번씩 저어. 또한 디 씹고라는이 단계는 친수성 ​​단백질 sericins에서 실크 피브로인 정화. (30 분) </li><l난&gt; 손으로 피브로인을 짜서 남아있는 세리신 및 화학 물질을 씻어 증류수에 적어도 세 번을 씻어. (5 분) </li><li> 페트리 접시에 젖은 피브로인을 놓고 흐름 후드 O / N에서 피브로인 추출물을 건조. </li><li> 다음 날은 건조 피브로인 질량의 무게와 유리 비커에 피브로인을 배치합니다. (15 분) </li><li> (4)에 의해 건조 피브로인의 질량을 곱하여의 LiBr 9.3 M의 LiBr 용액 (ML)에 필요한 양을 계산에 피브로인을 용해하기 위해 천천히 실크 피브로인 위에의 LiBr 용액을 붓고 주걱을 사용하여 모든 피브로인 섬유 젖어. 증발을 방지하고, 섬유를 용해 할 수 있도록 4 시간 동안 60 ° C에서 배치하는 비이커 커버. (15 분) </li><li> 주사기를 사용하여, 비커로부터 피브로인 용액을 수집하고 3500 MWCO 투석 카세트에 적재. 48 시간 동안 증류수에 대해 투석을 수행합니다. 물을 시간의 각 한 쌍을 변경합니다. </li><li> 주사기를 사용하여, 발 피브로인 수용액을 수집20 분 동안 4 ℃에서 9,000 RPM (~ 12,700 XG)에서 두 번 50 ML 원뿔 튜브와 원심 분리기에 카세트. 각각의 원심 분리 단계 이후에 새로운 튜브에 뜨는을 붓고 펠렛을 폐기합니다. (40 분) </li><li> 건조 중량을 추정하여 피브로인의 농도를 측정한다. 무게 배에 실크 용액 1 ㎖를 붓고. 2 시간 동안 60 ℃의 오븐에서 샘플을 건조. 건조 실크 피브로인을 달아 실크 (100)에 의해 용액의 농도를 예상 얻어진 가중치를 곱하여 실크 피브로인 수용액의 농도를 계산하는 단계 6-9%이다 (W / V). </li><li> 증류수에 희석하여 (V / W)은 6 %로 실크 농도를 조정한다. 정류소 : 액체 실크 피브로인은 밀폐 용기에서 한 달 동안 4 ℃에서 저장 될 수있다. </li></ol></li><li> 실크 솔루션에서 다공성 지지체의 제조. <ol><li> 과립은 이후 단계에서 사용될 500-600 μm의, 크기가 별도의 NaCl을 과립 체. 과립 폐기S는 500 μm의 아래에 600 μm의 위의 크기. (15 분) </li><li> 폴리 테트라 플루오로 에틸렌 (PTFE) 금형 (도 1)로 6 % 실크 용액 30 ㎖를 붓는다. 부드럽게 실크 통해 체질의 NaCl 60g을 산란. 소금의 균일 한 층을 얻기 위해 용기를 누릅니다. 실크 중합 실온에서 48 시간을 품어. </li><li> 중합을 완료하고 남은 액체를 증발시켜 60 ℃에서 1 시간 동안 오븐에서 골격 함유 PTFE 금형을 배치. </li><li> 염을 걸러 48 시간 동안 증류수 2 L를 넣은 비커에 PTFE 몰드의 콘텐츠를 배치했다. 물을 하루에 2 ~ 3 시간을 변경합니다. . 염이 완전히 정지 시점 침출시 몰드로부터 스폰지 지지체를 제거 스폰지가 탈수 비계 않도록 밀폐 용기 내에서 39 ° C의 물에 침지하여 저장 될 수있다. </li><li> 준비가되면, 5mm 직경 생검 펀치와 비계를 잘라. 높이 약 2mm 도달하는 발판을 중고 컷. 푸2mm 직경 생검 펀치 (그림 2A)와 발판의 중심을 NCH. (1 시간) </li><li> . 정지 점 (습식주기 121 ° C, 20 분)들을 살균 물에 침지하여 지지체를 압력솥 ​​: 스폰지가 탈수 비계 않도록 밀폐 용기 내에서 39 ° C의 물에 침지하여 저장 될 수있다. </li><li> 계획된 세포 파종하기 전에, 멸균 0.1 ㎎ / ㎖ 폴리 -D- 라이신 (PDL) 솔루션의 발판을 담그지. 37 ℃에서 1 시간 동안 품어. </li><li> 비계가 결합되지 않은 PDL을 제거하는 인산염 완충 식염수 (PBS)로 3 배 씻으십시오. (30 분) </li></ol></li></ol> 쥐 두피 뉴런 2. 분리 <ol><li> 승인 동물 프로토콜을 수득 한 후 이전과 Pacifici Peruzzi (27)에 의해 기술 된 바와 같이 태아 일부터 18 (E18) 흰쥐 피질을 해부. (2 시간) </li><li> (3)에서 20 분 동안 (소 췌장)에서 0.3 %의 DNase I 0.25 % 트립신 5 ml의 10 피질을 부화7 ° C. </li><li> 1 ㎎ / ㎖ 대두 단백질의 동일한 양을 첨가함으로써 트립신을 비활성화한다. </li><li> 단일 세포 현탁액이 생성 될 때까지 최대 10 ㎖ 피펫 팅 파스퇴르 피펫을 사용하여 피질 아래로 20 번 씹다. 부드러운하고 기포 형성을 피할 것. (10 분) </li><li> 5 분 동안 127 XG에 세포 현탁액을 원심 분리기. </li><li> 다시 일시 배지 10ml에 세포 펠렛을 (로 Neurobasal 배지, 1X B27 보충제, 1X 루타, 1 % 페니실린 / 스트렙토 마이신). 세포를 계산합니다. 예상 세포 농도는 약 2 × 7 / ㎖이다. (20 분) </li></ol> 3. 조립 및 문화를 구축 <ol><li> 세포와 비계 파종. <ol><li> 세포 배양 후드 내부 살균 비계와 필요한 모든기구를 이동합니다. 사용 멸균 집게는 96 웰 세포 배양 플레이트에 웰 당 골격은 하나의 골격을 할당하는 배치. (10 분) </li><li> 그들 전에 종자를 셀에 평형화 세포 배양 배지에 담가 비계ING. 37 ℃에서 1 시간 동안 품어. (10 분) </li><li> 지지체로부터 매체의 과잉 대기음. </li><li> 세포 현탁액 / 비계의 100 μl를 적용합니다. (10 분) </li><li> 지지체에 세포 부착을 허용하도록 37 ° CO / N에서 세포를 품어. </li><li> 다음 아침에 비 부착 세포를 대기음 200 μL / 웰의 신선한 배양액을 적용합니다. (10 분) </li></ol></li><li> 콜라겐 매트릭스에 포함 비계. (2 시간) <ol><li> 나는 얼음에 콜라겐 10 배 PBS, 물, 1 N NaOH를 쥐 꼬리를 놓습니다. 제조업체의 지침에 따라 콜라겐의 작업 솔루션을 준비합니다. 셀 시드 구조 (1 시간까지) 준비가 될 때까지 얼음에 유지한다. </li><li> 인큐베이터에서 셀 시드 실크 구조를 제거하고 매체의 초과를 대기음. </li><li> 사용 멸균 집게는 잘 판에 빈 우물에 비계를 전송하고 3 ㎎ / ㎖ 콜라겐 용액 100 ㎕를 각 발판을 담그지. 다시 조직 배양 플레이트를 배치인큐베이터에서 30 분 콜라겐의 중합을 허용하는. </li><li> 웰 / 예열 세포 배양 배지 100 μl를 적용한다. 일주일 배양 배지의 절반 체적을 대체하여 매일 배지를 변경하기위한 구조. </li></ol></li></ol> 4. 현미경 분석 <ol><li> 라이브 / 죽은 염색 (1 시간) <ol><li> 요오드화 프로피 듐 (PI) 증류수에 1 ㎎ / ㎖ (1.5 밀리미터)의 주식 솔루션을 준비합니다. </li><li> 아세톤 플루 오레 디 아세테이트 (FDA) 2 ㎎ / ㎖의 주식 솔루션을 준비합니다. </li><li> PBS에 희석하여 10 μm의 파이 0.15 μm의 FDA를 포함하는 작업 솔루션을 준비합니다. 수욕 중에서 37 ℃까지 용액을 미리 가온. </li><li> 혈청 에스 테라 제를 제거하기 위해 미리 예열 PBS로 세포를 씻으십시오. PBS를 대기음. </li><li> 2 분 동안 세포에 미리 예열 작업 솔루션을 적용하고 PBS로 씻어. </li><li> FDA 전 λ = 490 N; 이미지에 epifluorescent 현미경을 사용하여 세포 (PI의 전 λ = 490 nm의은, EM λ는 570 nm의 =m은, EM λ)는 514 nm의 =. 얼룩은 40 분에 걸쳐 안정적으로 유지. 장시간 염색 PI의 세포에 의한 흡수와 세포에서 PI / FDA의 공동 지방화에 따른 불특정 오염의 원인이 될 수 있습니다. </li></ol></li><li> 면역 염색 <ol><li> 배양 중 원하는 시점에서 RT에서 30 분 동안 4 % 파라 포름 알데히드 (PFA)으로 세포를 고정한다. 인해 PFA의 독성에 고정하는 단계는 화학 퓸 후드에서 수행되어야 발연. </li><li> . 배 PBS 중지 포인트 발판을 씻으 : PFA 고정 구조는 추가 단계를 진행하기 전에 며칠 동안 4 ° C에서 PBS에 저장 될 수있다. </li><li> 4 ℃에서 일차 항체 O / N을 함유하는 PBS에서 0.2 % 트리톤, 0.25 % 소 혈청 알부민 (BSA)의 골격을 부화. </li><li> 다음 날은 염색 액을 버리고 언 바운드 차 항체를 제거하는 PBS로 골격을 3 × 30 분을 씻는다. </li><li> 2 PBS에 트리톤 0.2 %에 희석 차 항체, 0.25 % BSA로 샘플을 품어실온에서 시간. </li><li> 염색 액을 제거하고 언 바운드 차 항체를 제거하는 PBS로 골격을 3 × 30 분을 씻는다. </li><li> 이미지 1 μm의 단계로 샘플의 100 μm의 Z-부분을 고려하여 20 배 목표와 공 초점 현미경을 사용하여 비계. 이미지 해상도가 최소 1,024 X 1,024 픽셀이어야 얇은 신경 돌기를 시각화합니다. </li></ol></li><li> RNA, DNA와 단백질 분리 주 : RNA, DNA 및 단백질 분리는 제조자의 지시에 따라 상용 AllPrep DNA / RNA / 단백질 미니 키트를 사용하여 수행된다. <ol><li> 사용 멸균 집게는 2 ml의 멸균 튜브에 문화 판에서 발판을 전송합니다. 튜브 당 하나의 발판을 놓습니다. </li><li> 각각의 튜브에 RLT 버퍼의 200 μl를 추가합니다. </li><li> 멸균 microscissors로 세분화하여 구조를 방해. </li><li> 분열 조직을 균일화하려면, 스핀 컬럼 튜브의 콘텐츠를 전송. 마이크로 원심에서 최고 속도로 2 분 동안 스핀.이 스핀 컬럼을 통과 할 해물 균질화된다. </li><li> 을 통해 흐름을 수집하고 RNA, 제조 업체 프로토콜 다음 DNA와 단백질을 분리하는 즉시 사용할 수 있습니다. </li><li> 핵산의 수율 호환되는 다른 방법 (예를 들면, NanoDrop)을 정량화. </li><li> 제조업체의 지시에 따라 BCA 단백질 분석을 이용하여 단백질을 정량 수율. 파장 562 nm에서 마이크로 플레이트 리더를 사용하여 분석의 결과를 측정한다. 주 : 핵산 및 세포 밀도와 관련 구조체 당 단백질의 예상 수율은도 4에 도시된다. </li></ol></li></ol>

<ol>
	<li>Dua, T., Janca, A., Kale, R., Montero, F., Muscetta, A., Peden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1,+Public+health+principles+and+neurological+disorders.">Chapter 1, Public health principles and neurological disorders.</a> Neurological Disorders. , (2005).</li><li>Ohtsuki, S., Hirayama, M., Ito, S., Uchida, Y., Tachikawa, M., Teresaki, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T.+Quantitative+targeted+proteomics+for+understanding+the+blood-brain+barrier:+towards+pharmacoproteomics.">T. Quantitative targeted proteomics for understanding the blood-brain barrier: towards pharmacoproteomics.</a> Expert. Rev. Proteomic. 11 (3), 303-313 (2014).</li><li>Hyder, F., Rothman, D. L., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+energy+demands+of+signaling+and+nonsignaling+components+in+brain+are+conserved+across+mammalian+species+and+activity+levels.">Cortical energy demands of signaling and nonsignaling components in brain are conserved across mammalian species and activity levels.</a> Proc Natl Acad Sci USA. 110 (9), 3549-3554 (2013).</li><li>Wesseling, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+metabonomic+and+proteomic+approach+identifies+frontal+cortex+changes+in+a+chronic+phencyclidine+rat+model+in+relation+to+human+schizophrenia+brain+pathology.">A combined metabonomic and proteomic approach identifies frontal cortex changes in a chronic phencyclidine rat model in relation to human schizophrenia brain pathology.</a> Neuropsychopharmacology. 38 (12), 2532-2544 (2013).</li><li>Fawcett, J. W., Housden, E., Smith-Thomas, L., Meyer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growth+of+axons+in+three-dimensional+astrocyte+cultures.">The growth of axons in three-dimensional astrocyte cultures.</a> Dev Biol. 135 (2), 449-458 (1989).</li><li>Cullen, D. K., Simon, C. M., LaPlaca, 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=Strain+rate-dependent+induction+of+reactive+astrogliosis+and+cell+death+in+three-dimensional+neuronal-astrocytic+co-cultures.">Strain rate-dependent induction of reactive astrogliosis and cell death in three-dimensional neuronal-astrocytic co-cultures.</a> Brain Res. 1158, 103-115 (2007).</li><li>East, E., de Oliveira, D. B., Golding, J. P., Phillips, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alignment+of+astrocytes+increases+neuronal+growth+in+three-dimensional+collagen+gels+and+is+maintained+following+plastic+compression+to+form+a+spinal+cord+repair+conduit.">Alignment of astrocytes increases neuronal growth in three-dimensional collagen gels and is maintained following plastic compression to form a spinal cord repair conduit.</a> Tissue Eng. Part A. 16 (10), 3173-3184 (2010).</li><li>Dubois-Dauphin, M. L., Toni, N., Julien, S. D., Charvet, I., Sundstrom, L. E., Stoppini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long-term+survival+of+in+vitro+engineered+nervous+tissue+derived+from+the+specific+neural+differentiation+of+mouse+embryonic+stem+cells.">The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells.</a> Biomaterials. 31 (27), 7032-7042 (2010).</li><li>Lancaster, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 501 (7467), 373-379 (2013).</li><li>Hogberg, H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+3D+model+of+human+brain+development+for+studying+gene/environment+interactions.">Toward a 3D model of human brain development for studying gene/environment interactions.</a> Stem Cell Res Ther. 4, S4 (2013).</li><li>Kato-Negishi, M., Morimoto, Y., Onoe, H., Takeuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millimeter-sized+neural+building+blocks+for+3D+heterogeneous+neural+network+assembly.">Millimeter-sized neural building blocks for 3D heterogeneous neural network assembly.</a> Adv Healthc Mater. 2 (12), 1564-1570 (2013).</li><li>Watanabe, K., Nakamura, M., Okano, H., Toyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+three-dimensional+culture+of+neural+stem/progenitor+cells+in+collagen+type-1+gel.">Establishment of three-dimensional culture of neural stem/progenitor cells in collagen type-1 gel.</a> Restor Neurol Neurosci. 25 (2), 109-117 (2007).</li><li>Aurand, E. R., Wagner, J. L., Shandas, R., Bjugstad, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+formulation+determines+cell+fate+of+fetal+and+adult+neural+progenitor+cells.">Hydrogel formulation determines cell fate of fetal and adult neural progenitor cells.</a> Stem Cell Res. 12 (1), 11-23 (2014).</li><li>Gurkan, U. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al..Simple+precision+creation+of+digitally+specified,+spatially+heterogeneous,+engineered+tissue+architectures.">et al..Simple precision creation of digitally specified, spatially heterogeneous, engineered tissue architectures.</a> Adv Mater. 25 (8), 1192-1198 (2013).</li><li>Mironov, V., Boland, T., Trusk, T., Forgacs, G., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+printing:+computer-aided+jet-based+3D+tissue+engineering.">Organ printing: computer-aided jet-based 3D tissue engineering.</a> Trends Biotechnol. 21 (4), 157-161 (2003).</li><li>Taylor, A. M., Blurton-Jones, M., Rhee, S. W., Cribbs, D. H., Cotman, C. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nature. 2 (8), 599-605 (2005).</li><li>Tang-Schomer, M. D., Davies, P., Graziano, D., Thurber, A. E., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuits+with+long-distance+axon+tracts+for+determining+functional+connectivity.">Neural circuits with long-distance axon tracts for determining functional connectivity.</a> J Neurosci Methods. 222, 82-90 (2014).</li><li>Tang-Schomer, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+functional+brain-like+cortical+tissue.">Bioengineered functional brain-like cortical tissue.</a> Proc Natl Acad Sci USA. 111 (38), 13811-13816 (2014).</li><li>Rockwood, D. N., Preda, R. C., Y&#252;cel, T., Wang, X., Lovett, M. L., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+fabrication+from+Bombyx+mori+silk+fibroin.">Materials fabrication from Bombyx mori silk fibroin.</a> Nat Protoc. 6, 1612-1631 (2011).</li><li>Lawrence, B. D., Pan, Z., Weber, M. D., Kaplan, D. L., Rosenblatt, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk+film+culture+system+for+in+vitro+analysis+and+biomaterial+design.">Silk film culture system for in vitro analysis and biomaterial design.</a> J. Vis. Exp. (62), e3646 (2012).</li><li>Maddah, M., Grimson, W. E., Warfield, S. K., Wells, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unified+framework+for+clustering+and+quantitative+analysis+of+white+matter+fiber+tracts.">A unified framework for clustering and quantitative analysis of white matter fiber tracts.</a> Med Image Anal. 12 (2), 191-202 (2008).</li><li>Vepari, C., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk+as+a+biomaterial.">Silk as a biomaterial.</a> Prog Polym Sci. 32 (8-9), 8-9 (2007).</li><li>Yao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt-leached+silk+scaffolds+with+tunable+mechanical+properties.">Salt-leached silk scaffolds with tunable mechanical properties.</a> Biomacromolecules. 13 (11), 3723-3729 (2012).</li><li>Kim, U. J., Park, J., Kim, H. J., Wada, M., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+aqueous-derived+biomaterial+scaffolds+from+silk+fibroin.">Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin.</a> Biomaterials. 26 (15), 2775-2785 (2005).</li><li>Gil, E. S., Mandal, B. B., Park, S. H., Marchant, J. K., Omentetto, F. G., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicoidal+multi-lamellar+features+of+RGD-functionalized+silk+biomaterials+for+corneal+tissue+engineering.">Helicoidal multi-lamellar features of RGD-functionalized silk biomaterials for corneal tissue engineering.</a> Biomaterials. 31 (34), 8953-8963 (2010).</li><li>Hopkins, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk+hydrogels+as+soft+substrates+for+neural+tissue+engineering.">Silk hydrogels as soft substrates for neural tissue engineering.</a> Adv Funct Mater. 23 (41), 5140-5149 (2013).</li><li>Pacifici, M., Peruzzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+embryonic+neural+cells:+A+quick+protocol.">Isolation and culture of rat embryonic neural cells: A quick protocol.</a> Vis. Exp. (63), e3965 (2012).</li></ol>

이 비디오 문서의 공개는 코닝 후원됩니다.

Here we described the guidelines to assemble a compartmentalized 3D brain-like tissue model. The model is characterized by dense polarized culture of neurons resulting in the development of two morphologically different compartments: one containing densely packed cell bodies, second containing pure axonal networks. Overall, the scaffold architecture and growth pattern mimic brain cortical tissue including the six laminar layers and white matter tracts21.
The donut-shaped silk protein-based tissue model allows for modifications and tuning of mechanical properties, versatile structural forms, hydrogel fillers and cellular components. Thus, this tissue model establishes a base platform for a wide range of studies. Silk is a favorable candidate for biomaterial platforms due to its biocompatibility, aqueous-based processing, and robust mechanical and degradation properties22. The silk matrix also serves as a stable anchor to reduce collagen gel contraction over time in culture. The properties such as silk concentration and porosity of the scaffold used in this model have been previously adjusted to achieve optimal cell growth and mechanical properties resulting in brain-like tissue elasticity18,23,24. We suggest to keep these parameters constant to ensure the successful outcome and reproducibility of the experiments.
The 3D neuronal network formation was achieved by combining two types of biomaterials with different mechanical properties: a stiff scaffold to provide neuronal anchoring and a softer gel matrix to permit axon penetration and connectivity in 3D. Selective material preferences of the silk scaffold and soft hydrogel provide the underlying principle for compartmentalizing the neurons from the axonal connections. Due to the inert nature of the silk scaffold, functionalization with poly-D-lysine is required for neuronal attachment. However, other cell adhesion promoting factors can be applied such as RGD25 or fibronectin26. To achieve the 3D network formation the silk scaffold needs to be filled with hydrogel in a timely manner as soon as neurons are attached to the scaffold. Likewise, the collagen matrix filler can be replaced by other hydrogels, thus allowing the study of the influence of 3D extracellular environments on axonal network formation to serve as a platform to evaluate novel hydrogels in terms of neuronal compatibility. Additionally, apart from rat cortical neurons other neuronal sources such as hippocampal neurons or induced pluripotent cell (iPSc)-derived neurons can be utilized. Moreover, the tissue model can be used to study heterocellular interactions by including glial cells along with neurons and to build more complex brain-like tissue.
As shown previously, our model can be utilized to evaluate neuronal functionality in 3D microenvironment with a variety of assays such as cell viability, gene expression, LC-MS/MS and electrophysiology, thus demonstrating physiological relevance of the model18. Other methods, which are frequently utilized to evaluate 3D tissue-engineered constructs, such as immunostaining and microscopy8,9,11 can also be used to assess cell distribution and extent of axonal network formation. However, it has to be noted, that due to the high density of the collagen matrix, the penetration of antibodies and depth of the imaging is limited to few hundred micrometers. Moreover, the signal to noise ratio may be affected by nonspecific background fluorescence. This can be overcome by using lipophilic cell tracers and genetically expressed fluorescent proteins which diminish the need for immunostaining11. Alternatively, the imaging can be performed with 2-photon microscopy instead of usual one-photon technique, which may reduce the signal loss, photobleaching, and can be extended to several hundred micrometers of depth.
Summarizing, the silk and collagen-based brain-like tissue offers a robust platform to study neuronal networks in 3D and is a starting point for the development of more advanced models of neurological disorders in the future. Independent from the mode of evaluation, the relative simplicity of this tissue model supports its utility, success and reproducibility.

중추 신경계 (CNS)의 구조적 기능적, 감염성 또는 퇴행성 혈관을 포함하는 다양한 질환에 의해 영향을받을 수있다. 추정 6백80만명 1은 전 세계적으로 성장하고 사회 경제적 부담을 나타내는 신경 질환의 결과로 매년 죽는다. 그러나, 장애의 몇 가지 가능한 치료를해야합니다. 따라서, 신경계 질환을 앓고있는 환자를위한 치료 전략의 혁신적인 중요한 필요성이있다. 불행히도, 많은 CNS 타겟 치료제 인해 생리 학적으로 관련 기능 판독과 급성 및 만성 영향 평가를 허용하지 않는 부적절한 전임상 연구 모델의 활용에 부분적으로 임상 시험 중에 실패합니다. 지난 수십 년간 상당한 연구 노력에도 불구하고, CNS의 구조와 기능에 대해 알 수없는 방대한있다. 더 많은 지식을 습득하기 위하여, 동물 모델은 종종 우리 아르ED 특히 전임상 연구에서, 이러한 외상성 뇌 손상 (TBI), 또는 치매 병리 상태를 모델링한다. 그러나 동물은 인간에서 모두 중추 신경계의 해부학에서뿐만 아니라, 기능, 유전자 발현 및 대사 2-4에 유의 한 차이. 한편, 2D 체외 배양 세포 생물학을 연구하는 일반적인 방법이고 정기적 약물 발견에 사용된다. 그러나, 2 차원 세포 배양은 복잡하고 인간의 두뇌 5-7에 비해 생체 적합성이 부족하다. 저가 및 단순성 2D 세포 배양 연구 또는 동물 모델에 의해 제공되는 복잡성을 대신 없지만, 3 차원의 조직 공학은 시험 관내 및 생체 내 접근법에서 2D 사이에 존재하는 간극을 폐쇄 개선 연구 모델을 생성 할 수있다. 3 차원의 조직 공학은 3D 세포 - 세포 상호 작용에 의해 제공되는 지지체 생체 외 단서에 의해 달성 이상의 생리 학적으로 관련 실험 조건을 제공한다. Desp3D 배양 값 뒤에 상당한 증거 ITE, 거기 이러한 줄기 세포 유래 organoid 배양 8-10 같은 몇 3D CNS 조직 모델 11 neurospheroids는 현재 및 하이드로 겔 배양 12,13 분산. 다층 리소그래피 (14), 및 3D 인쇄 (15) 등의 고급 기술 방법은 폐, 간 및 신장 조직을 연구에 이용되어왔다. 그러나, 대뇌 피질의 구조 생물학을 흉내 낸으로 구획 된 신경 세포의 성장을 허용 3D CNS 모델의 부족이있다. 신경 세포 기관에서 신경 돌기의 분리 된 성장은 이전 축삭 기관의 추적, 칼슘 유입, 네트워크 아키텍처 및 활동의 연구를 허용 미세 (16, 17)를 사용하여 2D 문화에서 증명되었다. 이 아이디어는 세포체와 축삭 책자는 뇌 (18)의 계층 구조를 흉내 낸 다른 구획에있는 3D 편광 신경 조직을 개발하기 위해 우리에게 영감을 </sup&gt;. 우리의 접근은 제한된 용적의 셀을 고밀도로 수용하고 콜라겐 겔에 치밀한 축삭 네트워크의 생장을 허용 고유 실크 골격 설계의 사용에 기초한다. 여기에서 우리는 인공 지지체 제조 및 신경 세포 배양을 포함한 뇌와 같은 조직의 전체 조립 과정을 보여줍니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Na2CO3</td>
			<td>Sigma-Aldrich</td>
			<td>223530</td>
			<td>-</td>
		</tr>
		<tr>
			<td>LiBr</td>
			<td>Sigma-Aldrich</td>
			<td>213225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MWCO 3500 dialysis cassettes&#160;</td>
			<td>Thermo Fisher</td>
			<td>66110</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>Fisher Scientific</td>
			<td>Isotemp</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Centrifuge 5804 R</td>
			<td>Eppendorf</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sieve</td>
			<td>Fisher Scientific</td>
			<td>-</td>
			<td>No. 270, No. 35, No. 30</td>
		</tr>
		<tr>
			<td>PTFE mold</td>
			<td>-</td>
			<td>-</td>
			<td>made in house (Figure 1) 10 cm diameter, 2 cm height</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71382</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 5mm</td>
			<td>World Precision Instrument</td>
			<td>501909</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 2mm</td>
			<td>World Precision Instrument</td>
			<td>501908</td>
			<td>-</td>
		</tr>
		<tr>
			<td>poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td>final concentration 10 ug/ml, dissolved in water</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D1283-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td>final concentration 0.3%</td>
		</tr>
		<tr>
			<td>Soybean protein</td>
			<td>Sigma-Aldrich</td>
			<td>T6522-100MG</td>
			<td>final concentration 1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>warm up to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>B27 supplement 50x</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Penicilin Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagen I, rat tail, 100 mg</td>
			<td>Corning</td>
			<td>354236</td>
			<td>final concentration 3 mg/ml</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>corrosive</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4170-10MG</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Fluorescein Diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378-5G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Olympus MVX10</td>
			<td>Olympus</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>toxic, final concentration 4%</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-10G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>anti-&#946;IIITubulin antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T2200</td>
			<td>rabbit 1:500</td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa-546</td>
			<td>Molecular Probes</td>
			<td>A11010</td>
			<td>goat 1:250</td>
		</tr>
		<tr>
			<td>goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023-5ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Leica SP2 confocal microscope</td>
			<td>Leica</td>
			<td>-</td>
			<td>objective 20x</td>
		</tr>
		<tr>
			<td>QIAshredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>-</td>
		</tr>
		<tr>
			<td>AllPrep DNA/RNA/Protein Mini Kit</td>
			<td>Qiagen</td>
			<td>80004</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethyl alcohol, Pure</td>
			<td>Sigma-Aldrich</td>
			<td>E7023&#160;</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>63689</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000c/2000 UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BCA protein assay</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Plate reader Spectramax M2</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>absorbance 562 nm</td>
		</tr>
		<tr>
			<td>Micro Scissors, Economy, Vannas-type</td>
			<td>TedPella</td>
			<td>1346</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

engineered 3d silk-collagen-based model of polarized neural tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

도넛 모양 솔리드 실크 스폰지 미리 절단은 신경 조직 (그림 2A)를 닮은 구획 구조를 달성하기 위해 독특하고 간단한 아이디어를 나타냅니다. 작은 부피 내에 시드 및 높은 세포 밀도의 증가를 허용하는 매우 높은 표면적이 약 500 μm의 결과 공경 지지체의 다공성 구조 (2 × 7 / ㎖) (도 2B). 또한, 발판의 높은 다공성 연장 시간 프레임을 통해 고밀도 세포 배양의 뛰어난 생존 능력의 결과로 영양분과 폐기물의 무제한 확산 수 있습니다. 신경 세포를 파종시 신속하고 균일하게 발판 기공의 표면에 부착합니다. 세포 부착이 완료되고 세포 실크 기판 상에 평형화 된 후, 스폰지 지지체가 축삭 네트워크 형성 (도 3 차원 환경을 제공하기 위해 부드러운 콜라겐 매트릭스로 채워진다 </strong&gt;). 뉴런은 단지 2 차원 표면 (도 3b)와 혼합 발견 네트워크 결과 기공의 표면에 확장을 성장 콜라겐 매트릭스의 부재 축삭과 세포체의 구획화는 불가능하다. 반면에, 콜라겐 젤은 신경 세포 기관이 실크 발판 (그림 3C)에 부착 된 상태로 유지하면서, 3D 광범위한 축삭 가지를 지원하는 메쉬 작업을 제공합니다. 이러한 성장 패턴은 세포 기관과 순수 축삭 네트워크 뇌의 피질의 회색과 흰색 물질과 유사합니다 (그림 3D)의 구획화에 연결됩니다. 그렇다 현미경에서, 구조물은 실험 뒤에 질문에 따라, 다른 방법 (18)의 다양한 평가 될 수있다. 예를 들어, 구조물에서 DNA, RNA 및 단백질의 분리가 용이하게 상업적 키트를 (도 4)을 사용하여 수행 할 수있다. 핵산 및 단백질 PE의 수율R 구조체는 초기에 실크 비계에 시드 셀의 개수에 의존한다. 이상의 셀 시드 DNA, RNA 및 단백질의 높은 수율. <img alt="그림 1" src="/files/ftp_upload/52970/52970fig1.jpg" /> 실크 스폰지 지지체의 제조에 사용 그림 1. PTFE 금형 크기 :.. 10cm 직경 2cm 높이 <a href="https://www.jove.com/files/ftp_upload/52970/52970fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/52970/52970fig2.jpg" /> 그림 2. 3D 뇌와 같은 조직 모델입니다. (A) 다공성 실크 스폰지 발판. 전에 콜라겐 (녹색 살아있는 세포, 붉은 죽은 세포)를 포함 실크 인공 지지체에 파종에 따라 1 일에서 신경 세포의 (B) 라이브 / 죽은 얼룩. 오른쪽 패널은 엄마를 표시빨간색 프레임에서 캡처 영역의 gnification. <a href="https://www.jove.com/files/ftp_upload/52970/52970fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/52970/52970fig3.jpg" /> 그림 3. 신경 세포의 3 차원 뇌와 같은 조직 모델에서 구획 가지 (A) 비계 구획 :. 레드 셀 바디 컴, 블루 축삭 함. (B) 콜라겐 삽입 전에 파종에 따라 1 일에서 신경 세포의 성장 패턴. (C). 세포체 구획 씨딩 7 일에 신경 생장. (D) 축삭 구획에 파종시 7 일에 신경 가지. 녹색 -. βIIITubulin <a href="https://www.jove.com/files/ftp_upload/52970/52970fig3large.jpg" target="_blank">이의 더 큰 버전을 보려면 여기를 클릭하십시오</a>그림. <img alt="그림 4" src="/files/ftp_upload/52970/52970fig4.jpg" /> (A) RNA와 DNA, 그리고 발판 당 시드 세포의 양에 관련 구조 당 (B) 단백질의 그림 4. 예상 수율 (± 표준 편차, N = 5). <a href="https://www.jove.com/files/ftp_upload/52970/52970fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오 .</a>

Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

편광 신경 조직의 엔지니어링 3D 실크 콜라겐 기반 모델

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

engineered-3d-silk-collagen-based-model-of-polarized-neural-tissue

Research

JoVE Journal

Bioengineering

12.4K 조회수.  Tufts University. Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

비디오: Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2D 및 3D 환경에서 신경 전구 세포의 전기장 제어 감독 마이 그 레이션

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

연구

생체공학

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

트라이 전단지 폴리머 밸브의 상대 유체 역학 평가를위한 프로토콜

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

인간의 3 차원의 조직 공학<em&gt; 시험관</em&gt; 종양 테스트 시스템

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

마우스 모델에서 조직 공학 혈관의 생성 및 손톱 확장

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

3 차원 조직 공학 인간의 식도 점막 모델의 제조, 특성 및 사용 가능성

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

결합 된 3 차원 조직은 엔지니어링<em&gt; 체외</em&gt; /<em&gt; 인 실리코</em특정 돌연변이 배경에서 약물의 효과를 예측&gt; 폐 종양 모델

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

연골과 피부 조직 아날로그 소설 수동 Bioink Precellularization에 대 한 단위 기술 혼합을 이용 하 여 Bioprinting

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

시드 및 마우스 모델에서 생 합성 조직 설계 Tracheal 이식의 이식

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

쥐의 원주 식도 재건을 위한 조직 공학 이식편

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

신경과 같은 조직을 위한 Murine 피질 성상 세포의 3D 생물 인쇄

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

Speed

Subtitles

편광 신경 조직의 엔지니어링 3D 실크 콜라겐 기반 모델