이 작품은 워싱턴 대학 근골격계 연구 센터 (NIH의 P30의 AR057235), 분자 이미징 센터 (NIH의 P50의 CA094056), 제어 공학 교육 그랜트 (NIH 5T32EB018266), NIH R21AR069804, 및 NIH K01AR069116에 의해 지원되었다. 저자는 데이터 수집에서 그의 공헌 패트릭 웡에게 감사의 말씀을 전합니다.

모든 동물 실험은 세인트 루이스 동물 연구위원회의 워싱턴 대학을 준수 하였다. 1. 동물 <ol><li> (N = 6, BALB-M, BALB / cAnNTac) 및 10 주령 핵 인자 카파 B - 루시퍼 라제 리포터 동물들 (NF-κβ 뤽)는에 자란 10 주령의 BALB / C : 마우스의 두 가지 변종을 구하는 BALB / C 배경 (N = 6, BALB / C-(TG RELA 뤽) 31Xen). </li><li> 해부하기 전에, 시간 거주지의 추가 2 분,이어서 5 분 동안 2.5-3 L / min의 유량으로 CO 2 과량으로 동물을 안락사. </li><li> 박리 전에 2 분 동안 70 % 에탄올 욕에서 입욕하여 동물의 외측 영역 소독. </li></ol> 2. 해부 <ol><li> 체강 노출되도록 절개 작은 가위를 사용하여 마우스의 등쪽 표면에 길이 방향의 수직 절단을 확인. </li><li> 부터 동물의 척추의 양쪽에 두 개의 길이 방향의 수직 절단을 확인제 요추 척추 꼬리로 (L1) (C8). </li><li> 메스 미세 겸자 미세 해부 가위를 사용하여 조심스럽게 동물의 체강 내에서 L1-C8의 척추를 제거한다. </li><li> 긁어 또는 복부 측 IVD을 손상하지 않도록 보장하면서 조심스럽게 척추 주변 초과 연조직을 제거한다. </li><li> 또한, L1 / L2, L3 / L4, L5 / L6, C3 / C4, C5 / C6 및 C7 / C8 디스크에서 척추 디스크 - 척추 기능 척추 유닛 (Fsus를)로 척추 해부. </li><li> 1 % 2 분 동안 페니실린 스트렙토 마이신이 보충 행크 평형 염 용액에 Fsus를 씻어. </li><li> 교양 및 치료 Fsus를 (신선한), 배양 그러나 치료 Fsus를 (제어), 배양 및 찔린 처리 Fsus를 (스탭) : 무작위로 세 그룹으로 Fsus를 할당합니다. </li><li> 스냅 즉시 -20 ° C의 냉동고에서 해부하고 저장 한 후 액체 질소로 신선한 FSU 샘플을 동결. </li></ol> 3. 장기 문화 조건<ol><li> 멸균 배지 500㎖의 준비 1 : 둘 베코 변형 이글 배지 : 영양 혼합물 F12 20 % 소 태아 혈청 (FBS) 및 1 % 페니실린 - 스트렙토 마이신이 보충 된 (DMEM F12). </li><li> 24 웰 배양 플레이트의 각 웰에 10 ㎖의 혈청 피펫, 피펫 배양액 2 ㎖의 사용. </li><li> 절개를 따라 린스 미디어 채워진 24 웰 플레이트의 개별 웰에 서로 FSU를 배치했다. </li><li> 37 ° C, 5 % CO 2, 20 % O 2 및&gt; 90 %의 습도를 유지하고 멸균 배양기에서 샘플을 인큐베이션. </li><li> 24 시간의 초기 배양 기간 후, 기계적으로도 1a에 도시 된 바와 같이 무균 27 게이지 바늘을 사용하여 섬유륜의 바늘 구멍을 통해 스터브 군 샘플 손상. </li><li> 새로운 사용 무균 핀셋 오래 플레이트로부터 새로운 미디어 채워진 24 웰 배양 플레이트를 준비하여 미디어마다 48 시간을 변경하고, 이전 샘플. 기존의 미디어와 dispos을 대기음생물 학적 폐기물 빈의 옛 문화 판의 전자. </li><li> 배양의 마지막 날에, 24 시간 동안 0.75 ㎎ / ㎖ 니트로 블루 테트라 졸륨 클로라이드를 함유하는 배지에서 모든 샘플을 배양한다. </li><li> 그 후, 동결 모든 Fsus를 기계적 시험 또는 조직학 (도 1b)에 대한 준비를 할 때까지 -20 ℃ 냉동고에서 액체 질소와 함께 점포. </li></ol> 4. 경도 측정 NF-κB <ol><li> 이미지는 1에서 NF-κβ - 루시 페라 동물 Fsus를, 5, 13, 19 일 NF-κB의 발현을 평가합니다. </li><li> 10 분 동안 37 ℃에서 멸균 배양기에서 각각 1 ㎎ / ㎖의 루시페린 용액 10 μL를 첨가하고 잘 배양한다. </li><li> 이미지 1 분의 노출 시간 및 (2)의 빈 설정으로 촬상 시스템을 이용하여 생물 발광 용 샘플. </li><li> 동시에, 사진을 가지고와 IVD에 발광의 해부학 적 위치를 결정하기 위해 생물 발광 이미지와 오버레이 기계를 사용합니다. </li></ol><p클래스 = &quot;jove_title&quot;&gt; 5. 기계 평가 <ol><li> 변위 제어 동적 압축 9를 사용하여, 사후 관리의 기계적 거동을 결정한다. </li><li> 손상 및 IVD에 부착 된 연골 종판 유지하면서 해부 현미경 메스와 핀셋을 사용하여, 각 시료에서의 FSU 뼈 추체를 제거한다. </li><li> 시아 노 아크릴 레이트 접착제를 이용하여 1cm X 1cm X 0.2 cm의 알루미늄 판으로 격리, 사후 관리를 첨부. </li><li> 레이저 마이크로 미터를 이용하여 각 디스크의 세로축에 3 개 회 측정의 평균을 취함으로써, 디스크의 높이 및 폭을 측정한다. 디스크의 최대 폭의 평균 디스크 높이를 나눈 디스크 높이 비율을 계산한다. </li><li> 기계적 시험에 사용되는 입력 스트레인 값을 계산하기 위해 측정 된 디스크 키를 사용한다. 디스크의 평균 높이는 약 690 ㎛의 ± 39 ㎛, 참고. </li><li> compressi 하에서 인산 완충 식염수 용기에 샘플을 놓고기계 0.02 N.에 디스크를 미리로드 </li><li> 순환 시험 3 20 1 %의 변형 수준 및 사이클 5 % 스트레인 레벨 1 Hz에서 각각 정현파를 사용하여 디스크를 압축 및 IVD의 하중과 변위 값을 기록한다. 긴장과 손상을 방지하기 위해 시험 디스크 사이의 휴식 시간이 10 분을 허용한다. </li><li> 최종 사이클의 로딩 단계로부터의 평균 강도를 계산하고, 변위 및 하중 데이터 사이의 위상 각의 정접을 계산한다. </li></ol> 6. 프로테오글리칸 및 콜라겐 정량화 <ol><li> 기계적 시험에 따라, 미리 칭량 한 원심 관에 분리 배치 IVD 및 분석 저울을 사용하여 중량을 측정함으로써 디스크 습윤 중량을 측정한다. </li><li> 250 μL의 파파인 소화 용액을 분리, 사후 관리 다이제스트 (0.1 M 아세트산 나트륨, 0.01 M EDTA, 0.005 M 염산 시스테인, pH를 6.4) 블록 히터를 사용하여 밤새 65 ° C에서. </li><li> 10 분 동안 원심 분리 샘플2,000 XG의 상청액을 수집하고. </li><li> 콘드로이틴 설페이트 표준과 디메틸 메틸렌 블루 (DMMB) 분석을 사용하여 IVD의 프로테오글리칸 함량을 측정. </li><li> DMMB 용액 (21 mg을 DMMB 5 mL의 무수 에탄올 1 L DDH 2 O 2 g을 포름산 나트륨)을 준비한다. </li><li> 각 웰에 피펫 DMMB 용액 250 μL와 함께 96 웰 플레이트에서 30 중으로 표준 μL 샘플. </li><li> 분광 광도계를 사용하여 525 nm에서 플레이트의 흡광도를 읽는다. </li><li> 제조업체의 지침에 따라 히드 록시 프롤린 분석 키트를 사용하여 콜라겐 함량을 측정한다. </li></ol> 7. 조직학 <ol><li> 24 시간 동안 4 % 파라 포름 알데히드의 샘플들의 서브 세트를 수정을 48 시간 동안 5 % 포름산 탈 석회 성으로 만들다, 에탄올로 탈수시키고, 파라핀에 삽입. </li><li> sagittally 10 개 μm의 두께 부, 샘플을 마이크로톰을 사용하여 유리 슬라이드에 적용 섹션. Safranin-O / 빨리 할머니를 사용하여 섹션을 얼룩EEN 및 DAPI. </li><li> 광학 현미경을 사용하여, 이미지는 10 배 배율로 슬라이드. </li></ol> 제 조영 증강 microComputed 단층 촬영 (microCT) <ol><li> 7 일의 시점에서 길이 Ioversol의 콘트라스트 향상 10 단자 인 몰리브덴 산 (PMA), 콘트라스트 강조를 사용하여 0, 2, 5, 7 일 시점에서의 샘플의 일부를 스캐닝. </li><li> microCT 스캔 시간에 앞서 4 시간, 4, 37 ° C에서 멸균 배양기에서 350 밀리그램 / 약 50 ㎎ / ㎖ 요오드 함유 Ioversol 용액의 최종 농도를위한 배양 배지에 ㎖의 요오드 Ioversol 용액 300 μL를 추가 부화 시간. </li><li> 인큐베이션 후, 1 ㎖의 마이크로 원심 튜브에의 멸균 거즈 장소에서 샘플 랩. </li><li> microCT 시스템 플레이스 샘플 45 keVp 177 μA, 10.5 μm의 복셀 사이즈, MS (300)에 통합 검색. </li><li> 수출 DICOM 파일로 microCT 데이터와 소프트웨어를 사용하여 시각화 ( <em&gt; 예를 들어, OsiriX). </li><li> 7 일 시점에서 3 일 동안 5 % PMA 용액을이어서 24 시간 동안 4 % 파라 포름 알데히드 용액을 사용하여 샘플을 고정하고, 단계 8.4에서 사용 된 것과 동일한 설정을 사용하여 검사한다. </li></ol> 제 3 차원 유한 요소 모델링 <ol><li> 세그먼트 수동 임계 값 (예를 들어, OsiriX)와 소프트웨어를 사용하여, 사후 관리의 DICOM 파일. </li><li> Meshlab를 사용하여 복셀 기반 유한 요소 메쉬로 IVD의 NP 및 AF 볼륨 변환. </li><li> 완전한 IVD를 형성하고 유한 요소 소프트웨어에서 실험적으로 결정 경계 조건을 적용 NP와 AF의 미세 구조를 결합합니다. </li></ol>

<ol>
	<li>Dagenais, S., Caro, J., Haldeman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+low+back+pain+cost+of+illness+studies+in+the+United+States+and+internationally.">A systematic review of low back pain cost of illness studies in the United States and internationally.</a> Spine J. 8 (1), 8-20 (2008).</li><li>Urban, J., Roberts, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degeneration+of+the+intervertbral+disc.">Degeneration of the intervertbral disc.</a> Arthritis Res Ther. 5 (6), 1-48 (2003).</li><li>Mirza, S. K., White, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+intervertebral+disc+and+pathophysiology+of+herniated+disc+disease.">Anatomy of intervertebral disc and pathophysiology of herniated disc disease.</a> J Clin Laser Med Surg. 13 (3), 131-142 (1995).</li><li>Acaroglu, E. R., 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=Degeneration+and+aging+affect+the+tensile+behavior+of+human+lumbar+anulus+fibrosus.">Degeneration and aging affect the tensile behavior of human lumbar anulus fibrosus.</a> Spine. 20 (24), 2690-2701 (1995).</li><li>Abraham, A. C., Liu, J. W., Tang, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+changes+in+the+structure+and+inflammatory+response+of+the+intervertebral+disc+due+to+stab+injury+in+a+murine+organ+culture+model.">Longitudinal changes in the structure and inflammatory response of the intervertebral disc due to stab injury in a murine organ culture model.</a> J Orthop Res. 34 (8), 1431-1438 (2016).</li><li>Ohtori, S., Inoue, G., Miyagi, M., Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathomechanisms+of+discogenic+low+back+pain+in+humans+and+animal+models.">Pathomechanisms of discogenic low back pain in humans and animal models.</a> Spine J. 15 (6), 1347-1355 (2015).</li><li>Pelle, D. W., 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=Genetic+and+functional+studies+of+the+intervertebral+disc:+A+novel+murine+intervertebral+disc+model.">Genetic and functional studies of the intervertebral disc: A novel murine intervertebral disc model.</a> PLoS ONE. 9 (12), (2014).</li><li>Zhang, 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=Time+course+investigation+of+intervertebral+disc+degeneration+produced+by+needle-stab+injury+of+the+rat+caudal+spine.">Time course investigation of intervertebral disc degeneration produced by needle-stab injury of the rat caudal spine.</a> J Neurosurg: Spine. 15 (4), 404-413 (2011).</li><li>Liu, J. W., Abraham, A. C., Tang, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+high-throughput+phenotyping+of+the+viscoelastic+behavior+of+whole+mouse+intervertebral+discs+using+a+novel+method+of+dynamic+mechanical+testing.">The high-throughput phenotyping of the viscoelastic behavior of whole mouse intervertebral discs using a novel method of dynamic mechanical testing.</a> J Biomech. 48 (10), 2189-2194 (2015).</li><li>Lin, K. H., Wu, Q., Leib, D. J., Tang, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+the+contrast-enhanced+microCT+imaging+of+murine+intervertebral+discs.">A novel technique for the contrast-enhanced microCT imaging of murine intervertebral discs.</a> J Mech Behav Biomed Mater. 63, (2016).</li><li>Pasparakis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+tissue+homeostasis+by+NF-kappaB+signalling:+implications+for+inflammatory+diseases.">Regulation of tissue homeostasis by NF-kappaB signalling: implications for inflammatory diseases.</a> Nat Rev: Immunol. 9 (11), 778-788 (2009).</li><li>O&#8217;Connell, G. D., Vresilovic, E. J., Elliott, D. M. <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+animals+used+in+disc+research+to+human+lumbar+disc+geometry.">Comparison of animals used in disc research to human lumbar disc geometry.</a> Spine. 32 (3), 328-333 (2007).</li><li>Lee, C. R., 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=In+vitro+organ+culture+of+the+bovine+intervertebral+disc:+effects+of+vertebral+endplate+and+potential+for+mechanobiology+studies.">In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies.</a> Spine. 31, 515-522 (2006).</li><li>Chan, S. C., Gantenbein-Ritter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Intact+Bovine+Tail+Intervertebral+Discs+for+Organ+Culture.">Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture.</a> J. Vis. Exp. (60), e3490 (2012).</li><li>Holguin, N., Aguilar, R., Harland, R. A., Bomar, B. A., Silva, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aging+mouse+partially+models+the+aging+human+spine:+lumbar+and+coccygeal+disc+height,+composition,+mechanical+properties,+and+Wnt+signaling+in+young+and+old+mice.">The aging mouse partially models the aging human spine: lumbar and coccygeal disc height, composition, mechanical properties, and Wnt signaling in young and old mice.</a> J Appl Physiol. 116 (12), (2014).</li></ol>

이 논문의 저자는 더 경쟁 재정적 이해 관계가 없음을 선언합니다.

이 프로토콜은 IVD의 생물학적 변화를 모니터링에 중점을두고 쥐의 FSU의 장기 문화를 설명합니다. 이러한 문화의 성공적인 유지 보수주의 멸균 기술을 필요로한다. 특히, 박리는 2.1-2.6 단계 배양은 3.1-3.6 무균 상태가 유지되도록하기 위해 특별한주의를 필요로 단계,이 단계는 오염을 최소화하는 HEPA 공기 흐름과 격리 절차 방에서 바람직하게 수행되어야한다. 절개 과정 조...

요통 (LBP)는 직장에서의 글로벌 장애에 대한 주요 요인 및 생산성 손실, 그리고 미국인들은 혼자 LBP 치료 한 50 억 달러의 초과 지출. 유행이지만, LBP의 원인은 복잡하고 다원적 남아있다. 그러나 척추 디스크 (IVD)의 변성은 LBP 2의 가장 중요한 위험 인자 중 하나입니다. IVD 세 마이크로 제조된다 : 외부 환형 섬유증 (AF), 내부 수핵 (NP), 및 근위부와 원위부 (3) 전체의 구조를 끼우는 두 연골 종판. 노화와 퇴행으로, IVD 구성 요소는 구성 적, 구조적 변화, 기계적 4. 이러한 변화가 NP의 프로테오글리칸 및 수분의 손실을 포함하는, 디스크 높이의 감소, 및 기계적 능력을 저하 5. 이러한 변화는종종 LBP 현상 6 이어질 사건 캐스케이드 절정 공동 공간으로 염증 반응뿐만 아니라, 호중구 및 후근 신경절 침입을 촉진 사이토킨 수반. 이 요통의 발생 전에 변성의 원인을 분리하는 것이 불가능하기 때문 IVD 변성의 메커니즘을 공부하는 것은 인간의 도전이다. 따라서, IVD 기관에 이르기까지 실험 시스템을 단순화의 환원 주의적 접근 방식은 인과 변수의 기계적 미세 조정을 할 수 있으며 자신의 다운 스트림 효과 (5) 검사. 이 시스템은 따라서 IVD 퇴화에 외부 자극 효과의 직접적인 해석을 가능하게 만 원시 세포 집단 및 세포 외 기질을 둘러싸는 감소된다. 또한, 낮은 비용과 생쥐 모델의 확장 성뿐만 아니라, 유전자 변형 동물 (7)의 많은 수의, t 허용IVD 퇴행성 메커니즘과 잠재적 치료의 그는 빠른 대상으로 심사. 여기서는 IVD 세포 및 조직 안정성, 사후 관리가 &#39;항상성 기계적 구조, 및 염증성 패턴 주어진 특정 포커스 21 일 이상 유지되는 뮤린 기관 배양 시스템을 설명한다. 이 방법을 사용하여, 우리는 디스크의 변성 뒤에 메커니즘을 이해하는 자상에 의한 손상 모델 (8)에서, 사후 관리 &#39;기능의 변화를 모니터링 할 수 있습니다. 더욱이, 우리는 조영 증강 microCT 이미징 및 IVD 입체 고해상도 모델링 포함한 추가 연구를 수행하는 기관이 배양 방법의 여러 애플리케이션을 설명한다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 well plate</td>
			<td>Midwest Scientific</td>
			<td>TP92096</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Midwest Scientific</td>
			<td>TP92024</td>
			<td>Used for organ culture</td>
		</tr>
		<tr>
			<td>25 ml pipettes</td>
			<td>Midwest Scientific</td>
			<td>TP94024</td>
			<td>Used for organ culture</td>
		</tr>
		<tr>
			<td>10 ml pipettes</td>
			<td>Midwest Scientific</td>
			<td>TP94010</td>
			<td>Used for organ culture</td>
		</tr>
		<tr>
			<td>5 ml pipettes</td>
			<td>Midwest Scientific</td>
			<td>TP94005</td>
			<td>Used for organ culture</td>
		</tr>
		<tr>
			<td>500 ml bottle top filters, 22um</td>
			<td>Midwest Scientific</td>
			<td>TP99505</td>
			<td>Used for filter media</td>
		</tr>
		<tr>
			<td>10 ul pipette tips</td>
			<td>Midwest Scientific</td>
			<td>NP89140098</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>200 ul pipette tips</td>
			<td>Midwest Scientific</td>
			<td>NP89140900</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>1000 ul pipette tips</td>
			<td>Midwest Scientific</td>
			<td>NP89140920</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>DMEM /F-12</td>
			<td>Invitrogen</td>
			<td>11330032</td>
			<td>Used for culture media</td>
		</tr>
		<tr>
			<td>Optiray 350</td>
			<td>Guebert</td>
			<td>19133341</td>
			<td>Used for contrast enhanced microCT</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F2442</td>
			<td>Used for culture media</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin&#160;</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td>Used for culture media</td>
		</tr>
		<tr>
			<td>Tetrazolium Blue Chloride</td>
			<td>Sigma</td>
			<td>T4375</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>D-Luciferin</td>
			<td>Sigma</td>
			<td>L6152</td>
			<td>Used for bioluminescence imaging</td>
		</tr>
		<tr>
			<td>Chondroitin Sulfate</td>
			<td>Sigma</td>
			<td>C9819</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>10% Phosphomolybdic Acid Solution</td>
			<td>Sigma</td>
			<td>HT152</td>
			<td>Used for contrast enhanced microCT</td>
		</tr>
		<tr>
			<td>Safranin O</td>
			<td>Sigma</td>
			<td>S8884</td>
			<td>diluted to .1% concentration (in water)</td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma</td>
			<td>F7258</td>
			<td>.001% concentration</td>
		</tr>
		<tr>
			<td>Papain from papaya latex</td>
			<td>Sigma&#160;</td>
			<td>P3125</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>Nucleic acid staining</td>
		</tr>
		<tr>
			<td>Cyanoacrylate Glue</td>
			<td>Loctite</td>
			<td>234790</td>
			<td>Adhesive&#160;</td>
		</tr>
		<tr>
			<td>1.5 ml Microcentrifuge Tubes&#160;</td>
			<td>Fischer Scientific</td>
			<td>S348903</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>Big Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioDent</td>
			<td>ActiveLife</td>
			<td></td>
			<td>For mechanical testing</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>Biotek</td>
			<td></td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>AxioCam503</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>VivaCT40</td>
			<td>Scanco</td>
			<td></td>
			<td>MicroCT</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Denver Instrument Company</td>
			<td>A-200DS</td>
			<td>Analytical balance</td>
		</tr>
		<tr>
			<td>Incubator HERAcell 150i</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Organ Culture</td>
		</tr>
		<tr>
			<td>Dissection Scope</td>
			<td>VistaVision</td>
			<td></td>
			<td>Used during dissection</td>
		</tr>
		<tr>
			<td>Laser Micrometer</td>
			<td>Keyence</td>
			<td>LK-081</td>
			<td>Measuring disc height</td>
		</tr>
		<tr>
			<td>Microcentrifuge 5810 R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica&#160;</td>
			<td>RM2255</td>
			<td>Used for histology</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 7</td>
			<td>GraphPad</td>
			<td></td>
			<td>For statistics</td>
		</tr>
		<tr>
			<td>MATLAB R2014a</td>
			<td>Mathworks</td>
			<td></td>
			<td>For modeling</td>
		</tr>
		<tr>
			<td>Osiri-LXIV</td>
			<td>Pixmeo</td>
			<td></td>
			<td>Open Source</td>
		</tr>
		<tr>
			<td>MeshLab v1.3.3</td>
			<td>Visual Computing Lab - ISTI - CNR</td>
			<td></td>
			<td>Open Source</td>
		</tr>
		<tr>
			<td>PreView/FEBio 2.3</td>
			<td>Utah MRL &#38; Columbia MBL</td>
			<td></td>
			<td>Open Source</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Windows</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cohan-Vannas Spring Scissors&#160;</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>15000-02</td>
			<td>Or any nice pair of spring scissors</td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp&#160; (small)</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp&#160; (larger)</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>14060-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>11252-40</td>
			<td>At least 2; can also use #3&#160;</td>
		</tr>
		<tr>
			<td>Extra Fine Graefe Forceps, serrated</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>11150-10</td>
			<td>At least 2</td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps, serrated</td>
			<td>World Precision Instruments</td>
			<td>503719-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps, teeth</td>
			<td>World Precision Instruments</td>
			<td>501244</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle - #3</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle - #4</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>10004-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blades - #23</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>10023-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Insect Pins , size 000</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>26000-25</td>
			<td></td>
		</tr>
		<tr>
			<td>27G Needle</td>
			<td>BD PrecisionGlide Needles</td>
			<td>BD305109</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro organ culture model of the murine intervertebral disc

척추 디스크 (IVD)의 변성은 요통에 크게 기여. IVD는 전송 및 척추에 부하를 저해하는 역할을하는 섬유 연골 관절이다. IVD는 프로테오글리칸 풍부한 수핵 (NP) 및 연골 단부 플레이트에 의해 협지 된 콜라겐이 풍부한 고리 섬유증 (AF)로 구성된다. 서로 인접 척추와 척추-IVD 구조는 기능 척추 유닛 (FSU)을 형성한다. 이 미세 고유 세포 유형뿐만 아니라 고유의 세포 외 매트릭스를 포함한다. 구소련의 전체 기관 문화는 원시 세포 외 기질, 세포 분화 표현형 및 세포 - 기질 상호 작용을 유지합니다. 따라서, 기관 배양 기술은 IVD의 복잡한 생물학적 메커니즘을 조사하는 데 특히 유용하다. 여기, 우리는 IVD에 대한 질병 메커니즘과 치료법을 연구하기위한 이상적인 플랫폼을 제공합니다 전체 요추 마우스 Fsus를 배양을위한 높은 처리량 방법을 설명합니다. 또한, 우리는의를 설명조영 증강 microCT 이미징 및 IVD 입체 고해상도 유한 요소 모델링을 포함하여 추가 연구를 수행이 장기 배양 방법을 이용하는 애플리케이션 everal.

도 2-3 배양 마우스, 사후 관리를위한 분배 프로테오글리칸, NF-κB의 발현 강도, 점도, 디스크 키, 습윤 중량의 대표적인 결과를 나타낸다. 제대로 배양하면, 제어 그룹의 IVD 매개 변수는 신선한 그룹에서 유의 한 차이를해서는 안됩니다. 문화가 손상 달리 감염 또는 경우, 대조군 특히 NF-κB의 발현과 프로테오글리칸 유통의 신선한 그룹에서 다른 것입니다 (결과는 도시하지 않음). IVD의 ...

Watch this Scientific Journal Video about An In Vitro Organ Culture Model of the Murine Intervertebral Disc at JoVE.com

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

척추 디스크 (IVD)의 전체 기관 배양 물은 세포 외 기질 원시 세포 표현형, 및 세포 - 기질 상호 작용을 보존한다. 여기에서 우리는 마우스 요추 및 기능적 척추 단위 꼬리, 사후 관리 및이 시스템을 이용한 여러 응용 프로그램을 사용하여 체외 배양 시스템을 설명합니다.

<em&gt; 체외</em쥐의 추간판의&gt; 장기 문화 모델

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

an-in-vitro-organ-culture-model-of-the-murine-intervertebral-disc

Research

JoVE Journal

Bioengineering

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

9.4K 조회수.  Washington University in St. Louis.  척추 디스크 (IVD)의 전체 기관 배양 물은 세포 외 기질 원시 세포 표현형, 및 세포 - 기질 상호 작용을 보존한다. 여기에서 우리는 마우스 요추 및 기능적 척추 단위 꼬리, 사후 관리 및이 시스템을 이용한 여러 응용 프로그램을 사용하여 체외 배양 시스템을 설명합니다. 

비디오: An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

난소와 Oviducts의 3 차원 조직 문화 알긴산의 Hydrogels

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

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생체공학

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

조직 문화에 대한 손상 소의 꼬리 척추 디스크의 준비

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

손쥐 모델에서 창자의 조직 공학

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

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 ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

복원 된 인간의 각막 모양의 상피 (RHCE) 조직 모델을 사용하여 눈에 자극성 화학 물질의 위험 식별을위한 눈 자극 시험 (EIT)

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

거꾸로 방향 가압된 주머니 안에 전체 인간의 마음의 decellularization

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

매트릭스 세포 대사 크로스토크 연구를 위한 인간 3D 세포외 세포외 매트릭스-세포세포 배양 모델

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

장기 나노 물질 노출 다음 체외 물질 독성 테스트를 위한 고급 3D 간 모델

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

하이드로겔 기반 3D 문화 모델을 자동화및 표준화방식으로 제조하는 오픈 소스 기술 플랫폼

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

심근 경색의 뮤린 모델에서 3D 생체 인쇄 패치의 이식

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...