이 연구는 CounterACT와 프로그램, 국립 보건원 (NIH), 이사의 사무실 및 환경 보건 과학 연구소 (NIEHS) 부여 번호 U54 ES015678 (CWW)에 의해 지원된다. SA는 어린이 병원 광산 공동 파일럿 보너스 번호의 G0100394 및 아동 병원 콜로라도 연구 Institue 파일럿 상 # 1 G0100471 콜로라도 / 콜로라도 학교에서 지원됩니다.

1. 쥐 심근 문화 <ol><li> 모든 실험은 기관의 동물 관리 및 사용위원회, IACUC 승인 프로토콜에 따라 수행되었다. </li><li> 이전에 4 설명한 방법을 사용하여 수컷 쥐의 마음 (심실) (2백40~2백60g)에서 쥐의 심근 세포를 얻습니다. (100 ㎎ / ㎏, 발가락 핀치 방법으로 마취를 확인) 간단히, 펜토 바르 비탈의 복강 내 주사를 사용하여 동물을 마취 한 후 10.0 ㎖, 크렙스 링거 버퍼, 산도 7.4를 포함하는 1 ㎜ 칼슘 2 +에 마음을 제거합니다. </li><li> 혈액을 제거하고 0.02 %의 단백질 분해 효소와 0.06 %의 콜라게나 제 (5.0 ㎖ / 마음)를 포함하는 칼슘 2 + 무료 크렙스 링거 버퍼로 전환 마음에게 5 - 6 배를 씻어. 가끔 흔들면서 37 ° C에서 10 ~ 15 분 동안이 솔루션의 마음을 품어. </li><li> 15 분 후, 추가로 5 분 동안 칼슘 2 + 무료 크렙스 링거 버퍼 효소 솔루션을 세척 할 것. 이완 된 조직을 우리로부터 세포를 해제위아래로 여러 번 현탁액을 피펫으로 25 ㎖의 피펫을 보내고. </li><li> 70 μm의 나일론 메쉬를 필터링하여 조직에서 세포를 분리하고 0.1 밀리미터 칼슘 2 +를 포함 크렙스 링거 버퍼에 정착 할 수 있습니다. 0.2 mM의 칼슘 2 +를 함유하는 1.0 ml의 크렙스 링거 완충액 세포 펠렛을 중지. </li><li> 조심스럽게 nonmyocytes에서 심근를 분리하고 30 분 동안 정착 할 수 있도록, 15 ML의 원심 분리기 튜브에 5.0 ㎖의 60 ㎍ / ㎖의 소 혈청 알부민, BSA를, 층. 심근은 무거워 튜브의 하단으로 이동합니다. 조심스럽게 뜨는 세포와 용지를 제거합니다. 또한 세포를 정화 한 번이 단계를 반복합니다. </li><li> 점차적으로 심실 근세포 / 심근 1.0 밀리미터 칼슘 2 + 포함하는 버퍼에 (0.25 ㎜, 0.5 ㎜, 0.75 ㎜, 1.0 밀리미터 칼슘 2 + 포함 된 버퍼를 사용하여 단계)의 세척 및 둘 베코의 수정 된 이글 매체로 구성된 ACCT 매체에 재현 탁으로 전환 , DMEM의 containiNG 2 ㎎ / ㎖ BSA, 2 MM의 L-카르니틴, 5 MM의 크레아틴, 5 mM의 타우린, 100 IU / ㎖ 페니실린, 100 ㎍ / ml의 스트렙토 마이신. </li><li> 100mm 또는 35mm의 라미닌에 100-150 세포 / mm 2의 밀도로 ACCT 매체에서 심근 플레이트 라미닌 (1 ㎍ / cm 2)와 프리 코트 플라스틱 배양 접시 또는 40 X 22mm 유리 커버 슬립을 입혔다. 1 시간 후, 부착되지 않은 세포를 제거하기 위해 2.0 ml의 ACCT와 설거지. 신선한 매체의 10.0 ML을 추가하고 세포를 품어. </li></ol> 인간기도 상피 기저 세포의 2. 차별화 된 공기 - 액체 인터페이스 (ALI) 문화 <ol><li> 기관 평가위원회에서 국가 질병 연구 IC에서 인간의 기관지 조직을 조달 프로토콜을 승인했다. 세포 수확 및 게시 절차 5,6를 사용하여 배양을 수행합니다. 필요한 경우가 틱에 인간의 흡입 노출에 대한 정확한 모델이기 때문에이 과정은 세포 배양을 사용하지만, 하나는 분리하고 ALI 모 성장할 수및 / 또는 쥐의기도 상피 세포 7,8 사용합니다. </li><li> 간단히 말해서, 모든 결합 조직과 림프절을 제거한 후 조직의 작은 조각 (~ ¼ 인치)를 확인합니다. 락 테이트 링거액에 조직을 여러 번 씻는다. 50 ML 원뿔 튜브에 조직을 추가하고 (최소한의 필수적인 매체, MEM, 유체 비율 (1시 10분)에 조직을 1 % Protease/0.01 %의 DNA 분해 효소) 단백질 분해 효소 솔루션을 추가합니다. </li><li> 밤새 4 ° C에서 조직 록. 10 % 소 태아 혈청을 첨가하여 조직의 해리를 끝낸다. 수술 메스 상피 표면을 긁고 원심 분리 (10 분 2,000 RPM)에 의해 세포를 수집합니다. </li><li> 펄쳐 그 외 여러분 (9)에 의해 상세히 기술 된 바와 같이 제조 된 특수 ALI 매체 콜라겐 코팅 snapwells에 통과 한 곳에서 플레이트 세포. 혀끝의 미디어를 제거하여 공기 - 액체 인터페이스 (ALI)를 변경하기 전에 5 일 동안 문화. </li><li> 모든 다른 일 ALI 미디어를 사용하여 세포를 공급하고 세포가 추가 2~3주 (위해 차별화 할 수노출을 수행하기 전에 다수의 박동 섬모와 점액 분비를) 관찰합니다. 미디어의 변화와 함께 따뜻한 PBS로 혀끝의 표면을 세척 할 것. </li></ol> 3. 염소 노출 <ol><li> 우발적 염소 누수 발생 인원의 노출을 방지하기 위해 필요한 이차 봉쇄를 제공 100 FPM의 동작면 속도로 정규화 화학 후드 안쪽 염소 노광 시스템 (CES)를 함유한다. </li><li> 약간의 정압 (물 0.5 인치)에서 시스템을 운영하고 있습니다. 건조 공기는 15 L / 분 및 염소가 원하는 최종 염소 농도를 달성하기 위해 공급된다의 적절한 수준으로 공급된다. 챔버는 4 미니 BCU 잠금 및 압력 밀봉을 제공하기 위해 낮은 경도의 실리콘 가스켓 잠금 뚜껑이 있습니다. </li><li> 질량 유량 제어기를 통해 망할 CIA 2 혼합물을 제공합니다. CES는 건조 질소에 Cl2 1.0 %를 포함하는 압축 가스 실린더를 사용합니다. </li><li> 사용하여 희석 공기 흐름을 조절하는지정 조작부를 설계 마찬가지로 노광 챔버로 전달에 Cl2 농도를 조절한다. 저용량 샘플링 펌프는 분석기에 접속 된 데이터 기록 장치에 기록 된 농도를 모니터링하는 염소 분석기로 챔버로부터 배기를 당긴다. </li><li> 노광에 앞서 동일한 배송 및 배기 속도를 보장하는 유량계를 이용하여 챔버 내에서 유량을 측정한다. </li><li> 뜨는 매체를 제거하고 신선한 미디어 (ALI 문화의 기저 미디어)를 추가하여 노출을위한 세포 배양을 준비합니다. 에이전트와 모든 전처리이 시간에 수행 될 수있다. </li><li> 두 개의 밀봉 된 폴리 설폰 biocontainment 실에서 Cl2 가스 (50, 100, 또는 30 분 동안 300 PPM)에기도 상피 세포의 ALI 문화를 노출합니다. 심근 (침수 또는 점막에 문화를 컨 플루 언트)가 15 분 동안 50 또는 100 PPM에 Cl2에 노출되어 있습니다. </li><li> 노광 후에 C까지 공기 실 플러시L 2 레벨은 1 PPM 이하로 안전하게 (5 분 이내) 세포를 제거하기 위해 열 수 있습니다. </li></ol> 4. Transepithelial 전기 저항 (TER) 측정 <ol><li> 공기 - 액체 계면의 TER을 측정 ALI 염화은 &quot;젓가락&quot;한 쌍의 전극과 상피 voltohmmeter를 사용하여 배양. </li><li> 사용하기 전에 ALI 미디어 15 분에 젓가락 전극을 평형. </li><li> 표면 (2.0 ㎖) (1.0 ㎖)을 정점 및 기저에 따뜻한 미디어를 추가하고 voltohmmeter의 젓가락 전극을 사용하여 TER을 측정합니다. </li><li> 혀끝의 미디어 전극의 짧은 팔과 기저 미디어에서 더 이상 팔을 찍어. 전기 저항을 평가하는 voltohmmeter에서 &#39;측정&#39;버튼을 클릭합니다. </li><li> voltohmmeter는 옴 K 옴을 측정 할 수있는 옵션이 있습니다. 거래를 수득 각 전지 층을 가로 질러 측정 된 저항에서의 무 세포 배양 지원 걸쳐 저항 빼기상피 저항 (TER). </li></ol> 5. 카스파 측정 <ol><li> 기저 표면 포스트 노출에 신선한 ALI 미디어 (2.0 ㎖)에 추가하고 실온에서 세포를 배양한다. 4, 24 시간에 뜨는 미디어를 수집합니다. </li><li> 상업적 카스파 3 / 7 분석 키트를 사용하여 미디어 상청액에서 카스파 3 / 7 활성을 측정한다. </li></ol> 6. 서쪽 오점 및 면역 세포 화학 <ol><li> 앞에서 설명한 10로 세포 용 해물을 사용하여 서양 말을 수행합니다. 5X 감소 샘플 버퍼에 단백질 용 해물 (20 μg)를 중단하고 5 분 동안 끓인다. SDS-PAGE (4 % -15 %)에 단백질 용 해물을 주제 및 전기 블로 팅에 의해 니트로 셀룰로오스 막으로 분리 된 단백질을 전송합니다. </li><li> 세척 버퍼 5 % 우유 (PBS + 0.1 % 세제)를 가진 막을 배양하여 비 특정 바인딩을 차단하고 4 ° C에서 하룻밤, 1:1,000 희석에 SERCA2 또는 sarcomeric 액틴에 대한 기본 항체 세포막을 조사. 다음, 세척 및 배양 세포막을 각각의 퍼 옥시 데이즈 - 복합 이차 항체 및 상업 과산화 효소 검출 키트를 사용하여 10 전에 설명한 바와 같이 검출을위한 개발. </li><li> 면역 세포 화학을 위해 PBS로 세척 한 후 20 분 동안 10 mM의 시트르산 나트륨 완충액에서 0.4 % 트리톤-X-100으로 10 - 웰 플레이트에 삽입하거나, 유리 커버 슬립 상에 성장 생균은 치료. </li><li> 블록 비특이적 20 분 동안 5 % 당나귀 혈청 세포를 치료하고 sarcomeric 말라 또는 KI-67에 특정 비 특이 IgG 또는 개별 차 항체와 세포를 배양하여 바인딩. </li><li> PBS로 세포를 씻어 차 항체 및 핵 얼룩 (1 ㎍ / ㎖의의 DAPI)을 감지하는 형광 - 복합 이차 항체와 부화. </li><li> PBS로 세척 및 상업 설치 미디어를 사용하여 커버 슬립을 탑재합니다. </li><li> 형광 현미경을 사용하여 염색을 시각화. </li></ol>

<ol>
	<li>Mohan, 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=Acute+accidental+exposure+to+chlorine+gas:+clinical+presentation,+pulmonary+functions+and+outcomes.">Acute accidental exposure to chlorine gas: clinical presentation, pulmonary functions and outcomes.</a> Indian J Chest Dis Allied Sci. 52, 149-152 (2010).</li><li>Kose, 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=Myocardial+infarction,+acute+ischemic+stroke,+and+hyperglycemia+triggered+by+acute+chlorine+gas+inhalation.">Myocardial infarction, acute ischemic stroke, and hyperglycemia triggered by acute chlorine gas inhalation.</a> Am J Emerg Med. 27, 1021-1024 (2009).</li><li>Das, R., Blanc, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorine+gas+exposure+and+the+lung:+a+review.">Chlorine gas exposure and the lung: a review.</a> Toxicol Ind Health. 9, 439-455 (1993).</li><li>Claycomb, W. C., Palazzo, 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=Culture+of+the+terminally+differentiated+adult+cardiac+muscle+cell:+a+light+and+scanning+electron+microscope+study.">Culture of the terminally differentiated adult cardiac muscle cell: a light and scanning electron microscope study.</a> Dev Biol. 80, 466-482 (1980).</li><li>Ahmad, S., 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=Bcl-2+suppresses+sarcoplasmic/endoplasmic+reticulum+Ca2+-ATPase+expression+in+cystic+fibrosis+airways:+role+in+oxidant-mediated+cell+death.">Bcl-2 suppresses sarcoplasmic/endoplasmic reticulum Ca2+-ATPase expression in cystic fibrosis airways: role in oxidant-mediated cell death.</a> Am J Respir Crit Care Med. 179, 816-826 (2009).</li><li>Ahmad, S., 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=Tissue+factor+signals+airway+epithelial+basal+cell+survival+via+coagulation+and+protease-activated+receptor+isoforms+1+and+2.">Tissue factor signals airway epithelial basal cell survival via coagulation and protease-activated receptor isoforms 1 and 2.</a> Am J Respir Cell Mol Biol. 48, 94-104 (2013).</li><li>Lam, H. C., 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=Isolation+of+mouse+respiratory+epithelial+cells+and+exposure+to+experimental+cigarette+smoke+at+air+liquid+interface.">Isolation of mouse respiratory epithelial cells and exposure to experimental cigarette smoke at air liquid interface.</a> J Vis Exp. (48), (2011).</li><li>Hosokawa, 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=Differentiation+of+tracheal+basal+cells+to+ciliated+cells+and+tissue+reconstruction+on+the+synthesized+basement+membrane+substratum+in+vitro.">Differentiation of tracheal basal cells to ciliated cells and tissue reconstruction on the synthesized basement membrane substratum in vitro.</a> Connect Tissue Res. 48, 9-18 (2007).</li><li>Fulcher, M. L., 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=Well-differentiated+human+airway+epithelial+cell+cultures.">Well-differentiated human airway epithelial cell cultures.</a> Methods Mol Med. , 107-183 (2005).</li><li>Ahmad, S., 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=SERCA2+regulates+non-CF+and+CF+airway+epithelial+cell+response+to+ozone.">SERCA2 regulates non-CF and CF airway epithelial cell response to ozone.</a> PloS One. 6, e10 (2011).</li><li>Martin, J. G., 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=Chlorine-induced+injury+to+the+airways+in+mice.">Chlorine-induced injury to the airways in mice.</a> Am J Respir Crit Care Med. 168, 568-574 (2003).</li><li>Evans, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorine:+state+of+the+art.">Chlorine: state of the art.</a> Lung. 183, 151-167 (2005).</li><li>Vliet, 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=Formation+of+reactive+nitrogen+species+during+peroxidase-catalyzed+oxidation+of+nitrite.+A+potential+additional+mechanism+of+nitric+oxide-dependent+toxicity.">Formation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite. A potential additional mechanism of nitric oxide-dependent toxicity.</a> J Biol Chem. 272, 7617-7625 (1997).</li><li>Ahmad, S., 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=Lung+epithelial+cells+release+ATP+during+ozone+exposure:+signaling+for+cell+survival.">Lung epithelial cells release ATP during ozone exposure: signaling for cell survival.</a> Free Radic Biol Med. 39, 213-226 (2005).</li><li>Allen, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+system+for+exposure+of+cultured+cells+and+other+materials+to+ozone.">An automated system for exposure of cultured cells and other materials to ozone.</a> Inhal Toxicol. 15, 1039-1052 (2003).</li></ol>

The authors declare that they have no competing financial interests.

하나는 폐에 유독 한 화학 물질을 호흡 할 때 급성 독성 노출의 가장 일반적인 유형은 발생합니다. 이러한 화학 물질은 신속하게 혈류에 흡수 될 수 있고, 뇌와 심장 등 다른 장기에 영향을 미칠 수 있습니다. 동물 모델을 사용하여 여러 에이전트의 흡입 독성을 연구하고 널리보고, 그러나 메커니즘은 덜 잘 이해된다. 이는 효과적인 치료 개발에 주요 장애물이다. 체외 노출 시스템의 부재는...

독성 화학 물질 흡입 (TICS)에 노출 / 염소 (망할 CIA 2) 등의 가스 사고 피폭뿐만 아니라 화학 물질의 위협으로 자신의 잠재적 인 사용의 지속적인 건강 관심사 남아있다. 폐는 기본 목표이지만, 심장과 뇌 등의 장기는 생체 내 모델이 일반적으로 틱에서 테스트 독성에 사용됩니다. 1-3에 영향을하지만, 독성 평가를위한 시험 관내 분석에서 비용 효과, 간단한 빠르고 더 있습니다됩니다.에서 체외 모델은 생체 내에서 평가하기 어려울 수 있습니다 에이전트 세포 상호 작용의 광범위한 조사를 할 수 있습니다. 이러한 체외 노출 시스템은 드물고 또한, 독성 에이전트가 세포가 침수되는 배양 배지에 첨가되는 일부 기존 모델에, 에이전트의 속성으로 인해 상호 작용과 매체의 구성 요소에 바인딩을 변경할 수 있습니다. 같은 이러한 시나리오 세포 배양 시스템에서 공기 - 액체 계면 (ALI직접 가스 에이전트에 노출 될 수 있습니다 여기에 제안 된 기본 인간기도 상피 세포의) 문화가 유망 될 수 있습니다. 기도를 안 감 상피 세포 흡입 독성 화학 물질에 대한 방어의 첫 번째 라인이다. 인간기도 상피는 폐의 루멘 및 기본 세포 사이의 물리적 장벽을 형성하고 폐의 응답에 참여하고 있습니다. 그것은 사이토 카인 및 기타 직업 및 항염증제의 수뿐만 아니라 상피 세포를 덮고있는 점액 /기도 표면 액체 (ASL)을 분비를 생성합니다. 체외 배양 시스템에 빠져들 기존의 한계 중 하나는 상피 표면을 커버 ASL과 점액을 제거하거나 희석하는 것도있다. 이것은 공기에 노출되어 폐 상피 세포의 생리 학적 상태를 반영하지 않는다. 따라서, TIC의 독성 시험에 대한 시험 관내 시스템에 이상적으로는이 아키텍처를 복제해야합니다. 빠른 심사 M을 개발에 큰 관심이 있습니다생체 내 독성 예측 ethods. ALI에서 성장 상피 세포는 차별화가 아니라 차별화 된 구조와 기능을 침수 성장 세포에 비해기도의 우수한 모델을 제공합니다. 이 연구에서, 우리는 인간의기도의 공기 - 액체 인터페이스 문화 유독 흡입 가스의 독성을 테스트 (기관지) 상피 세포의 사용을 설명하고, 따라서 독성의 또 다른 중요한 목표를 공부하고, 심근 세포의 침수 세포 배양과 비교.

<table border="0" cellpadding="0" cellspacing="0" width="1128">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Name</td>
			<td style="width:329px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Rats</td>
			<td style="width:329px;">Harlan Laboratories</td>
			<td>Sprague-Dawley&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Pentobarbital</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P3761</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Chlorine</td>
			<td style="width:329px;">AirGas, Inc</td>
			<td style="width:147px;">X02NI99CP163LS1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Caspase 3/7 kit&#160;</td>
			<td style="width:329px;">Promega</td>
			<td style="width:147px;">G8091</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Epithelial voltohmmeter and chopstick electrode</td>
			<td style="width:329px;">World Precision Instruments</td>
			<td style="width:147px;">EVOM and STX2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Snapwell inserts</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">07-200-708</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">70 micron nylon cell strainer</td>
			<td style="width:329px;">Corning</td>
			<td style="width:147px;">#352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysulfone biocontainment chambers&#160;</td>
			<td style="width:329px;">BCU, Allentown Cage Equipment</td>
			<td style="width:147px;">BCU</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td style="width:329px;">Life technologies</td>
			<td style="width:147px;">12491-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sarcomeric actin antibody</td>
			<td style="width:329px;">Abcam Cambridge, MA</td>
			<td style="width:147px;">ab28052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SERCA2 antibody</td>
			<td style="width:329px;">&#160;Affinity Bioreagents, Golden, CO</td>
			<td style="width:147px;">MA3-9191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ki-67 antibody</td>
			<td style="width:329px;">&#160;Dako, Carpinteria, CA</td>
			<td style="width:147px;">M7248</td>
			<td></td>
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			<td height="21" style="height:21px;">Alexa-488-conjugated secondary antibody</td>
			<td style="width:329px;">&#160;Invitrogen, Grand Island, NY</td>
			<td style="width:147px;">A11029</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:487px;">BSA</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Carnitine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C0283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Taurine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">T8691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Creatinine</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Krebs Ringer Buffer</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">K4002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Protease</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagenase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C6885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; DNAase</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">DN-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Lactated Ringer solution</td>
			<td style="width:329px;">Abott Laboratories</td>
			<td style="width:147px;">7953</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Donkey serum</td>
			<td style="width:329px;">Fisher Scientific</td>
			<td style="width:147px;">017-000-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PBS, phosphate buffered saline</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">4-15% SDS-PAGE gels</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">456-1083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Nitrocellulose membrane</td>
			<td style="width:329px;">Bio-Rad</td>
			<td style="width:147px;">162-0115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Dergent, Tween&#160;</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Peroxidase detection kit</td>
			<td style="width:329px;">Pierce</td>
			<td align="right" style="width:147px;">3402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">DAPI</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Mounting media, Fluormount G</td>
			<td style="width:329px;">eBiosciences</td>
			<td style="width:147px;">00-4958-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium citrate</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">71497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagen</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">C7521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">MEM</td>
			<td style="width:329px;">Sigma-Aldrich</td>
			<td style="width:147px;">M8028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Laminin</td>
			<td style="width:329px;">BD biosciences</td>
			<td style="width:147px;">354259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Penicillin/Streptomycin</td>
			<td style="width:329px;">Life Technologies</td>
			<td style="width:147px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">FBS</td>
			<td style="width:329px;">Gibco</td>
			<td style="width:147px;">200-6140AJ</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro cell culture model for toxic inhaled chemical testing

세포 배양은 이전의 동물 모델에서의 사용에 대한 치료제의 효능을 개발하고 연구하는 것이 필수적이다. 우리가 잘 분화 된 인간기도 상피 세포와 심장 근육 세포를 모델링 할 수있는 독특한 기능이 있습니다. 이것은 일반적으로 세포 표면과 상호 작용하고, 물과 반응하고, 침수 문화에 미치는 영향을 제한에 따라 다양한 부산물을 형성 할 수있는 염소 등의 유해 화학 물질 흡입,의 해로운 효과를 연구하는 귀중한 도구가 될 수 있습니다. 공기 liqiuid 인터페이스에서 잘 분화 된 인간기도 상피 세포 배양을 이용하여 우리의 모델은 이러한 제한을 우회뿐만 아니라 잠재적 인 독성 흡입 화학 물질의 독성의 중요한 메커니즘을 평가하는 기회를 제공한다. 우리는 막 무결성의 강화 손실, 염소 노출 같은 독성 화학 물질 흡입시 카스파 릴리스과 죽음에 대해 설명합니다. 이 문서에서는, 우리는 포유 동물의 마음과기도 상피 C 염소 노출을 모델링하는 방법을 제안한다문화와 이러한 종류의 세포에 미치는 영향을 평가하는 간단한 테스트에서 ELL 학생.

기본 막대 모양의 심근은 라미닌 행렬에 연결하고 (그림 1A 및 삽입)를 확산하고 융합 문화로 분화. 이들 세포는 더 sarcomeric 액틴 및 SERCA2 식 (도 1B 및 1C)에 기초하여 특징으로 하였다. 쥐의 심근은 라미닌 코팅 된 멤브레인 (그림 1D)에 성장 세포에 잠긴 문화와 합류 층의 중단에 광범위한 세포 반올림과 죽음을 야기 100 ppm의 염소에 15 분 노출로 염?...

Watch this Scientific Journal Video about In vitro Cell Culture Model for Toxic Inhaled Chemical Testing at JoVE.com

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

이 프로토콜은 흡입 독성 화학 물질을 세포 배양의 노출 방법을 설명하기 위해 설계되었습니다. 차별화 된 공기 - 액체 인터페이스기도 상피 세포 (ALI) 문화의 노출은 염소와 같은 독성 가스에기도 노출의 독특한 모델을 제공합니다. 이 논문에서 우리는 염소 상피 세포의 공기 - 액체 인터페이스의 문화에 대한 노출과 심근의 침수 문화의 영향을 설명합니다. 체외 노출 시스템이 중요한 기전 연구는 다음 새로운 치료제를 개발하는 데에 이용 될 수있는 경로를 평가할 수 있습니다.

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 ...

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In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

10.6K 조회수.  University of Colorado.  이 프로토콜은 흡입 독성 화학 물질을 세포 배양의 노출 방법을 설명하기 위해 설계되었습니다. 차별화 된 공기 - 액체 인터페이스기도 상피 세포 (ALI) 문화의 노출은 염소와 같은 독성 가스에기도 노출의 독특한 모델을 제공합니다. 이 논문에서 우리는 염소 상피 세포의 공기 - 액체 인터페이스의 문화에 대한 노출과 심근의 침수 문화의 영향을 설명합니다. 

비디오: In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

종양 Microenvironment를 재현을위한 마이크로 유체 장치<em&gt; 체외에서</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

연구

생체공학

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

활성 세포 배양을위한 형상 기억 폴리머

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

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

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

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

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

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

인간 세포 배양을위한 회전 세포 배양 시스템 : 모델로 인간 Trophoblast 셀

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

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 ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

세포 배양 응용 프로그램에 대한 히드로 겔의 빛 매개 형성 및 패터닝

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

<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 ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

쥐 망막 신경 전달 물질 글루타민 산 염에 체 외에서 의 생체 모방 화학 Neuromodulation 위한 방법론

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

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 ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

거대 분자 군중을 사용하여 인간의 절단 비대 흉터의 시험관 내 모델

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...