이 연구는 로렌스 리버모어 국립 연구소 LDRD 18-ERD-062 (C.R.)의 자금 지원을 받았습니다. DgKO 및 DgKI 세포를 선물해 주신 John Muschler에게 감사드립니다. 전자 현미경 샘플 준비 및 데이터 수집에 대한 조언과 도움을 주신 University of California Berkeley Electron Microscope Laboratory의 직원에게 감사드립니다.

1. 해동 및 분취 라미닌-111
참고: 정제된 라미닌-111은 상업적으로 이용 가능하지만(일반적으로 Matrigel로 판매되는 EHS 매트릭스에서 정제됨) 모든 공급원이 온전하고 분해되지 않은 단백질을 제공하는 것은 아닙니다.
<ol><li>Ln1 및 실행 크기 배제 크로마토그래피(26 ) 또는 폴리아크릴아미드 겔 전기영동(26)을 소싱하여 Ln1이 분해되지 않았는지 확인합니다. 변성된 Ln1은 337kDa, 197kDa 및 177kDa의 대역을 가져야 하며 네이티브 Ln1은 711kDa의 단일 대역을 가져야 합니다.</li>
	<li>Ln1을 냉장고의 얼음 위에서 밤새 해동하고, 저단백질 결합 팁과 멸균 기술을 사용하여 저단백질 결합 마이크로 원심분리기 튜브(일반적으로 배양 오버레이의 경우 100μg/튜브 또는 코팅의 경우 10μg의 부분 표본)에 분취한다27. 부분 표본을 얼려서 -80 °C에서 보관하십시오.</li>
</ol>2. 낮은 pH 완충액을 이용한 라미닌 중합

<ol><li>polyLM 중합 완충액 만들기: 무게를 측정하여 1mM CaCl2 및 20mM 아세트산 나트륨을 초순수에 혼합합니다. 그런 다음 HCl 또는 아세트산을 사용하여 pH를 4.0으로 조정합니다. 0.2μm 필터를 통해 제균 필터를 사용하고 4°C에서 보관합니다.</li>
	<li>대조군 중합 완충액 만들기: 1mM CalCl2 및 20mM Tris를 혼합하고 필요에 따라 10N HCl 또는 12N NaOH를 사용하여 pH를 7.0으로 조정합니다. 0.2μm 필터를 통해 제균 필터를 사용하고 4°C에서 보관합니다. 참고: 여기에서 프로토콜을 일시 중지할 수 있습니다.</li>
	<li>필요한 수의 Ln-1 부분 표본을 냉장고에서 밤새 해동합니다.</li>
	<li>멸균 세포 배양 기법을 사용하여 적절한 완충액(2.1단계의 polyLM 중합 완충액 또는 2.2단계의 대조 중합 완충액)을 최종 농도 100μg/mL(즉, 0.5mL에서 50μg 분취액 추가)로 라미닌 분취액에 첨가하고 피펫팅으로 부드럽게 혼합합니다.</li>
	<li>시각화를 위해 형광 태그가 부착된 라미닌을 사용하는 경우 1:50 wt/wt 비율(예: 2 μg/100 μg)로 재구성 및 추가하고 피펫팅으로 부드럽게 혼합합니다. 알림: 세포 부착을 위한 기질로 라미닌을 사용하는 경우 2.6-2.8단계를 따르십시오.</li>
	<li>적절한 완충액으로 희석한 직후 라미닌을 배양 플라스틱 또는 유리 용기에 옮기고 37°C의 세포 배양 인큐베이터에서 밤새 배양합니다. 커버슬립을 코팅할 때는 평형 방울이 생성될 수 있을 만큼 충분히 큰 부피를 추가하고(일반적으로 13mm 원형 커버슬립의 경우 50μL 사용) 37°C의 가습 챔버에서 밤새 보관합니다.</li>
	<li>멸균 기술27을 사용하여 1x 인산염 완충 식염수(PBS)로 세척합니다.</li>
	<li>액체를 조심스럽게 제거하십시오. 표면이 완전히 건조되지 않도록 하십시오. 알림: 우유 단백질 유도를 위해 세포를 덮기 위해 라미닌을 사용하는 경우 2.9-2.12단계를 따르십시오.</li>
	<li>유방 상피 세포 배양. 2% FBS, 0.01mg/mL 인슐린, 0.005μg/mL EGF, 0.05mg/mL 겐타마이신 및 0.05g/mL 노르모신이 보충된 DMEM-F12의 MepG 디스트로글리칸 녹인(DgKI) 또는 MepG 디스트로글리칸 녹아웃 세포(DgKO) 세포를 배양합니다.</li>
	<li>웰 플레이트 또는 커버슬립 바닥 접시에서 10,500 cells/cm2 의 라미닌 자극을 위해 DgKO 및 DgKI 세포를 종자합니다.</li>
	<li>희석된 라미닌을 37°C의 세포 배양 인큐베이터에서 30분 동안 배양합니다.</li>
	<li>원심분리기 polyLM은 2,000 x g 에서 20분 동안 분주하지만, 단백질 개체의 크기가 작고 효과적으로 수집하기 위해 더 빠른 속도가 필요하기 때문에 LM을 20,000 x g 의 중성 pH에서 20분 동안 원심분리합니다.</li>
	<li>멸균 기법27을 사용하여 상층액을 부드럽게 제거하고 3μg/mL 프로락틴, 2.8μM 하이드로코르티손 및 5μg/mL 인슐린이 보충된 DMEM-F12의 적절한 부피의 유도 배지에 재현탁시킵니다</li>
	<li>세포 배양 배지에 라미닌을 즉시 전달하여 세포를 자극합니다. 50-800 μg/mL Ln1로 세포를 성공적으로 자극했습니다.</li>
</ol>3. 분리된 당단백질을 이용한 라미닌 중합
<ol><li>재조합 니도겐-1과 Ln1을 냉장고의 얼음 위에서 밤새 부드럽게 해동합니다.</li>
	<li>세포 배양 배지에서 저단백질 결합 팁과 튜브를 사용하고 멸균 기술을 사용하여 Ln1과 니도겐-1을 1:1 wt/wt 비율로 혼합합니다. 세포 배양 배지에서 순수 Ln1을 대조군으로 사용하십시오.</li>
	<li>시각화를 위해 형광 태그가 부착된 라미닌을 사용하는 경우 1:50 wt/wt 비율(예: 2 μg/100 μg)로 재구성 및 추가합니다. 피펫팅으로 부드럽게 혼합합니다.</li>
	<li>Ln1-nidogen을 배양하거나 37°C의 세포 배양 인큐베이터에서 30분 동안 대조군 Ln1을 배양합니다.</li>
	<li>2,000 x g 에서 30분 동안 Ln1-니도겐 공중합체를 원심분리하고 대조군 Ln1을 20,000 x g 에서 30분 동안 원심분리합니다.</li>
	<li>상층액을 부드럽게 제거하고 세포 배양 배지에서 최종 농도 100-800μg의 총 단백질/mL로 재현탁시킵니다.</li>
	<li>즉시 사용하여 세포를 자극하거나 커버슬립으로 옮겨 가습된 세포 배양 인큐베이터에서 37°C에서 밤새 부착할 수 있습니다.</li>
	<li>커버슬립에서 배양 배지를 제거하고 1x PBS로 커버슬립을 세척한 다음 실온에서 10% 포르말린(즉, PBS의 4% 포름알데히드)으로 15분 동안 고정합니다.</li>
</ol>4. 디스트로글리칸 발현 세포를 이용한 라미닌 중합
<ol><li>유방 상피 세포 배양. 2% FBS, 0.01mg/mL 인슐린, 0.005μg/mL EGF, 0.05mg/mL 겐타마이신 및 0.05g/mL 노르모신이 보충된 DMEM-F12의 MepG 디스트로글리칸 녹인(DgKI) 또는 MepG 디스트로글리칸 녹아웃 세포(DgKO) 세포를 배양합니다.</li>
	<li>성장률의 차이로 인해 DgKI 세포를 5,000 cells/cm2로 종자하고 DgKO 세포를 8,000 cells/cm2 로 종자합니다. 가습 인큐베이터에서 37°C에서 2-3일마다 배지를 교체하고 엄격한 멸균 기술을 사용하여 4-5일마다 통과합니다. 적절한 성장 속도를 보장하기 위해 혈구계 또는 자동 세포 카운터(27 )로 세포를 주의 깊게 계수합니다.</li>
	<li>10,500 cells/cm2 에서 라미닌 자극을 위한 시드 세포를 웰 플레이트 또는 커버슬립 바닥 접시에 넣고 DgKI 세포를 사용하여 음성 대조군으로 프랙탈 Ln1 및 DgKO 세포를 생성하여 2일 동안 배양합니다. Ln-1을 냉장고의 얼음 위에서 밤새 부드럽게 해동한 다음 3μg/mL 프로락틴, 2.8μM 하이드로코르티손, 5μg/mL 인슐린이 보충된 DMEM-F12 유도 배지와 혼합합니다. 35mm 접시의 경우 50-800μg의 Ln1과 함께 1mL의 유도 배지를 사용합니다.</li>
	<li>형광 Ln1을 사용하는 경우 50:1 wt/wt 비율로 혼합합니다.</li>
	<li>세포에서 배지를 제거하고 3μg/mL 프로락틴, 2.8μM 하이드로코르티손 및 5μg/mL 인슐린이 보충된 DMEM-F12의 라미닌 및 유도 배지로 덮습니다.</li>
	<li>2일 동안 세포를 배양한 후 10% 포르말린으로 고정합니다.</li>
</ol>5. 광학 현미경을 통한 라미닌 미세구조의 특성화
<ol><li>고정 샘플을 수집하고 실온에서 1x PBS로 5분 동안 3회 세척합니다.</li>
	<li>0.5% Triton X-100, 3% 소 혈청 알부민, 10% 염소 혈청 및 40μg/mL 항마우스 차단 항체 단편을 사용하여 가습 챔버에서 실온에서 1시간 동안 1x PBS의 샘플을 차단 및 투과시킵니다. 알림: Triton X-100은 위험합니다. 장갑과 보안경을 착용하고 적절하게 폐기하십시오.</li>
	<li>1:100으로 희석하여 항-라미닌 항체를 0.5% Triton X-100 및 3% BSA가 포함된 1x PBS와 혼합하고 가습 챔버에서 실온에서 2시간 동안 또는 4°C에서 밤새 샘플에 첨가합니다(일반적으로 바닥에 젖은 실험실 물티슈가 있는 팁 상자 사용).</li>
	<li>1차 항체를 제거하고 0.5% Triton X-100 및 3% BSA가 함유된 1x PBS로 3회 세척합니다.</li>
	<li>0.5% Triton X-100 및 3% BSA가 포함된 1x PBS의 샘플에 1:500으로 희석하여 적절한 2차 항체를 추가하고 가습 챔버의 어두운 실온에서 배양합니다.</li>
	<li>2차 항체를 제거하고 0.5% Triton X-100 및 3% BSA가 함유된 1x PBS로 3회 세척합니다.</li>
	<li>세포가 포함된 샘플의 경우 6μM 팔로이딘을 45분 동안 첨가한 다음 0.5% Triton X-100 및 3% BSA가 포함된 PBS로 3회 세척한 다음 PBS로 3회 세척합니다. 그런 다음 PBS에서 0.1μg/mL DAPI로 5분 동안 염색합니다.</li>
	<li>필요한 경우 샘플을 장착합니다.</li>
	<li>고해상도 컨포칼 현미경을 사용한 이미지 ln-1 구조. 침지 대물렌즈(Water Immersion Plan Apochromat 40X/1.1NA 또는 Oil Immersion Plan Apochromat 63X/1.4NA)가 장착된 레이저 스캐닝 현미경을 사용하십시오.</li>
</ol>6. 박스 카운팅 치수를 통한 라미닌 구조물의 특성화
<ol><li>Matlab 툴박스 또는 ImageJ에서 사용할 수 있는 상자 계수 차원 알고리즘을 사용하여 프랙탈 속성을 분석합니다. 이 방법은 라미닌의 이미지를 임계값으로 표시하고 로그-로그에 플로팅하여 Ln1을 포함하는 상자의 개수를 상자 크기와 비교하여 플로팅합니다. 이러한 파라미터 사이의 관계의 기울기는 박스 카운팅 차원입니다: 비프랙탈 기하 도형은 0, 1 또는 2의 차원을 갖는 반면, 프랙탈 기하 도형은 정수가 아닌 차원28을 갖습니다. 참고: 산 처리, 당단백질 처리 및 DgKI 세포 부착 Ln1은 모두 ~1.7의 상자 계수 프랙탈 차원을 갖는 반면 대조군 Ln1은 2.0의 프랙탈 차원을 갖습니다.</li>
</ol>7. 전자 현미경을 통한 라미닌 구성물의 특성 분석
<ol><li>세포 배양 배지에 라미닌을 재현탁시키고 습한 37°C 환경에서 90분 동안 실리카 웨이퍼에 흡착시킵니다.</li>
	<li>pH 7.2에서 0.1M 나트륨 카코딜레이트 완충액의 2.5% 글루타르알데히드에 매트릭스를 고정합니다. 알림: 글루타르알데히드는 위험하므로 장갑과 보안경이 있는 흄 후드에서 처리하고 유해 폐기물로 처리해야 합니다.</li>
	<li>pH 7.2에서 0.1M 나트륨 카코딜레이트 완충액의 1% 오스뮴 테트라옥사이드의 후미자 매트릭스. 알림: 사산화오스뮴은 위험하므로 장갑과 보안경이 있는 흄 후드에서 취급하고 적절하게 폐기해야 합니다. 카코딜레이트는 위험하므로 장갑과 보안경이 있는 흄 후드에서 취급하고 적절하게 폐기해야 합니다.</li>
	<li>pH 7.2의 카코딜산나트륨 완충액으로 3회 세척합니다.</li>
	<li>에탄올 농도를 증가시켜 탈수하십시오.</li>
	<li>얇은 금색 층이 있는 스퍼터 코트 매트릭스.</li>
	<li>주사 전자 현미경을 사용한 이미지.</li>
</ol>

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이 연구는 미국 에너지부의 후원 하에 로렌스 리버모어 국립 연구소(Lawrence Livermore National Laboratory)가 DE-AC52-07NA27344 계약에 따라 수행했습니다. IM 릴리스 LLNL-JRNL-799443.

라미닌은 상피, 근육 및 신경 장기 시스템에서 ECM의 핵심 요소를 나타내며 세포의 기능적 분화를 조절하는 데 필수적인 역할을 하는 것으로 체외에서 밝혀졌습니다. 세포에 표시되는 라미닌의 구조가 신호 전달 및 결과 세포 표현형15,16을 조절한다는 증거가 증가하고 있으므로 Ln1 미세 구조를 제어하는 방법은 이 분야에서 일하는 기초 생물학자와 체외...

성장 인자나 사이토카인과 달리 세포외 기질(ECM) 단백질은 구조적 네트워크로 조립될 수 있습니다. ECM 기능 장애는 많은 질병 상태의 핵심 요소이기 때문에 세포가 ECM 구성, 미세 구조 및 생체 역학의 신호를 감지하고 전달하는 메커니즘은 치료제 개발을 위한 매력적인 표적입니다. 이러한 네트워크의 미세 구조가 이러한 단백질이 세포에 신호를 보내는 방식에 중요한 역할을 한다는 증거가 증가하고 있습니다. 현재까지 ECM 미세 구조와 세포 기능 사이의 이러한 연관성은 콜라겐 I1, 피브로넥틴2 및 피브린3에 대해 나타났습니다.
Laminin-111 (Ln1)은 상피 세포4, 근육 세포5 및 신경 세포6와 접촉하는 세포외 기질의 핵심 구조 구성 요소인 삼중 십자형 단백질입니다. 유선에서 Ln1은 유선 상피 세포가 우유 생산 세포로 기능적으로 분화하는 데 필요하며 7,8,9 Ln1은 종양 복귀 유도에 중요합니다10,11. Ln1 및 기타 라미닌은 근육12,13에서 대뇌 피질 액틴 네트워크 및 육종 조직의 발달과 신경 세포 이동 및 신경돌기 성장에 필요하다13 . 따라서 라미닌이 세포에 신호를 보내는 메커니즘을 이해하는 것은 활발한 연구 분야입니다.
Ln1에는 인테그린, 신데칸, 디스트로글리칸, LBP-110 및 LamR을 포함한 광범위한 수용체에 대한 여러 접착 도메인이 포함되어 있습니다(그림 1A)4,14. 라미닌 α1의 c-말단 구형 도메인을 포함하는 E8 단편은 디스트로글리칸과 인테그린 α6β1 및 α3β115의 결합에 필요하며, 상피 세포의 기능적 분화에 필요하다15,16. 대조적으로, 짧은 팔은 상이한 생물학적 활성을 나타내는데(17), 이는 네트워크 형성 후 이들 상이한 Ln1 도메인을 인식할 수 있는 가용성이 세포 거동을 변화시킬 수 있음을 의미한다.
우리는 최근에 고분자 네트워크로 배열된 Ln1이 프랙탈 특성(즉, 여러 길이 스케일에 걸쳐 자체 유사한 구조)을 나타낸다는 것을 입증했습니다18. 프랙탈 네트워크는 이 배열이 결여된 Ln1과 비교하여 상피 세포에 다르게 신호를 보낸다19. 이 프랙탈 네트워크는 특정 조립체 구조를 나타내는데, 여기서 긴 팔은 세포(18)에 우선적으로 노출된다. 이러한 상이한 긴 팔 디스플레이의 결과로, 상피 세포는 잘 조직된 단단한 접합부와 액틴 스트레스 섬유의 억제를 가진 우유 생산 세포로 기능적 분화를 겪는다19.
Ln1 긴 팔의 높은 디스플레이를 보여주는 짧은 팔-짧은 팔 결합 상호 작용에서 Ln1 네트워크를 생성하는 3가지 방법을 설명합니다: 1) 산성 완충액을 사용한 cell-free 조립, 2) 당단백질과의 공동 배양 또는 3) 세포 표면 디스트로글리칸과의 상호 작용. 이 세 가지 방법은 세 가지 매우 다른 메커니즘을 통해 유사한 라미닌 미세 구조를 생성하여 실험 설계에 유연성을 허용합니다. 이전 연구는 이 세 가지 방법이 생물학적 강건성을 나타낼 수 있음을 시사한다: 유선상피에서 세포 표면 디스트로글리칸, 당단백질 또는 인위적으로 구조화된 라미닌은 기능적 분화를 유도할 수 있다19. 따라서, 연구자들은 연구 주제에 따라 이러한 방법들 중에서 선택할 수 있다: 세포막의 디스트로글리칸이 프랙탈 네트워크로 올바른 중합을 유도하기 때문에, 디스트로글리칸 발현 세포는 Ln-1 미세구조에 대한 민감도를 나타내지 않는다 19,20. 라미닌과 당단백질(21)의 혼합물인 마트리겔(Matrigel)은 Ln-1의 가장 경제적인 공급원이며, 디스트로글리칸 녹아웃(dystroglycan knockout) 유선세포가20을 분화하도록 유도하는 데 필요한 미세구조를 제공하지만, 로트 간 변동성이 있는 복잡한 단백질 혼합물을 포함하고 있다. PolyLM은 이 기능을 제공하는 가장 깨끗하고 환원주의적인 시스템이지만 Matrigel19에 비해 유방 세포 분화를 유도하는 데 비용이 증가하고 효능이 낮습니다.
이들 3가지 방법은 확산 제한 응집(diffusion limited aggregation)18,19의 프랙탈 네트워크 구조 특성을 가지며, 다중 실험 시스템(22,23,24,25)에서 응집된 라미닌(laminin)과 비교하여 변화된 생물학적 활성을 나타낸다. 이러한 방법을 사용하는 향후 연구는 상피 분화에 필요한 수용체와 신호를 확인하고 종양과 같은 비정상적인 미세 환경20에서 세포에서 정상적인 세포-기질 상호 작용을 복원하도록 결정할 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:665px;" width="665">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% formalin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">HT501128</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Laminin primary antibody</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">L9393</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-Rabbit Alexafluor 488 secondary antibody</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">A32731</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">C4901 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Culture Incubator</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">Heracell 150 or similar</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge for microcentrifuge tubes</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:161px;">5418 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal Microscope</td>
			<td style="width:121px;">Zeiss</td>
			<td style="width:161px;">LSM 710 or equivalent</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coverslips</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">25CIR-1 or similar</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DMEM-F12, Liquid, High Glucose, +HEPES, L-glutamine, +Phenol red</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">11330107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EGF (Epidermal Growth Factor)</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">11376454001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescently Labeled Laminin</td>
			<td style="width:121px;">Cytoskeleton Inc</td>
			<td style="width:161px;">LMN02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gentamicin</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">VWRV0304-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutaraldehyde</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">G5882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCl</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">320331 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrocortisone</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">H0888-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">16634-50mg</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MepG Dystroglycan Knockout Cells</td>
			<td style="width:121px;">LBNL</td>
			<td style="width:161px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">S8045 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normocin</td>
			<td style="width:121px;">Invivogen</td>
			<td style="width:161px;">ant-nr-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmium Tetroxide</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">75633</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Ovine Prolactin</td>
			<td style="width:121px;">Los Angeles Biomedical Research Institute</td>
			<td style="width:161px;">Ovine Pituitary Prolactin</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phalloidin</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">A22287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">10010023 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Purified Laminin-111</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">L2020-1MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Recombinant human Nidogen-1, carrier free</td>
			<td style="width:121px;">R&#38;D systems</td>
			<td style="width:161px;">2570-ND-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Acetate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">S2889 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Cacodylate</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">C0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile filters</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">SLGP033RS or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:161px;">252859 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td style="width:161px;">X100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra-low protein binding tubes</td>
			<td style="width:121px;">VWR</td>
			<td style="width:161px;">76322-516, 76322-522 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrapure water</td>
			<td style="width:121px;">Thermo</td>
			<td style="width:161px;">15230253 or similar</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating a fractal microstructure of laminin-111 to signal to cells

Laminin-111(Ln1)은 상피, 근육 및 신경계의 세포외 기질의 필수적인 부분입니다. 우리는 이전에 Ln1의 미세 구조가 세포에 신호를 보내는 방식을 변경한다는 것을 입증했는데, 이는 네트워크로의 Ln1 조립이 다른 접착 영역을 노출시키기 때문일 수 있습니다. 이 프로토콜에서는 중합된 Ln1을 생성하는 세 가지 방법을 설명합니다.

Laminin-111은 짧은 팔 끝에 3개의 자가 조립 도메인이 있는 삼량체 단백질입니다(그림 1A). 적절하게 처리하면 짧은 암이 육각형 격자(그림 1B,1C)로 중합될 수 있으며, 이는 더 큰 공간 규모에서 확산 제한 응집체 프랙탈 특성을 나타냅니다(그림 2). 칼슘 함유 중성 pH 완충액에서 배양된 라미닌은 항-Ln1 항체(<strong class="xfig"...

Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

중합되지 않은 Ln1과 비교하여 세포에 다르게 신호를 보내는 프랙탈 특성을 가진 Ln1 중합체를 생성하는 세 가지 방법을 설명합니다.

세포에 신호를 보내는 Laminin-111의 프랙탈 미세구조 생성

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the ...

generating-a-fractal-microstructure-of-laminin-111-to-signal-to-cells

Research

JoVE Journal

Bioengineering

918 조회수.  Texas Heart Institute. 중합되지 않은 Ln1과 비교하여 세포에 다르게 신호를 보내는 프랙탈 특성을 가진 Ln1 중합체를 생성하는 세 가지 방법을 설명합니다.

비디오: Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

연구

생체공학

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

기판에 연결된 전지의 Micropipette의 열망은 셀 강성을 추정하기 위해

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

전체 피부 결함 모델은 생체의 혈관 신생을 평가하기<em&gt; 생체</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

원격으로 업 컨버전 나노 중재 니 Photoactivation에 의해 세포내 신호 변환 하는 통합된 시스템

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

미생물 행동을 연구하기 위한 제어된 동적 화학 적 경관 생성

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

소프트 리소그래피의 변경을 사용하여 루트 표면 미세 구조의 생물 적 복제

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

인간 유도 만능 줄기 세포 유래 심근 세포에서 단세포 광학 작용 잠재적 측정

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

정지 인간 조혈 줄기 세포를 유지하는 배양 방법

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.