This work was supported by the Belgian National Foundation for Scientific Research (F.R.S.-FNRS) through &#8220;MIS Confocal Microscopy&#8221;, &#8220;Cr&#233;dit aux Chercheurs&#8221; grants and the &#8220;Nanomotility FRFC project&#8221; (no. 2.4622.11). T.G. doctoral fellowship is supported by the Foundation for Training in Industrial and Agricultural Research (FRIA). The authors gratefully acknowledge Sylvain Desprez for mechanical characterization and G&#233;raldine Circelli for confocal imaging.

1 유리 Coverslips는의 표면을 활성화 <ol><li> 페트리 접시 스미어 (권장 화학 흄 후드) 5 분 정도에 0.1 M NaOH 용액에 넣어 유리 원형 커버 슬립 (25mm 직경). </li><li> NaOH 용액을 제거하고 부드럽게 멸균 문화 후드에 흔들 접시에 락을하는 동안 완벽하게 20 분 동안 멸균 DDH 2 O로 된 커버를 체험. </li><li> 멸균 DDH 2 O를 배출하고 단계 1.2를 반복합니다. </li><li> 멸균 핀셋으로 된 커버를 제거하고 활성화 된 얼굴 위로 새로운 배양 접시에 배치합니다. </li><li> 고순도 질소 가스의 지속적인 흐름하에 건조 된 커버. </li><li> 멸균 배양 후드에서 1 시간 동안 커버 슬립의 활성화 측면에서 3 - (트리 메 톡시 실릴) 프로필 아크릴 레이트 (92 %)의 얇은 층을 얼룩. </li><li> 광범위 살균 DDH 2 O 3 세척과 유리 된 커버를 세척하고 새로운 배양 접시에 무균 DDH 2 O에서 그들을 몰입. </li><li> 페트리 접시를 누릅니다및 파라 필름으로 10 분 동안 교반과에서 로커 접시에 놓습니다. </li><li> 좋은 팁을 멸균 핀셋 DDH 2 O의 커버 슬립을 제거하고 활성화 된 얼굴 위로 새로운 배양 접시에 배치합니다. </li><li> 커버 슬립에 달라 붙는 먼지를 방지하기 위해 알루미늄 호일로 건조한 장소에서 실온에서 보관하십시오. </li></ol> 히드 록시 PAAm 히드로 겔의 2 준비 <ol><li> 1.5 ㎖의 에펜 도르프 튜브에 N-하이드 록시 에틸 아크릴 아미드 (HEA) 65 mg의 중량을 준비한다. 이는 신선한 HEA 용액을 제조하는 것이 중요하다. </li><li> HEA에 50 mM의 HEPES 버퍼의 1 ML을 추가하고 전체 HEA 용해 할 때까지 vortexer를을 사용하여 혼합한다. </li><li> 바람직한 하이드로 겔 강성에 도달 / w HEPES 아크릴 아미드 용액 및 / HEPES 비스 - 아크릴 아미드 용액에 w (표 1 참조) w 2 % w 필요한 부피 40 % 400 μl를 추가. 5 ML의 최종 볼륨에 50 mM의 HEPES로 조정합니다. </li><li> vortexer를 탈기하고 그것을 사용 용액을 섞어하이드 록시 PAAm 중합을 방지 용액 내 산소 농도를 감소시키기 위해 20 분 동안 진공 챔버에서. </li><li> 무균 후드 하에서, 소독하기 위해 0.2 μm의 세공 크기 필터를 탈기 용액을 필터. </li><li> 7 분 동안 UV / 오존 클리너 원형 유리 커버 슬립 (22mm 직경) 활성화. </li><li> 10 % 황산 암모늄 (APS) 용액 100 μl를 준비, 즉 100 μL의 DDH 2 O.에서 10 mg의 APS입니다 그것은 새로운 APS 용액을 제조하는 것이 중요하다. </li><li> 중합을 개시 테트라 메틸렌 디아민의 2.5 μL (TEMED) 및 살균 히드 록시 PAAm 솔루션 (단계 2.5)에 APS 솔루션의 25 μl를 추가합니다. 멸균 조건 하에서, 거품의 도입없이 3 연속 pipettings 의해 용액을 혼합한다. </li><li> 멸균 후드, 22mm의 GL을 배치 즉시 (단계 1.9에서 가능) 및 25mm coverslip에의 히드 록시 PAAm 솔루션의 25 μL 강하를 배치방울의 상단에 엉덩이를 커버 슬립 (단계 2.5에서 준비)는 히드 록시 PAAm 솔루션을 짜내합니다. 멸균 핀셋 22mm 유리 커버 슬립을 중앙에 모든 거품을 부드럽게. </li><li> 하이드 록시 PAAm 하이드로 겔은 실온에서 15 분 동안 중합하도록 허용합니다. 중합 공정의 완료를 수행하는 에펜 도르프 튜브에서 직접 잔여 하이드 록시 PAAm 용액을 전환. </li><li> 완전히 멸균 DDH 2 O로 커버 슬립을 담그고 신중 22mm 유리 커버 슬립 및 하이드 록시 PAAm 하이드로 겔 층 사이에 면도날의 에지를 도입하여 22mm 유리 커버 슬립을 분리. </li><li> 멸균 PBS로 히드 록시 PAAm 하이드로 겔을 씻으 (PBS의 3 교류)와 완벽하게 수분을 유지하기 위해 멸균 PBS에 열중 젤을 할 수 있습니다. </li><li> 최대 3 일 동안 4 ° C에서 멸균 PBS에서 히드 록시 PAAm 하이드로 겔을 저장합니다. </li></ol> 3 폴리 디메틸 실록산 (PDMS) Microstamp 제작 NOTE : 실리콘 마스터의 제조는 STA에 앞서 필요PDMS에게 microstamp 제조를 실온. 실리콘 마스터의이 마이크로는 전문 장비와 훈련이 필요 리소그래피 기술에 의해 수행 할 수 있습니다. 나노 제조 시설과 협력은 실리콘 마스터를 제작하는 것이 좋습니다. 또한, 필요에 따라 맞춤형 미세 실리콘 마스터를 제조하고 회사에 문의하십시오. 이것은 실리콘 마스터의 제조에만 필요 한번 수행되어야한다는 것이 중요하다. 실제로, 미세 실리콘 주인은 탄성 중합체 스탬프를 생성하기 위해 무기한 사용할 수 있습니다. <ol><li> 믹스 및 PDMS (10) 경화제 : 플라스틱 비커 1 비율 10 분간 피펫을 사용하여 충분히 혼합한다. </li><li> 3.1 공정 중에 형성된 기포를 제거하기 위해 진공 하에서 PDMS 혼합물을 탈기. </li><li> 페트리 접시에서 미세 실리콘 마스터를 놓고 거품을 형성하지 않고 그 위에 가스가 제거 된 PDMS 혼합물의 10-mm 두께의 층을 캐스팅. </li><li> 60 ° C에서 2 시간 동안 PDMS를 치료하자오븐. </li><li> 메스와 먼지가없는 환경에서, 필 오프 PDMS 층과 소비세 1cm 2 microstamps. </li><li> 집게를 사용하여, 장소 PDMS는 페트리 접시에 패턴 업을 microstamps. </li></ol> (4) 미세 히드 록시 PAAm 히드로 겔 <ol><li> 15 분 동안 에탄올 / 물에 장소 PDMS의 microstamps (50 분의 50) 솔루션 및 초음파 처리. </li><li> 질소 기류의 흐름과 함께 우표를 건조하고 그들에게 7 분의 UV / 오존 청소기 (λ &lt;200 nm의)의 패턴 업을 배치합니다. </li><li> 멸균 후드에서 1 cm 2 PDMS 스탬프의 미세 표면에 원하는 단백질 용액 (예를 들어, 100 μg / ml의 라미닌 PBS 또는 25 μg / ml의 피브로넥틴 PBS에서)의 150 ㎕의 드롭을 배치합니다. </li><li> 선택적 : 세포 리간드 밀도를 변조하는 단백질 용액의 농도를 수정한다. </li><li> 각각의 모서리를 향해 멸균 피펫 팁으로 이동하여 도장면에 걸쳐 단백질 용액 확산스탬프입니다. </li><li> 멸균 후드 아래에 60 분 동안 PDMS 스탬프에 흡착 단백질 용액을 둡니다. 단백질 손상을 방지하기 위해 램프를 끄고. </li><li> 멸균 후드, 페트리 접시에 (단계 2.13에서 구입 가능) 히드 록시 PAAm 코팅 된 커버를 전송할 수 있습니다. </li><li> 멸균 조건 하에서 낮은 질소 스트림과 하이드 록시 PAAm 기판의 표면으로부터 과량의 PBS를 제거한다. 즉시 겔 표면에 물을 서의 증거가 관찰되지로 절차를 중지. 겔이 단계에서 충분히 건조되어서는 안된다. </li><li> 드라이 신중 PDMS의 구조화 된 표면은 고순도 질소 가스의 정상류와 스탬프. </li><li> 드레싱 조직 겸자와 단백질 코팅 된 스탬프를 잡고 건조 된 하이드로 겔 표면과 접촉 구조화 된 표면을 배치합니다. 스탬프 마이크로 피처 및 하이드로 겔 표면 사이의 양호한 접촉을 보장하기 위해 PDMS 스탬프의 위에 핀셋의 끝 간단한 압력 점을 적용한다. </li><li> 일에 PDMS 스탬프를 남겨실온에서 1 시간 동안 전자 하이드로 겔 표면. </li><li> 부드럽게 드레싱 티슈 포셉 히드 록시 PAAm 하이드로 겔에서 PDMS 스탬프를 제거하고 스탬프를 청소하는 단계 4.1을 따릅니다. </li><li> 환율 당 10 분 동안 멸균 조건에서 PBS의 3 교류 (산도 = 7.4)에 의해 광범위하게 각인 히드 록시 PAAm 하이드로 겔을 씻으십시오. </li><li> 선택적 : 다른 ECM 단백질의 추가의 미세 패턴은 단계 4.5에서 4.11에 따라 하이드 록시 PAAm 표면에 첨가 될 수있다. </li><li> 흔들 접시에 부드러운 교반하면서 4 ° C에서 하룻밤 동안 PBS에서 5 ㎎ / ㎖에서 BSA의 무균 용액으로 보호막을 비 인쇄 영역. </li><li> 환율 당 10 분 동안 멸균 조건에서 PBS의 3 교류 (산도 = 7.4)에 의해 광범위하게 씻으십시오. 이 단계에서, 스탬프 히드 록시 PAAm 하이드로 겔은 일주까지 4 ° C에 저장할 수 있습니다. </li></ol> 미세 패턴 히드 록시 PAAm 히드로 겔에 5 셀 증착 <ol><li> 이전 도금 C에 30 ~ 45 분 동안 셀 미디어에 커버 슬립을 품어ELL 학생. </li><li> 37 ° C에서 멸균 PBS와 75cm 두 문화 플라스크에서 배양 부착 세포를 세척하고 10 분 동안 3 트립신-EDTA의 ML 또는 accutase로 분리. </li><li> g 떨어져있는 세포를 포함하는 플라스크로 세포주를 위해 미리 예열 완전 성장 배지에 적절한 원하는 금액을 전송 및 X 650에서 3 분 동안 세포 현탁액을 원심 분리. </li><li> 마이크로 피펫으로 상층 액을 제거하고 15,000-20,000 세포 / ml에서 완전 배지에서 세포를 재현 탁. </li><li> 1 ~ 2 시간 동안 습도를 (단계 5.1에서 얻은) 미세 패턴 커버 슬립에 셀 솔루션의 4 ML을 추가하고 배치 셀 덮인 커버 슬립을 문화 인큐베이터에서 37 ° C에서 5 %. </li><li> 대기음 부드럽게 부착되지 않은 세포와 문화 매체를 교체합니다. 인큐베이터에 부착 된 세포를 반환하고가 (세포 유형에 따라 3-6 시간) 완전히 전파 할 수 있습니다. </li></ol>

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No conflicts of interest declared.

현대 세포 생물학 체외 관측 많은 종종 ECM 단백질 또는 RGD 서열을 함유하는 합성 펩티드의 얇은 층으로 코팅 된 경질 유리 커버 슬립 상에 수행되었다. 그러나, 이러한 기본 배양 기질 세포 mechanotransduction의 프로세스 연구를위한 정확한 모델을 제공하지 않는, 따라서 ECM의 전체 물리 복잡도 요점을 되풀이하지 않는다. 이 문제를 해결하기 위해, 우리는 원하는 금액과 ECM 단백질의 자?...

지방 세포의 미세 환경의 많은 부분 (예를 들면, 강도, 기공 크기, 단백질의 성질, 또는 세포 리간드 밀도)가 이러한 운동성, 세포 증식, 분화 및 유전자 발현과 같은 세포 과정을 조절 규제 큐들의 좌표를 제공한다. 세포 외 환경의 물리 화학적 특성의 수정은 세포에 의해 감지 및 세포 편광, 마이그레이션 및 분화의 변형 등 다양한 생리 학적 결과를 야기 할 수 있습니다. 그것은 세포가 세포 생화학 적 신호로 ECM 수정을 번역하는 방법을, 그러나 확실하지 않다. 따라서 주요 중요성 mechanotransduction의 경로를 공부 셀과 미세 환경 사이의 상호 작용을 재현 할 수있는 시험 관내 미세 환경 제어 엔지니어이다. 이 문제를 해결하기 위해, 우리는 최근에 쉽게 두 푼을 생성하기 위해, 히드 록시 PAAm 하이드로 겔이라는 새로운 방법 하나를 소개했다nsional 소프트 독립적으로 중요한 mechanotransduction의 단서를 제어 할 수 있도록 매트릭스 : 매트릭스 강성, 셀 형상과 감금, 단백질 및 세포 리간드 밀도의 자연입니다. ECM은 morphogens에서 그라디언트를 통해 세포 과정 (화성), 접착 단백질 (haptotaxis), 강성 (durotaxis)을 지시합니다. 지난 몇 년 동안, 시험관 플랫폼에서 첨단 세포 생리 프로세스 2-5로 생화학 및 생물 물리학 적 기능을 변환 할 수있는 방법을 애타게하기 위해 이러한 세포 외 신호를 분리하기 위해 개발되었다. 전자빔 (6), (7), 포토 리소그래피, 광 화학적 고정화 8 또는 플라즈마 - 보조 기술도 9는 기판 상에 미세 패턴 살아있는 세포의 성장을 유도하기 위해 개발되었다. 이러한 기술이 중요한 결과를 산출하고 있지만, 이들 중 대부분은 세포 행동에 상이한 큐들의 개별적인 영향 사이의 차별을 허용하지그들은 몇 가지 실험실이 감당할 수있는 기술 설비를 필요로한다. 이러한 기술 중 미세 접촉 인쇄 (μCP)는 세포 접착 마이크로 섬 열을 만들 수있는 강력하고 접근 가능한 방법으로 떠오르고있다. 최근에, 광범위한 노력 11-14은 생체 조직에서 관찰 강성의 넓은 범위를 재현하기 위해 가변 강성과 하이드로 겔에 μCP을 개발되었습니다. 이들 작업 중, 폴리 아크릴 아미드 (PAAm)는 15 대중적 이미 세포 생체 역학 분석을 위해 가장 일반적으로 사용되는 중합 체계 매트릭스 중 하나입니다. PAAm 표면은 일반적으로 N-sulfosuccinimidyl -6- [4&#39;-아지도-2&#39;-nitrophenylamido] (술포 SANPAH) 및 ECM 단백질 술포 SANPAH의 니트로의 UV 활성화함으로써 표면으로 연결되어 헤테로 가교제로 작용된다 아 지드 그룹 16. 또 다른 기술은 심한 산화 된 단백질에 결합 히드라진에 이루어져요오드 17. Hynd 및 동료 acroyl - 스트렙 타비 딘 단량체 (18)의 존재 광중합을 필요로 단백질과 펩타이드 생체 모방 하이드로 겔 표면을 패터닝하는 기술을 소개했다. 최근 챙은 외. 일 에틸 3 - [3 - 디메틸 아미노 프로필] 카르 보디이 미드 히드로 클로라이드로 활성화 된 PA 젤을 배양하는 데 필요한 광학 석영 마스크를 통해 PAAm 깊은 UV 노출에 기초한 새로운 미세 가공 방법 19 (EDC)를보고했다 및 N-히드 록시 숙신이 미드 (NHS) 수용액 전에 단백질을 추가한다. 긴 합성 과정 (예, 투석, 동결 건조, 등), 비싼 화학 물질 (예를 들면, 히알루 론산, 술포 SANPAH) 또는 깊은 UV : 균일하고 재현성 단백질 미세 패턴을 생성하기 위해 이러한 기술의 능력에도 불구하고, 대부분의 주요 한계 고통 조사. 또한, 이들 기술은 강성 기판, 미세 패턴의 독립적 인 변조를 허용하지 않는다기하학, ECM 단백질 자연, 그리고 세포 리간드 밀도. 계정에 이러한 한계를 가지고 가서, 우리는 부드러운 하이드로 겔의 단백질 및 생체 분자의 다양한 고정 수 및 세포 기능에 미치는 역할을 해독하기 위해 mechanotransduction의 큐의 독립적 인 튜닝을 허용하는 새롭고 간단한 아크릴 아미드 기반의 접근 방식을 개발했다. 대신에 강한 화학 화합물 PAAm 하이드로 겔을 치료, 우리 PAAm 중합 중에 수산기와 상업용 아크릴 모노머를 도입. 이 간단한 조작으로 다른 기술적 요구 사항없이 PAAm 하이드로 겔의 고유 안티 - 접착 성을 극복한다. 수산기의 존재는 수소 결합 상호 작용을 형성하는 단백질 및 생 분자를위한 히드 록시 PAAm 하이드로 겔의 높은 친화력으로 이끈다. μCP와 함께, 하이드 록시-PAAm 하이드로 겔은 독립 제어와 이차원 배양 플랫폼의 신속한 생성을 가능하게mechanotransduction의 연구를위한 강력한 플랫폼으로 구상하는 매트릭스 강성, ECM 단백질의 종류, 세포 리간드 밀도와 밀폐 된 접착에. 이 프로토콜의 목적은 쉽게 재료 과학에서 어떠한 지식없이 록시-PAAm 하이드로 겔을 제조하기위한 필요한 정보를 제공하는 것이다. 연구자 병태 생리 학적 기전에 관여 mechanotransduction의 경로를 더 잘 이해 될 수있는 세포 및 조직 수준에서 생리 관련 질문을위한 궁극적 목적은 수단을 제공하는 것이다.

<table border="0" cellpadding="0" cellspacing="0" width="573">
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	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">UV/Ozone Photoreactor</td>
			<td style="width:71px;">Ultra-Violet Products</td>
			<td style="width:119px;">Model PR-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Rocking plate</td>
			<td style="width:71px;">IKAcWerke</td>
			<td style="width:119px;">Model KS 130 Basic</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Vortexer</td>
			<td style="width:71px;">Scientific Industries</td>
			<td style="width:119px;">Model Vortex Genie2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Vacuum degassing chamber</td>
			<td style="width:71px;">Applied Vacuum Engineering</td>
			<td style="width:119px;">DP- 8-KIT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Parafilm</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7793-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Stainless steel forceps with fine tip</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z225304-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Dressing tissue forceps</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F4392-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Petri dishes in polystyrene</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5731-500EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Aluminium foil, thickness 0.5&#160;mm</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">266574-3.4G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Isopore membrane filter (0,2 &#181;m pore size)</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">GTTP Filter code</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Round glass coverslip (22 mm diameter)</td>
			<td style="width:71px;">Neuvitro</td>
			<td style="width:119px;">GG-22</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Round glass coverslip (25 mm diameter)</td>
			<td style="width:71px;">Neuvitro</td>
			<td style="width:119px;">GG-25</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Variable volume micropipette</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z114820</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Protein microcentrifuge tubes</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z666505-100EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Scalpel handles</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2896-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Scalpel blades</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2771-100EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Cell culture flasks (75 cm2)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS430641</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ultrasonic bath tray, solid (stainless steel)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z613983-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of the Reagent</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalogue Number</td>
			<td style="width:167px;">Comments</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Polydimethylsiloxane&#160;</td>
			<td style="width:71px;">Dow Corning</td>
			<td style="width:119px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Acrylamide (powder)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3553</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N,N&#8217;-Methylenebis(acrylamide)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">146072</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N-Hydroxyethylacrylamide</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">697931</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N,N,N&#8217;,N&#8217;-Tetramethylethylenediamine</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9281</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Amonium PerSulfate (APS)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3678</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">3-(Trimetoxysilyl)propyle acrylate</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">1805</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Human Plasma Fibronectin</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">FC010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Laminin from EHS</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">L2020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium hydroxyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">221465-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Double-distilled water (ddH2O)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Endothelial cell growth medium</td>
			<td style="width:71px;">Cells Applications</td>
			<td style="width:119px;">211K-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C-003-5C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Accutase</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">L11-007</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">HEPES buffer solution 1M in H20</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">83264-500ML-F</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Antibiotics-antimycotics</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">P11-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Phosphate Buffer Saline solution</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">H15-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Alexa Fluor 488 Phaloidin</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">A12379</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-vinculin antibody produced in mouse</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">V9131</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Goat anti-mouse antibody-tetramethylrhodamine</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">T-2762</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Fibronectin</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">F3648</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Streptavidin&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">41469</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Laminin antibody</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">L9393</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-rabbit IgG-FITC</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F7512</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Trypsin-EDTA solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3924-100ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Absolute ethanol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">459844-2.5L</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

preparation of hydroxy-paam hydrogels for decoupling the effects of mechanotransduction cues

그것은 지금 잘 많은 세포 기능은 자신의 세포 외 기질 (ECM) 환경의 물리 화학적 및 기계적 신호와 세포의 상호 작용에 의해 조절되는 것으로 설정됩니다. 진핵 세포는 지속적으로 생화학 적 신호로 ECM의 물리적 변화를 형질 도입하기 위해 표면 mechanosensors 통해 로컬 미세 환경을 감지하고, 유전자 발현의 특정 변화를 달성하기 위해 이러한 신호를 통합. 흥미롭게도, 서로 ECM 캔 부부의 물리 화학적 및 기계적 매개 변수는 세포의 운명을 조절합니다. 따라서, mechanotransduction의를 이해하는 열쇠는 세포 기능에 ECM 큐의 상대적 기여도를 분리하는 것입니다. 여기에서 우리는 신속하고 쉽게 체외에서 mechanotransduction의 큐의 독립적 인 튜닝을위한 생물학적으로 관련 하이드로 겔을 생성하는 자세한 실험 프로토콜을 제시한다. 우리는 화학적으로 변성 폴리 아크릴 아마이드 하이드로 겔 (PAAm)는 자신의 본질적 비 ADHES을 극복하기중합시 히드 기능화 아크릴 모노머를 포함함으로써 속성 필자. 우리는 ECM 단백질의 원하는 성질의 고정화를 허용 록시-PAAm 불리는 신규 PAAm 하이드로 겔을시켰다. 마이크로 콘택트 프린팅은 독립적으로 단일 세포의 형태학, 매트릭스 강성 및 자연 ECM 단백질의 농도를 제어 할 수 있도록하여 하이드 록시 PAAm의 조합은 하이드로 겔. 우리는 시험 관내 세포 mechanotransduction의 과정에서 공부하는 모든 생물학 실험실에서 설정할 수있는 간단하고 빠른 방법을 제공한다. 우리는 ECM의 강성과 핵 사이의 기계적 커플 링을 보여 내피 세포에 대한 실험을 실시하여이 소설 두 차원 플랫폼을 검증합니다.

도 1a와 아크릴 아미드의 공중합 (AAM) 및 비스 아크릴 아미드 (비스 AAM)를 제시 N-hydroxyethylacrylamide 임베디드 히드록시기로 임의 라디칼 중합에 의해 폴리 아크릴 아미드의 친수성 네트워크를 형성 일차 수산기를 함유하는 (HEA) 모노머 (히드 록시 PAAm) . 이 프로토콜에서, HEA의 중량 65 ㎎을 HEPES 1 ㎖의 부피로 희석해야한다. HEA의 밀도가 한 대략 같다는 것을 알고, 우리는 우리가 1065 μL (HEA...

Watch this Scientific Journal Video about Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues at JoVE.com

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

우리는 최소한의 비용 또는 전문 지식을 갖춘 ECM 단백질의 직접적인 결합을 허용 히드 록시 PAAm라는 새로운 폴리 아크릴 아미드 하이드로 겔을 제시한다. 마이크로 콘택트 프린팅 휴대 mechanostransduction을 연구 천연 세포 미세 환경의 많은 큐들의 독립적 제어를 용이하게하여 히드 록시 PAAm의 조합은 하이드로 겔.

mechanotransduction의 큐의 영향 디커플링에 대한 히드 록시 PAAm 히드로 겔의 제조

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

preparation-hydroxy-paam-hydrogels-for-decoupling-effects

Research

JoVE Journal

Bioengineering

13.2K 조회수.  Université de Mons.  우리는 최소한의 비용 또는 전문 지식을 갖춘 ECM 단백질의 직접적인 결합을 허용 히드 록시 PAAm라는 새로운 폴리 아크릴 아미드 하이드로 겔을 제시한다. 마이크로 콘택트 프린팅 휴대 mechanostransduction을 연구 천연 세포 미세 환경의 많은 큐들의 독립적 제어를 용이하게하여 히드 록시 PAAm의 조합은 하이드로 겔. 

비디오: Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

액슨 스트레치 성장의 연결을 성장의 Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

연구

생체공학

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 ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Biomarker 디스커버리를위한 Mesoporous 실리카 박막의 낮은 분자량의 단백질 농축

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

치료 응용 프로그램을위한 도구로 RGD-작용 히드로 겔의 합성

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify 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 ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

물 정화를위한 막으로 방오 특성을 가진 히드로 겔의 합성

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

캡슐화 및 3d에서 셀의 Immunostaining에 대 한 엘라 스 틴 같은 단백질 Hydrogels의 생산

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

글로벌 세포 매트릭스 Metalloproteinase 및 3D Hydrogels 신진 대사 활동 측정

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical environment. During this process, mechanosensitive osteocytes regulate the opposing responses between the catabolic osteoclasts and anabolic osteoblasts. To better understand the highly intricate signaling pathways that regulate this process, our lab has developed a foundationary lab-on-a-chip (LOC) platform for analyzing functional outcomes (formation and resorption) of bone remodeling within a small scale system. As bone remodeling is a lengthy process that occurs on the order of weeks to months, we developed long-term cell culturing protocols within the system. Osteoblasts and osteoclasts were grown on functional activity substrates within the LOC and maintained for up to seven weeks. Afterward, chips were disassembled to allow for the quantification of bone formation and resorption. Additionally, we have designed a 3D printed mechanical loading device that pairs with the LOC platform and can be used to induce osteocyte mechanotransduction by deforming the cellular matrix. We have optimized cell culturing protocols for osteocytes, osteoblasts, and osteoclasts within the LOC platform and have addressed concerns of sterility and cytotoxicity. Here, we present the protocols for fabricating and sterilizing the LOC, seeding cells on functional substrates, inducing mechanical load, and disassembling the LOC to quantify endpoint results. We believe that these techniques lay the groundwork for developing a true organ-on-a-chip for bone remodeling.

Here, we present protocols for analyzing bone remodeling within a lab-on-a-chip platform. A 3D printed mechanical loading device can be paired with the platform to induce osteocyte mechanostransduction by deforming the cellular matrix. The platform can also be used to quantify bone remodeling functional outcomes from osteoclasts and osteoblasts (resorption/formation).

골연세포 메카노트랜스덕션 을 자극하고 뼈 리모델링의 기능적 결과 분석을 위한 Lab-On-A-Chip 플랫폼

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

라이브 현미경 이미징 에서 3D 하이드로겔의 제어 변형

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...