The authors thank the Fraunhofer ISC for the collaboration concerning the construction of the automated wounding device. The project was founded by Fraunhofer internal project &#8220;M&#228;rkte von &#220;bermorgen&#8221; (SkinHeal).

주 : 프로토콜 24 전층 피부 등가물의 제조를 위해 설계된다. 피부 섬유 아세포와 표피의 각질 형성 세포 인간 21, 22 동의서 사전 얻었다. 이전에 출판 된 프로토콜에 따라 피부 생검에서 분리하고, 연구는 율리우스 막시밀리안 대학 뷔르츠부르크의 인간 연구에 대한 기관 윤리위원회에 의해 승인되었다 (182 투표 / 10). 피부 구성 요소 1. 생산 <ol><li> 6 mg / ml의 최종 농도로 0.1 % 아세트산과 콜라겐 녹인다. 겔 중화 용액의 경우, 232.5 ml의 DMEM 배, 7.5 ml의 소 태아 혈청, 7.5 ㎖의 3 M HEPES, 2.5 ml의 콘드로이틴 설페이트 (5 ㎎ / ㎖)를 혼합한다. 얼음 젤 중화 솔루션과 콜라겐 솔루션을 유지합니다. 삽입 / 피부 당량 당 겔의 500 μl를 계산합니다. 주 : 겔 중화 용액 콜라겐 용액 처리장의 두 부분과 혼합 될 수있는 바와 같이콜라겐의 양을 두 번 다시. </li><li> 24 웰 플레이트에 24 삽입을 놓습니다. </li><li> 10 ml의 DMEM, 270 XG에 5 분 동안 원심 분리기에 인간의 피부 섬유 아세포 (HDF)를 재현 탁하고 세포를 계산합니다. g X 270에서 5 분 동안 필요한 HDF의 양 (500 μL 콜라겐 젤 당 5 × 10 4 세포)와 원심 분리기 세포를 추출합니다. </li><li> 4 ml의 기포를 생성하지 않고 겔 중화 용액 냉각에 재현 탁 HDF를 조심스럽게 상층 액을 제거하고. 이 단계에서 콜라겐 젤의 조기 응고를 방지하기 위해 가능한 한 빨리 작업을해야합니다. </li><li> 콜라겐 용액 8ml를 가진 용액을 재현 탁. </li><li> 다단계 피펫으로 콜라겐 세포 혼합물을 가지고 각 삽입 빠르게 혼합물의 500 μl를 입력합니다. 인큐베이터에서 20 분 동안 젤을 품어 (37 ° C, 5 % CO 2) 젤 굳어진합니다. </li><li> 웰당 2ml의 둘 베코 변형 이글 DMEM 배지 + 10 % 소 태아 혈청 (FCS)에서 겔을 담근다및 인큐베이터에서 24 시간 (37 ° C, 5 % CO 2)를 위해 그것을 품어. </li></ol> 인간의 표피 각질 형성 세포의 2. 추가 (HEK) <ol><li> 24 시간 후 배지를 제거합니다. 50 ㎍ / ml의 최종 농도로 초순수 인간 피브로넥틴 단백질을 녹인다. 피브로넥틴 용액 25 μL에 해당하는 각각의 피부를 커버. 배양기에서 30 분 동안 젤을 인큐베이션 (37 ℃, 5 % CO 2). </li><li> 한편, 10 ㎖ KGM-2를 재현 탁 및 HEK KGM-2 + 5 %의 FCS 준비로 1 × 106 세포 / ml의 세포 농도를 설정한다. 각 겔에 씨앗 1 × 10 5 HEK. 인큐베이터에서 45 분 (37 ° C, 5 % CO 2) 세포가 준수 할 수 있도록 젤을 품어. </li><li> 인큐베이터에서 KGM-2 + 준비 5 % FCS와 문화를 (37 ° C, 5 % CO 2)와 젤을 담그지. </li></ol> 전체 두께 피부 등가물 3. 문화 (FTSE) <ol><li> 6~7일에 대한 문화 FTSEFCS 농도 (5 %, 2 %, 0 % FCS)을 내림차순. 다음으로 낮은 FCS 농도가 2-3 일마다 매체를 변경합니다. 이를 위해, 완전히 매체를 제거하고 신선한 매체의 1.6 ml를 추가합니다. </li><li> 7 일에서 완전히 매체를 제거합니다. 참고 : 그렇게하는 동안 피펫의 끝 FTSE 표면을 만지지 마십시오. </li><li> 모든 멸균 집게로 6 웰 플레이트의 한 우물에 삽입 놓습니다. FTSE 표면을 젖은 가지 않도록 잘 당 1.5 ml의 공수 매체를 추가합니다. 추가로 14 일 동안 2-3 일마다 중간 변경합니다. 주 : 배지의 충전 레벨은 FTSE의 메 니스 커스에 달려있다. </li></ol> 4. 조직 학적 분석 <ol><li> 고정 및 FTSE의 파라핀 삽입 <ol><li> 실온에서 2 시간 동안 조직을 신선한 24 웰 플레이트에 삽입을 전송하고 잘 1.6 ml의 각 % 파라 포름 알데히드 (4)를 추가하여 조직을 수정하고 배양, 배양 배지를 제거합니다. 주의! Paraformaldehyd이 독성, 흄 후드를 조작 할 수 있습니다. 이전조직 포함 카세트 FTSE. 물로 세척하여 남아있는 고정 제를 제거하고 상행 에탄올 농도를 사용하여 조직을 탈수. </li><li> 중간에 수직 절단에 의한 피부 등가를 나눈다. 아래쪽 절단면과 파라핀 채워진 금속베이스 몰드의 두 조각을 놓는다. 백업으로 금형의 상단에있는 조직 카세트를 추가합니다. </li><li> 적합한 현미경 슬라이드에 슬라이드를 장착, 3-5 μm의 조각을 잘라 straitening에 대한 40 ° C의 물을 욕조에 그들을 떠. 완전히 건조 조각. </li></ol></li><li> 헤 마톡 실린 및 에오신 (H &amp; E)과 면역 조직 화학 (IHC) 염색 <ol><li> 장소는 랙에 슬라이드와 60 ° C에서 1 시간 동안 배양한다. 파라핀이 용융되어 있는지 확인합니다. 얼룩은 표준화 개요 염색으로 H &amp; E와 조각을 재수. </li><li> H &amp; E 염색을 위해 에탄올 50 %, 4 배 탈 이온수, 1 배 헤 마톡 실린, 1X 염산-E 1X와 에탄올로 2 배, 에탄올로 3 배, 크 실롤으로 70 %를 96 %를 4 배 유리 염색 큐벳을 준비thanol (13.7 ml의 1 M 염산 광고 200 ml의 50 % 에탄올), 1X 탭 물, 1X 에오신 및 배 2- 프로판올 (100 ㎖ 탈 이온수에 1g의 에오신). </li><li> 장소는 크 실롤의 I에서 10 분 deparaffinize과 슬라이드를 재수하는 크 실롤 II에서 10 분을 슬라이드. 딥 에탄올 50 % 에탄올 70 %와 3 배에서 3 배, 에탄올에 에탄올 96 % I, 배에 96 % II를 3 배를 슬라이드. 탈 이온수에서 슬라이드를 돌립니다. 헤 마톡 실린 염색, 염산 - 에탄올 딥 배를 구분합니다. 탈 물에 씻어. 청소법를 들어, 장소는 수돗물에 5 분을 슬라이드. 에오신 염색의 경우, 장소는 에오신에서 1 분 슬라이드 탈 이온수에 나중에 씻으십시오. 탈수 법, 딥 슬라이드 에탄올 96 % 에탄올에 70 %, 2 × 2 배 및 2- 프로판올 II에서 5 분 동안 2- 프로판올 I에서 5 분 동안 놓으 크 실롤에서 5 분 I 및 크 실롤 II에서 5 분. H &amp; E 염색 한 후, 세포 핵은 빨간색으로 염색 블루, 세포질과 세포 외 기질에 염색. </li><li> 항원 검색을 위해, 자일 렌으로 파라핀을 제거하고 염색에 대한 슬라이스 재수. 장소 조각증기 밥솥에 미리 가열 된 10 mM에서 20 분 구연산 완충액 (pH 6)에 대한 탈 파라핀과 재수 조각을 요리하고. </li><li> 액체 구충제 슬라이드 마커 펜 또는 다이아몬드 스타일러스 세척 버퍼에 넣어 슬라이드 (PBS 버퍼 + 0.5 % Polysorbat 20)과 원 조각. </li><li> 수분 챔버에서 슬라이드를 놓습니다. 각 슬라이스에 100 ㎕의 3 % 과산화수소 용액을 첨가하여, 내인성 퍼 옥시 다제를 차단. 주의! 피부와 눈에 접촉 한 경우, 대단히 유해 함. 개인 보호 장비를 처리합니다. 워시는 세탁 버퍼에 슬라이드. </li><li> 차 항체를 적용하고 실온에서 1 시간 동안 배양한다. 세척은 세척 버퍼로 3 회 슬라이드. 여기부터, 샘플은 어둠 속에서 유지되어야한다. </li><li> 결합 특이 항원 - 항체의 검출을 위해 비오틴 - 스트렙 타비 딘 탐지 시스템을 사용합니다. </li><li> 1 분 동안 헤 마톡 실린 핵을 Counterstain과. 탈수 및 슬라이드를 탑재합니다. </li><li> 역 현미경을 사용하여 이미지 샘플. </li></ol></li></ol> &lt;P 클래스 = &quot;jove_title&quot;&gt; 5. 자동 상처 장치의 사용에 의한 상해 (AWD) <ol><li> 70 % 에탄올 및 자외선과 작업 영역을 소독하여 AWD를 준비한다. 원하는 크기의 드릴 헤드를 확인합니다 (예를 들어, 1mm)가 부착되어있다. </li><li> 멸균 집게를 사용하여 멸균 샘플 캐리어 플레이트의 지정된 영역으로 피부 등가물을 놓습니다. 드릴링 헤드 아래의 소켓에 샘플 캐리어 플레이트를 놓습니다. </li><li> 스핀 속도 : 다음과 같이 상처 매개 변수를 설정하는 제어 소프트웨어를 사용하여 15,000 RPM을; 침투 : 1.5 mm; 추진 : 100 Hz에서. 이들 파라미터는 개별적으로 각각의 샘플 유형에 대해 정의 될 필요가있다. 전송 AWD에 매개 변수 부상을 입었습니다. </li><li> 절차를 상처 시작합니다. 소켓에서 샘플 캐리어 플레이트를 제거합니다. 전송 멸균 집게를 사용하여 배양에 사용되는 인서트로 다시 FTSE 부상. </li><li> 중생의 조사를 위해 인큐베이터에 상처 FTSE의 문화 (37 ° C, 5 % CO 2)를 진행합니다. 알대체적으로, 언제든지 조직 학적 분석을위한 모델을 사용합니다. </li></ol>

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+model+of+wound+healing+based+on+transplanted+tissue-engineered+skin.">In vivo model of wound healing based on transplanted tissue-engineered skin.</a> Tissue Eng. 10 (7-8), 1006-1017 (2004).</li><li>Bell, E., 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=The+reconstitution+of+living+skin.">The reconstitution of living skin.</a> J Invest Dermatol. 81 (1 Suppl), 2s-10s (1983).</li><li>Asselineau, D., Prunieras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+'simplified'+skin:+control+of+fabrication.">Reconstruction of 'simplified' skin: control of fabrication.</a> Br J Dermatol. 111, 219-222 (1984).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nat Rev Mol Cell Biol. 8 (10), 839-845 (2007).</li></ol>

The authors have nothing to disclose.

시험 관내 세포에서 세포가 보통 플라스틱 표면에 부착하는 이차원 세포 배양 물에서 확장된다. 그러나 이러한 배양 조건은 세포가 생체 내에서 성장하는 생리 학적 입체 조건을 반영하지 않습니다. 입체 조건에서 세포는 자연 세포 - 세포 및 세포 - 매트릭스 첨부 파일을 형성 할 수 및 3 차원으로 이동한다. 세포 이동 및 행렬 생성은 상처 치유의 핵심 요소로서, 특히 생체 내 ...

피부는 신체에서 가장 큰 기관이다. 그것은 외부 환경과 내부 장기 사이에 장벽을 만듭니다. 또한, 피부는 유체 손실, 환경 적 영향, 상처 감염으로부터 몸을 보호하고, 체온을 조절 한 것을 돕는다. 인해 노출 된 위치에, 피부는 종종, 기계적, 열적 또는 화학적 외상에 의해 영향을 받는다. 피부 자체 수리의 일반적으로 사용하는 것은 아니지만, 감염, 산소, 정맥 자족 여러 지역 요인 손상된 상처 치유 될 수 있습니다. 상처 치유는 비만, 알코올 중독, 흡연, 약물, 영양, 당뇨병과 같은 질환 등의 전신적인 요인에 의해 방해 할 수있다. (2) (I) 염증 상, (II) 및 증식 (III) 리모델링 단계 : 상처 치유 과정은 3 단계로 나눌 수있다. 피부에 손상되면, 복잡한 신호 캐스케이드 상처의 폐쇄로 이어지는 시작합니다. 3손상 후 상처 출혈과 혈병이 형성된다. 섬유 아 세포는 혈액 응고로 이동하고 이후 년 동안 리모델링되어 새로운 조직으로 대체. 생물학적 과정의 기본 피부 복구 전류 이해는 제한된다. 작은 동물과 돼지 모델은 상처 치유를 연구하는 데 사용되었다. 그러나, 이러한 결과는 직접 종 특이 차이에 의해 인체에 전달 될 수 없다. 이러한 생체 내 모델에 추가하여, 상처 치료의 일부 양태들은 4 선택된 긁 모델 고도로 표준화. 불멸화 세포주 또는 일차 세포에 기초한 생체 외 단층 배양 스크래칭을 통해 상처 상황을 시뮬레이션에 의해 연구 할 수 있지만, 충분히 반영되지 않는다 복잡한 생체 내 생리. 5 이차원 모델은 또한, 삼차원 인간 피부 등가물 피부병 연구 개발되어왔다. 이 모델의 피부 부분은 GE 있습니다nerated 탈세 포화 진피 6 콜라겐 히드로 겔, 7,8 글리코 9 또는 합성 재료 등 다양한 지지체를 사용. (10)이 피부 등가물을 채용, 상피 간엽 상호 작용 (11)의 역할을, 재 상피화, 섬유 아세포 및 각질 세포 및 간 세포 누화 다른 성장 인자의 영향이 연구 될 수있다. 또한,이 모델은 섬유 아세포가 부상 영역으로 마이그레이션하는 방법과 화학 주성 인자가 조직 재생에 영향을 미치는 방법에 대한 새로운 지식을 습득하는데 유용하다. (12) 상처 치유 모델 자체의 생성뿐만 아니라 도전이지만, 또한 모델의 고도로 표준화 상처 문제가 확립. 일반적인 기술은 상처 스크래치 테스트, 13 화상, 14 테이프 마모, 15 열 손상, 16 흡입 물집, 17 액체 질소, 18 레이저, 19 있습니다 만들 수 있습니다 </SUP&gt; 메스, 18 메쉬 작업자 6 생검 펀치. 이러한 방법 20 대부분 같은 함정이있다. 수동으로 구현 부상 표준화 및 여러 테스트를 사이에 재생하기 어렵습니다. 크기, 형상 및 상처의 깊이 연구 사이에서 변화하고, 따라서 연구 데이터의 품질이 저하. 정의 피부 상처 용 레이저의 사용은 비교적 용이하지만 표준화 화상 상처를 흉내 낸 상황에 이르게 할 수있다. 레이저에 의해 적용 열 괴사 조직으로 이어질 수 있습니다 단백질 변성, 혈소판 응집 또는 혈관 수축을 일으킬 수 있습니다. 대안적인 접근법에서, 우리는 우리가 멸균 조건 하에서 정확한 정의 및 피부 상처를 생성 할 수있는 자동화 된 상처 소자 (AWD)를 개발 하였다. 깊이 및 속도의 침투뿐만 아니라 드릴 헤드의 회전 수 등을 파라미터 상흔을 규제 할 수있다. 이 연구에서 우리는 집에서 개발 한 전체 두께 피부 등가물과 AWD를 결합 (피트Gangatirkar 등에 의해 발표 된 프로토콜에 비교할 SE). (8) 피부 상당의 피부 층은 내가 하이드로 콜라겐에 포함 된 인간 피부 섬유 아세포 (HDF)로 구성되어있다. 진피층에서 인간의 표피 각질 형성 세포 (HEK)은 시드입니다. 공기 - 액체 계면에서 2 주 이내에 HEK 여러 중요한 세포층과 각질층 이루어지는 표피를 구축. 이 모델의 생성 외에도,이 연구는 FTSE에 정의 된 정확한 상처를 만들 수있는 AWD를 사용하는 방법을 보여줍니다.

<table border="0" cellpadding="0" cellspacing="0" width="750">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Trypsin EDTA (1:250) 0.5 % in DPBS&#160;</td>
			<td style="width:135px;">PAA</td>
			<td style="width:119px;">L11-003</td>
			<td align="right">0.05%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Collagen (6 mg/ml in 0,1 % acetic acid)</td>
			<td style="width:135px;">Produced in house</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fibronectin Human Protein, Plasma (50 &#181;g/ml)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">33016-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Inserts</td>
			<td style="width:135px;">Nunc</td>
			<td align="right" style="width:119px;">140627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">6 well plate&#160;</td>
			<td style="width:135px;">Nunc</td>
			<td align="right" style="width:119px;">140685</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">24 well plate</td>
			<td style="width:135px;">Nunc</td>
			<td align="right" style="width:119px;">142485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Microscope slides</td>
			<td style="width:135px;">R. Langenbrinck</td>
			<td style="width:119px;">03-0070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fibroblasts culturing (500 ml):</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DMEM, high glucose</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td>89% (445 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fetal bovine serum</td>
			<td style="width:135px;">Bio &#38; Sell</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td>10% (50 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinozyten culture medium (500 ml):&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-20111</td>
			<td>89% (445 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2 SupplementPack</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-39011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Gel neutralization solution (250 ml):</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Dulbecco&#8217;s Modified Eagle Medium, high Glucose Powder with L-Glutamine</td>
			<td style="width:135px;">PAA</td>
			<td style="width:119px;">G0001,3010</td>
			<td>93% (232,5 ml)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Chondroitin sulfate sodium salt from shark cartilage</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">C4384-1g</td>
			<td>1% (2,5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fetal bovine serum</td>
			<td style="width:135px;">Bio &#38; Sell</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td>3% (7,5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">HEPES</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td style="width:119px;">H3375-1kg</td>
			<td>3% (7,5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Skin model submers medium (500ml):&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-20111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2 SupplementPack</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-39011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fetal calf serum</td>
			<td style="width:135px;">Bio &#38; Sell</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td>5%-2% (25 ml-10 ml)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Skin model air-liquid interface medium (500 ml):&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Keratinocyte Growth Medium 2 Supplement Pack</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-39011</td>
			<td>adding only supplements: Insulin, Hydrocortisone, Epinephrine, Transferrin, CaCl2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:329px;">CaCl2 (300 mM)</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td style="width:119px;">C7902-500g</td>
			<td>&#160;0,62% (3,1 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Histology:</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">IHC-Kit DCS SuperVision 2 HRP</td>
			<td style="width:135px;">DCS</td>
			<td style="width:119px;">PD000KIT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Vimentin antibody</td>
			<td style="width:135px;">Abcam</td>
			<td>ab92547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">CK14 antibody</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td>HPA023040-100&#181;l</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">CK10 antibody</td>
			<td style="width:135px;">Dako</td>
			<td style="width:119px;">M7002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Filaggrin antibody</td>
			<td style="width:135px;">Abcam</td>
			<td>ab81468</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">H&#38;E staining</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Mayer&#180;s Haemalaun</td>
			<td style="width:135px;">AppliChem</td>
			<td style="width:119px;">A0884,2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Xylol</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>296325-4X2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Ethanol</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>32205-4X2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">HCl</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>H1758-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Eosin</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>E4009-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2-Propanolol</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>I9516-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Mounting Medium</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>M1289-10ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of a three-dimensional full thickness skin equivalent and automated wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

고립 된 HDF와 HEK는 형태와 일반적인 마커의 발현 모두 다르다. HEK의 형태가 볼일 형태에 의해 설명 될 수있는 반면 HDF는 전형적인 스핀들 형상의 형태를 보였다. 세포 FTSE 위해 사용하기 전에 면역 조직 화학 염색 (도 1)을 특징으로 하였다. HDF는 멘틴 (그림 1A), 섬유 아 세포 마커에 대한 긍정적이다. 차 HEK 매우 초기 분화 단백질 아세포-14 (그림 1B)하지만 거의?...

Watch this Scientific Journal Video about Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding at JoVE.com

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

3 차원 전체 두께 피부의 세대 동등한 및 자동화 부상

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

generation-three-dimensional-full-thickness-skin-equivalent-automated

Research

JoVE Journal

Bioengineering

18.4K 조회수.  University Hospital Würzburg. The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility. 

비디오: Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

3 차원의 인간 피부 모델을 재구성 : 도구는 일반 피부와 흑색증의 진행을 공부하기

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

연구

생체공학

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

전도성 잉크의 평면 및 3 차원 인쇄

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

모델 보컬 폴드 폴립에 의해 유도된 3차원 유동 분리 조사

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

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 ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

세 가지 차원 콜라겐 매트릭스 내에서 세포의 이동 분석

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

세포 외 매트릭스 내에서 임베디드 공학 3 차원 상피 조직

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an&#160;additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

인쇄를 만드는 열가 소성 재료의 3 차원 미세 응용 프로그램에 대 한 피드백 제어와 주사기 펌프를 자동

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

대규모로 3차원 재건 된 인간 표피 를 육성

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

마이크로머신 마이크로유체 플랫폼의 간소화된 3차원 스킨 온-어칩 모델 생성

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.