우리는 크게 셰릴 Schaeberle에 의해 편집 기여를 주셔서 감사합니다. 이 연구는 에이 보조금 # 02-2009-093과 02-2011-095, 및 AMS에 NIEHS / NIH ES 08314에 의해 지원되었다. 내용은 전적으로 저자의 책임이며 반드시 환경 보건 과학 연구소 또는 국립 보건원의 공식 견해를 대변하지 않습니다.

시약 1. 준비 <ol><li> 10-3 M의 재고 솔루션을 만들 수있는 합성 progestagen의 promegestone (R5020) 및 에탄올 17-β-에스트라 디올 (E2)를 녹인다. 1 ㎎ / ㎖ 모액을 증류하여 탈 이온수 프로락틴을 녹인다. 10 -2 M 주식 솔루션을 만들기 위해 DMSO에 항 에스트로겐 ICI 182,780을 녹인다. 최대 6 개월 동안 -20 ° C에서 이러한 솔루션을 저장합니다. </li><li> 숯불 덱스 트란 (CD)의 혈청 및 CDFBS 매체를 제거 : <ol><li> 30 분 동안 57 ° C의 물 중탕에 소 태아 혈청 (FBS)을 열 - 불 활성화. 참고 : 사용이 프로토콜의 나머지 부분에 대해서만 산 세척 유리합니다. </li><li> 필요한 숯의 양을 계산한다. 상기 활성탄에 의한 변위를 보상 5 % 중량 / 부피의은 분말 활성탄 무게 혈청 량보다 10 % 더 많은 양을 사용. </li><li> 혈청 양과 동일한 양을 사용하여 차가운 탈 증류수로 두 번 세척 숯. 각 세척 C에 대한숯 펠렛 2 분 50 XG에 entrifuge. 세척 사이에 물을 제거합니다. 마지막 숯 펠렛 0.5 % 중량 / 부피의 덱스 트란 T-70을 추가합니다. 5 분 100 XG에 증류수, 탈 이온수와 원심 분리기에 다시 일시 중지합니다. 물을 대기음. </li><li> 펠렛에 혈청의 적절한 볼륨을 추가하고 혈청에 숯 덱스 트란 펠렛을 다시 일시 중지합니다. 60 분 37 ºC에서, (6 RPM) 압연 동안 품어. 20 분 동안 2,000 XG에 원심 분리기. 상층 액이 흐린 나타나는 경우, 원심 분리를 반복; 그러나, 혈청은 현재 완전히 명확하지 않을 수 있지만, 여과시 삭제됩니다. </li><li> 큰 입자상 물질을 제거하기 위해 0.45 미크론 필터를 통해 다시 0.80 미크론 필터를 통해 여과하고. 마지막으로, 0.20 미크론 필터로 멸균 필터. 스토어는 -20 ºC에서 유리 튜브 또는 T-25 배양 플라스크에 CDFBS 필터링. </li><li> 숯 덱스 트란은 태아 미디어 (CDFBS 미디어) 소 함유 박탈 : 페놀 레드가없는 DMEM의 375 ml의 DMEM의 125 ㎖의 혼합/ F-12. 37.5 ml의를 제거하고 숯 덱스 트란 박탈 소 태아 혈청의 동일한 볼륨으로 교체. 2 mM의 최종 농도 106 U / ㎖ 페니실린 및 L- 글루타민을 추가합니다. 그 결과 매체는 75 % DMEM, 25 % DMEM / F-12, 7.5 %의 숯불 덱스 트란 제거-FBS, 2mM의 L 글루타민, 10 (6) U / ㎖ 페니실린이다. </li></ol></li><li> 10X PBS, 1N의 NaOH와 증류수 탈 이온수를 준비합니다. 필요한 콜라겐 용액의 부피에 따라 각각의 충분한 양을 준비한다. 필터링 모든 솔루션을 소독. 최대 1 개월 4 ° C에서 10 배 PBS와 멸균 증류수 탈 이온수를 저장합니다. 1N NaOH 용액을 보관하지 마십시오. </li><li> 제조업체에서 제공하는 &quot;콜라겐 I, 쥐 꼬리의 대체 겔화 절차&quot;에 대한 프로토콜에 따라 1 ㎎ / ㎖ 최종 농도 콜라겐 솔루션을 준비합니다. 각 겔 콜라겐 용액 1.5 ㎖로한다. 예를 들어, 제조 콜라겐 용액 (피펫 동안 손실을 고려하여 0.5 mL)을 18.5 mL의 제조12 젤. 바로 콜라겐 솔루션을 사용하거나 최대 2 ~ 3 시간 동안 얼음에 개최. </li><li> 카마 졸업생 염료 : <ol><li> 증류수 500 ㎖에 1g 카민 2.5 g 알루미늄 칼륨 설페이트 결합. 20 분 동안 교반하는 동안 끓인다. 이상 끓는 않도록주의하세요. 추가 증류수로 다시 500 ml의에 최종 볼륨을 조정합니다. </li><li> 항균 활성에 대한 티몰의 결정을 추가하고 빛으로부터 보호하기 위해 알루미늄 호일로 병을 커버합니다. 각 사용하기 전에 실험실 등급의 종이 필터를 통해 필터링합니다. 최대 3 개월 카민 용액을 다시 사용합니다. 실온에서 저장, 빛으로부터 보호. </li></ol></li><li> 콜라게나 제 용액 / V w 0.125 %의 농도로 DMEM / F12 배지에서 콜라겐을 분해. 이 솔루션을 보관하지 마십시오. </li></ol> 쥐의 꼬리 콜라겐 타입 1 젤과 호르몬 치료 2. 3D 문화 T47D의 세포 참고 : 4 ° C에서 멸균 혈청 피펫과 피펫 팁을 유지합니다. <ol><li> 처리장단일 세포 현탁액을 Re 및 세포 수를 수행한다. 분할 T47D 세포 배양은 70 %의 합류에 도달하기 전에. 필요한 셀의 양을 포함하는 양을 원심 분리기. 한 젤은 약 75,000 T47D 세포를 포함하는 것을 고려하십시오. </li><li> 초기 피펫을 사용하여 콜라겐의 적절한 부피 세포 펠렛을 다시 일시. 각 겔은 콜라겐의 1.5 ml의로 구성되어 있음을 고려한다. 거품을 도입 피하기 위해 조심스럽게 세포와 콜라겐을 섞는다. </li><li> 세포의 분포에 정전기의 영향을 방지 판을 풀기 후 정전기 감소 브러시​​로 12 웰 플레이트의 경계, 상단과 하단 브러시합니다. 후드의 작업 표면과 접촉시 정전기의 발생을 방지하기 위해, 또한 정전기 감소 brush.Gently로 닦았 된 다른 12 웰 플레이트 (빈 접시)의 상단에이 판을 배치 믹스의 1.5 ml에 부어 12 웰 플레이트의 각 웰에. </li><li> 30 분 동안 37 ° C에서 인큐베이터에서 접시를 놓습니다. 일을 제거전자 인큐베이터에서 플레이트와 후드에 다시 배치합니다. </li><li> P200 멸균 팁을 사용하여 조심스럽게 잘 국경에서 부드럽게 원을 그리 젤을 분리합니다. 겔은 웰의 바닥으로부터 분리한다. </li><li> CDFBS 배지에서 희석 호르몬 웰에 첨가한다. 10 -7 M 최종 농도에서 10 -10 M 최종 농도와 프로락틴에서 E2 및 R5020을 사용합니다. 대조군으로 추가 호르몬하지만 여전히 사용되는 호르몬 주식과 동일한 DMSO의 높은 볼륨을 포함하지 않고 CDFBS 미디어를 사용합니다. 각 잘 미디어 1.5 ml에 포함되어 있는지 확인합니다. 37 ° C, 5 % CO 2, 100 % 내부의 3D 문화를 배치 습도 조직 문화 인큐베이터. 2 일마다 미디어를 변경, 1 ~ 2 주 동안 문화를 유지한다. </li></ol> 전체 마운트 3. 젤 처리 <ol><li> 문화 미디어를 제거하고 각 웰에 PBS 1.5 ml에 추가 할 수 있습니다. </li><li> 슬라이드에 젤을 전송합니다. 곡선을 사용하여 반 겔을 잘라D 수술 블레이드. 절반으로 처리를 계속 조직 학적 분석을 위해 나머지 절반을 저장합니다. </li><li> 전체 슬라이드에 마운트의 절반 젤을 전송합니다. 5 분 동안 젤 건조하자 다음 10 % 포르말린을 포함하는 코 플린 항아리에 슬라이드를 전송합니다. 참고 : 모든 배양은 실온에서 수행해야합니다. </li><li> 통 O의 /의 N에 코 플린 항아리에 젤을 수정합니다. </li><li> 세척 통에 10 분마다 두 번 PBS와 슬라이드. 전송은 70 %의 EtOH에 슬라이드; 통에 1 시간 동안 품어. 카마 졸업생 O / N로 전송 슬라이드. </li><li> 다음 날, 1 시간마다 70 % EtOH로 95 % EtOH로 100 %의 EtOH에 통에 슬라이드를 탈수. 이전 두 번 1 시간마다 자일 렌 통에 유지하는 슬라이드. 젤 1.5 mm 두께 된 커버를 탑재 톨루엔 기반 장착 매체를 사용합니다. </li><li> 일반 빛 (현미경 입체경)에서 전체 마운트를 검사합니다. 형상 및 루멘의 자세한 분석을 위해 공 초점 현미경을 사용하여 633 nm의 오펜 레이저와 카민 염료를 시각화. </li></ol>조직학 분석 처리 4. 젤 처리 <ol><li> 10 % 포르말린을 함유하는 20 mL 유리 바이알에 겔의 나머지 절반을 전송하고 진탕 O / N에 고정한다. </li><li> 통에 10 분마다 PBS로 두 번 젤을 씻으십시오. 파라핀 삽입에 대한 처리 카세트에 배치 젤. </li><li> 70 %의 EtOH로 전송 겔은, 1 시간 동안 배양하고 1 시간 동안 95 % EtOH로 다음 1 시간 동안 신선한 95 % EtOH로 변경, 100 %의 EtOH를 1 시간, 1 시간 동안 신선한 100 %의 EtOH로 변경합니다. 1 시간 동안 신선한 크실렌 다시 이때 겔은 실온에서 100 % EtOH로 O / N에 저장 될 수 있거나, 1 시간 동안 배양하고, 크실렌. </li><li> 전사 카세트 회 이상 신선한 파라핀 반복 진공 배양기에서 60 ℃에서 1 시간 동안 파라핀. </li><li> 깨끗한 파라핀 플라스틱 금형을 입력합니다. 카세트를 열고 젤을 제거하고 플라스틱 금형 포함 된 파라핀에 넣습니다. 파라핀과 함께 작성하고 상단에 라벨을 포함하는 카세트의 상단을 추가하고 블록이 식지. </li><li> 비접촉식를 제거IC에서 금형 및 섹션 마이크로톰을 사용하여 블록. 그는 염색 및 면역 세포 5 음 두꺼운 부분을 얻습니다. </li></ol> 콜라게나 치료를 사용하여 젤의 세포에서 세포의 5. 추출 <ol><li> 웰에서 겔을 제거하고 PBS를 함유하는 페트리 접시로 옮긴다. 린스 젤 잠시. </li><li> 0.125 % 콜라게나 제 2 ㎖를 함유하는 15 ㎖의 튜브 겔을 전송. 작은 조각으로 젤을 파괴하기 위해 최대 피펫 아래로 4-5 시간. </li><li> 25 분 동안 37 ° C에서 품어 (겔 용해 세포 현탁액에 때까지) </li><li> 혈청 보충 배지 2 ㎖를 추가합니다. 원심 분리 (1,000 XG, 5 분), 폐기 뜨는하여 세포를 수집, 세포를 다시 일시 중지하고 계산합니다. </li></ol>

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The authors have nothing to disclose.

Here, we describe a hormone-sensitive 3D culture model to test the action of hormones on breast epithelium. The response to hormones can be assessed at the tissue morphology, cell proliferation and gene expression levels 12. One limitation of this technique is that visualization during the culture period is restricted to light microscopy since the cultures are grown in a plastic bottom plate. The 3D culture system could be adapted to glass bottom plates to allow for live imaging of the cultures 1.</...

표준 2D 배양과는 달리, 3 차원 세포 배양 대리 모델 생리 관련 컨텍스트 번 유사한 조직에서 상피 세포 행동 연구 허용한다. 유선의 3D 문화는 유선 개발 및 신 생물의 여러 측면을 명료하게 도왔다. 그러나, 현재 3D 배양 모델의 대부분은 작업에 사용되는 인간 상피 세포주 호르몬 수용체 발현 6,7,9 부족하기 때문에 호르몬 작용을 연구하는 것이 적합하다. 여기서, 우리는 호르몬 작용 (12) 연구에 적합 인간의 유방 상피 세포의 3 차원 문화 모델을 설명합니다. 이 모델은 원래 유방 침투 유관 암종 54 세의 여성 환자로부터 얻은 흉수에서 파생 된 시판 호르몬 응답 인간 유방 상피 세포주 T47D 3,11,13을 사용한다. 우리는 매트릭스 쥐 꼬리 콜라겐 타입 1을 사용합니다. 이 3D 숭배URE 모델은 인간 유방 상피 세포에 대한 세 가지 mammotropic 호르몬 (에스트라 디올, promegestone (황체 호르몬의 유사체) 및 프로락틴)의 작용의 연구에 적합하다. 호르몬에 의한 상피의 형태는 형태 계측 학적 분석 (12)에 의해 시간이 지남에 따라 정량적으로 평가 될 수있다. 적절한 파종 밀도는 이러한 3D 문화 2 주 동안 유지 될 수 있습니다. 현재까지 구조물의 발달은 상피 형태에서 호르몬 작용 강력한 정량 평가 충분하다. 겔은 세포 증식에​​ 대해 이전 시점에서 수확 될 수 있고, 유전자 발현 분석. 또한,이 모델은 순차 호르몬 치료의 효과를 시험하기에 적합하다; 예를 들어, 다음 주 중에 호르몬의 다른 호르몬 / 조합으로 첫 주 동안 에스트라 디올 치료 및 교체 후. 예컨대 ICI 182,780 같은 항 에스트로겐 화합물 및 에스트로겐의 효과도 STU 수이 3D 문화 모델 (12)를 사용하여 죽었다.

<table border="0" cellpadding="0" cellspacing="0" width="1344">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">12-well Tissue Culture Plates (Falcon)</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">08-772-29</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">15 ml polystyrene conical Tubes</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">14-959-49D</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Activated Charcoal</td>
			<td style="width:249px;">Sigma</td>
			<td style="width:171px;">C-5510</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Carmine Alum</td>
			<td style="width:249px;">Sigma</td>
			<td style="width:171px;">C1022-100G</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Collagenase, Type 3</td>
			<td style="width:249px;">Worthington</td>
			<td style="width:171px;">S0C11784</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Confocal Microscope</td>
			<td style="width:249px;">Zeiss</td>
			<td style="width:171px;">LSM510</td>
			<td style="width:488px;">Equiped with HeNe 633nm laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Dextran T-70</td>
			<td style="width:249px;">Abersham/Pharmacia</td>
			<td style="width:171px;">17-0280-01</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">DMEM/F-12, HEPES, no phenol red&#160;</td>
			<td style="width:249px;">Life Technologies</td>
			<td style="width:171px;">11339-021</td>
			<td style="width:488px;">Phenol red-free media for hormone use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:436px;">DMEM, low glucose, pyruvate, no glutamine, no phenol red</td>
			<td style="width:249px;">Life Technologies</td>
			<td style="width:171px;">11054-020</td>
			<td style="width:488px;">Phenol red-free media for hormone use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">17-&#946;-Estradiol</td>
			<td style="width:249px;">EMD Millipore</td>
			<td style="width:171px;">3301</td>
			<td style="width:488px;">Dissolved in Ethanol</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Ethanol</td>
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			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Fetal Bovine Serum</td>
			<td style="width:249px;">Hyclone</td>
			<td style="width:171px;">SH30070.03</td>
			<td style="width:488px;">For use with hormones, must be Charcoal Dextran stripped&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:436px;">Filters (115ml)</td>
			<td style="width:249px;">Nalgene</td>
			<td style="width:171px;">380-0080, 245-0045, 120-0020</td>
			<td style="width:488px;">0.88, 0.45, 0.20 micron, respectively&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Formalin, 10%</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">SF93-20</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">L-Glutamine (200 mM)</td>
			<td style="width:249px;">Life Technologies</td>
			<td style="width:171px;">25030-081</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">ICI 182,780 (fulvestrant)</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">I4409-25MG</td>
			<td style="width:488px;">Dissolved in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Microtome&#160;</td>
			<td style="width:249px;">Leica</td>
			<td style="width:171px;">RM2155</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Tissue embedding media</td>
			<td style="width:249px;">McCormick Scientific</td>
			<td style="width:171px;">39502004</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Penicillin</td>
			<td style="width:249px;">Sigma</td>
			<td style="width:171px;">7794-10MU</td>
			<td style="width:488px;">Dissolved in 10 ml of distilled deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Permount</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">SP15-500</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Phosphate Buffered Saline pH 7.4</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">P3813-10PAK</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Prolactin</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">L4021-50UG</td>
			<td style="width:488px;">Dissolved in distilled deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Promegestone</td>
			<td style="width:249px;">Perkin Elmer</td>
			<td style="width:171px;">NLP004005MG</td>
			<td style="width:488px;">Dissolved in Ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Rat-Tail Collagen&#160;</td>
			<td style="width:249px;">Corning&#160;</td>
			<td style="width:171px;">354236</td>
			<td style="width:488px;">Lots may contain varying concentrations, note accordingly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Scalpel</td>
			<td style="width:249px;">Miltex</td>
			<td style="width:171px;">4311</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Semi-enclosed Benchtop Tissue Processor</td>
			<td style="width:249px;">Leica</td>
			<td style="width:171px;">TP1020</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Sodium Hydroxide</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">S5881</td>
			<td style="width:488px;">Prepare 1N NaOH stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">StaticMaster Anti-static brush&#160;</td>
			<td style="width:249px;">Amstat</td>
			<td style="width:171px;">C3500</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Stripette Serological Pipettes&#160;</td>
			<td style="width:249px;">Corning&#160;</td>
			<td style="width:171px;">4101</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">T-25 flasks</td>
			<td style="width:249px;">Corning&#160;</td>
			<td style="width:171px;">430168</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Tissue Cassettes</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">15-200-403E</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Wheaton Vials, Glass, 20mL</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">03-341-25D&#160;</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Xylene&#160;</td>
			<td style="width:249px;">VWR</td>
			<td style="width:171px;">95057-822</td>
			<td style="width:488px;"></td>
		</tr>
	</tbody>
</table>

a hormone-responsive 3d culture model of the human mammary gland epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

도 1은 호르몬 성 3 차원 배양 물을 제조하는 절차를 요약 한 것이다. 상피 구조 단독 E2의 존재하에 2 주 동안 배양 및 다른 호르몬과 조합하여 겔 마운트 전체에서 관찰된다. 어떤 호르몬은 배양 배지 (CDFBS 매체) (그림 2)에 추가되지 않은 경우에만 단일 세포 또는 2-3 셀 그룹이 존재한다. 이 상태는 음성 대조군으로 작용한다. 모양, 크기, 루멘 존재 다...

Watch this Scientific Journal Video about A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium at JoVE.com

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

인간의 유선 상피의 호르몬 응답 3D 문화 모델

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

a-hormone-responsive-3d-culture-model-human-mammary-gland

Research

JoVE Journal

Bioengineering

8.7K 조회수.  Tufts University School of Medicine. We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

비디오: A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

3D Spheroids의 생성을위한 간단한 한깅 드롭 세포 배양 프로토콜

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

연구

생체공학

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

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

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

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

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

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

인간의 3 차원의 조직 공학<em&gt; 시험관</em&gt; 종양 테스트 시스템

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

3 차원 조직 공학 인간의 식도 점막 모델의 제조, 특성 및 사용 가능성

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D 상호 작용의 연구에 대한 3D 콜라겐 젤과 마이크로 채널 준비<em&gt; 생체</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3 차원 세포 배양을위한 폐 세포 외 매트릭스에서 조정 히드로 겔

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

토끼 유선으로 관내 배달

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

인간의 췌 장 신경 섬 네트워크의 고해상도 3D 이미징

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3 차원 동적 문화 시스템과 다층된 중간 엽 줄기 세포 시트의 건설

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

유 방 땀 샘에서 조사 된 유기 체의 성장과 특성화

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

인간의 유선 상피의 호르몬 응답 3D 문화 모델

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