Name: a hormone-responsive 3d culture model of the human mammary gland epithelium
Uploaded: 2016-02-07T14:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium at JoVE.com

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.
The way in which cells are maintained appears to be critical for retention of hormone-responsive phenotypes. It is important to frequently check for E2 responsiveness in these cells. We recommend performing an E2 dose-response curve on the fourth passage after thawing a new vial, and routinely after every 10 passages thereafter. An E2 concentration of 10 -10 M will typically yield three times the control cell number in 2D culture and two times in 3D culture when compared to those grown in CDFBS medium for 1 week 12. Phenol red-free media should be used for all experiments involving hormone-responsiveness, due to the proven estrogenicity of phenol red 2. In addition, the brands listed in this protocol are current without hormonal activity. If the estrogenic response in 2D or 3D changes, the plastic materials used for tissue culture should be evaluated for leaching of hormonally-active compounds. 
The presence of static electricity during gel preparation may affect cell distribution. This may cause the cells to quickly sink to the bottom of the well during congealing and then grow as a 2D monolayer. In this case, the gel appears mostly empty of cells with them attached directly to the bottom plastic surface. To prevent this effect, it is recommended to brush the borders, top and bottom of the 12-well plate with a static-reducing brush after unwrapping the plate. To avoid static charge buildup during contact with the hood working surface, place this plate on top of another 12-well plate (blank plate) that has also been brushed with the static-reducing brush. These recommendations are especially useful during periods of low ambient humidity. 
As opposed to existing "on top" culture models9, the culture system that we describe here allows for the epithelial cells to form 3D structures which are embedded in a matrix. Furthermore, this culture system enables for the study of the influence of extracellular matrix components, such as collagen fibers, and mechanical properties, such as matrix stiffness, in hormone-instructed morphogenesis of 3D epithelial structures 1,12. 
The hormone-sensitive 3D culture model described herein also allows for the addition of cell types considered of relevance in breast tissue morphogenesis, such as fibroblasts, adipocytes and circulating blood cells 6,8,10. Finally, testing of hormonal influence on the breast epithelium in this 3D culture model is not limited to endogenous hormones alone. The response to environmental estrogenic compounds and other hormonally-active chemicals can also be tested to uncover their effects and those elicited when tested in the presence of the natural, endogenous steroidal and polypeptidic hormones.

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><tbody><tr><td>12-well Tissue Culture Plates (Falcon)</td><td>Fisher Scientific</td><td>08-772-29</td><td></td></tr><tr><td>15 ml polystyrene conical Tubes</td><td>Fisher Scientific</td><td>14-959-49D</td><td></td></tr><tr><td>Activated Charcoal</td><td>Sigma</td><td>C-5510</td><td></td></tr><tr><td>Carmine Alum</td><td>Sigma</td><td>C1022-100G</td><td></td></tr><tr><td>Collagenase, Type 3</td><td>Worthington</td><td>S0C11784</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM510</td><td>Equiped with HeNe 633nm laser</td></tr><tr><td>Dextran T-70</td><td>Abersham/Pharmacia</td><td>17-0280-01</td><td></td></tr><tr><td>DMEM/F-12, HEPES, no phenol red&amp;nbsp;</td><td>Life Technologies</td><td>11339-021</td><td>Phenol red-free media for hormone use</td></tr><tr><td>DMEM, low glucose, pyruvate, no glutamine, no phenol red</td><td>Life Technologies</td><td>11054-020</td><td>Phenol red-free media for hormone use</td></tr><tr><td>17-&amp;beta;-Estradiol</td><td>EMD Millipore</td><td>3301</td><td>Dissolved in Ethanol</td></tr><tr><td>Ethanol</td><td>Koptec</td><td>V1001</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30070.03</td><td>For use with hormones, must be Charcoal Dextran stripped&amp;nbsp;</td></tr><tr><td>Filters (115 ml)</td><td>Nalgene</td><td>380-0080, 245-0045, 120-0020</td><td>0.88, 0.45, 0.20 micron, respectively&amp;nbsp;</td></tr><tr><td>Formalin, 10%</td><td>Fisher Scientific</td><td>SF93-20</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>ICI 182,780 (fulvestrant)</td><td>Sigma Aldrich</td><td>I4409-25MG</td><td>Dissolved in DMSO</td></tr><tr><td>Microtome&amp;nbsp;</td><td>Leica</td><td>RM2155</td><td></td></tr><tr><td>Tissue embedding media</td><td>McCormick Scientific</td><td>39502004</td><td></td></tr><tr><td>Penicillin</td><td>Sigma</td><td>7794-10MU</td><td>Dissolved in 10 ml of distilled deionized water</td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-500</td><td></td></tr><tr><td>Phosphate Buffered Saline pH 7.4</td><td>Sigma Aldrich</td><td>P3813-10PAK</td><td></td></tr><tr><td>Prolactin</td><td>Sigma Aldrich</td><td>L4021-50UG</td><td>Dissolved in distilled deionized water</td></tr><tr><td>Promegestone</td><td>Perkin Elmer</td><td>NLP004005MG</td><td>Dissolved in Ethanol</td></tr><tr><td>Rat-Tail Collagen&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>354236</td><td>Lots may contain varying concentrations, note accordingly</td></tr><tr><td>Scalpel</td><td>Miltex</td><td>4311</td><td></td></tr><tr><td>Semi-enclosed Benchtop Tissue Processor</td><td>Leica</td><td>TP1020</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma Aldrich</td><td>S5881</td><td>Prepare 1N NaOH stock</td></tr><tr><td>StaticMaster Anti-static brush&amp;nbsp;</td><td>Amstat</td><td>C3500</td><td></td></tr><tr><td>Stripette Serological Pipettes&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>4101</td><td></td></tr><tr><td>T-25 flasks</td><td>Corning&amp;nbsp;</td><td>430168</td><td></td></tr><tr><td>Tissue Cassettes</td><td>Fisher Scientific</td><td>15-200-403E</td><td></td></tr><tr><td>Wheaton Vials, Glass, 20 ml</td><td>Fisher Scientific</td><td>03-341-25D&amp;nbsp;</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>VWR</td><td>95057-822</td><td></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 다를 3D 문화 양식 구조에서 셀. 겔 구조의 분포는 일반적으로 불균일하다. 이전에 나타낸 바와 같이, 중앙 4에서 볼보다 겔의 경계를 따라 일반적으로보다 구조가있다. 이 때문에 공간 이질의, 표본 간의 비교는 샘플에 걸쳐 지역 대응으로 제한되어야한다. 콜라겐 타입 1이 영향을 표현형을 유도하는 종결과 상피 구조 5 차 품질. 여기에 사용 된 쥐의 꼬리 콜라겐 타입 1은 조직을 파괴 셀 그룹 (그림 3)의 형성 결과 소 콜라겐 타입 1에 파종하기 때문에 선택되었다. 상피 구조의 상세한 분석은 공 촛점 현미경 (도 4)을 사용하여 달성 될 수있다. 둥근 긴 구조 (그림 4A)에서 E2 처리 결과. E2 더 많은 신진 구조 (그림 4B)에서 프로락틴 결과의 존재, 동안 측의 연상 세포 돌기 (그림 4C)을 분기와 불규칙한 구조에서 E2 및 promegestone 결과의 존재. Z 방향의 성장에 의해 측정했을 때, 및 E2 플러스 promegestone 관측과 비교할 때, E2 단독 및 E2 플러스 프로락틴 조직 구조를 두껍게. 호르몬 치료 후 세포 수를 정량하기 위하여, 세포는 아르 extracte콜라게나 제 처리를 사용하여 겔로부터 D. 예를 들어,이 방법을 이용하여, 우리는 항 에스트로겐 ICI 182780은 3D 배양에서 세포의 증식에 대한 E2의 효과 (도 5)를 억제하는 방법을 연구 할 수있다. <img alt="그림 1" src="/files/ftp_upload/53098/53098fig1.jpg" /> 도 호르몬 성 차원 배양의 제조 절차 1.이. 간략하게, T47D 세포의 단일 세포 현탁액을 래트 꼬리 콜라겐 타입 1로 혼합하여 12 웰 플레이트의 웰에 주입된다. 겔은 37 ℃ / 5 % CO2 배양기에서 30 분 동안 응결 수있다. 배양 배지를 첨가하고, 겔은 잘로부터 분리된다. 문화는 엔드 포인트에 따라 1 ~ 2 주 후에 수확 할 수있다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> . 카마 졸업생 염료로 염색 한 후 3D 문화의 그림 2. 전체 마운트 상피 구조, 형태 분석을위한 ROI의 예를 시각화하는 카민 명반으로 염색 반 겔 (A) 전체 마운트는 상자로 표시됩니다; 3D 문화의 WM은 2 주 (B)에 대한 성장은 E2 및 프로락틴 보충 미디어 (추가 더 호르몬)와 (C) CDFBS 미디어를 CDFBS 없습니다. 스케일 바는 5,000 μm의 (A) 500 μm의 (B &amp; C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/53098/53098fig3.jpg" /> 3D 배양에 사용되는 행렬 그림 3. 비교. (A) BOVI. 100 μm의 : 쥐의 꼬리 콜라겐 유형 1. 스케일 막대를 사용하는 경우 북동 콜라겐 유형은 조직 구조 (B)에 반대 세포 조직을 파괴 클러스터, 1 결과는 관찰 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/53098/53098fig4.jpg" /> (A) E2, (B) 및 프로락틴 E2 + (C) + E2 R5020과 이주. 처리 후 3-D 콜라겐 겔에서 관찰 T47D 구조도 4 공 촛점 이미지. 화살표는 세포질 돌기를 가리 킵니다. 상피 구조의 두께는 각각 24, 40 및 20 μm의했다. 스케일 바 :. 40 μm의 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 도 5 3D 배양 E2 단독으로 처리하거나 또는 E2 플러스 ICI 182,780 일주. 세포 수를 콜라게나 제 처리를 사용하여 세포의 추출 후. (A)는 매체 CDFBS, (B)는 -10 M, 10-7 M 1X 플러스 E2에서 1 × -10 M 및 (D)에 1 × ICI 182,780 -8 M 플러스 E2의 (C)에 1 × ICI 182,780 매체 플러스 E2를 CDFBS 1 × -10 M.에서 바 :. SEM <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53098/53098fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

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

Nanyang Technological University

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

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

마우스 유방 상피 HC11 및 EpH4 세포의 분화

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

연구

암 연구학

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

차 사람 골수 및 악성 종양을 연구하는 세 가지 차원 조직 배양 모델

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

의학

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

인지질 매개체는 3차원 배양에서 형질전환을 유도했습니다.

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

세 가지 차원 문화 : 유방 세포의 악성 변형 대 일반 선포 건축을 연구하는 도구

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

3 차원 (3D) 모델을 사용하여 유방암 세포 침윤의 정량화

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

계대증 없이 정상 및 종양 유방 조직에서 마우스 및 인간 상피 오가노이드 생성 및 이미징

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

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, ...

생체공학

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

차동 시도에 의한 모자이크 유방 오르가노이드 생성

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

발생학

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

<em&gt; 생체 내</em&gt; 모델은 인간의 유방암에서 호르몬 행동을 연구하기 위해

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

세 가지 차원 혼합 셀 회전 타원체 공동 문화에서 Mammary 상피 세포의 분리

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...