Name: transfer of mammary gland-forming ability between mammary basal epithelial cells and mammary luminal cells via extracellular vesicles/exosomes
Uploaded: 2017-06-03T14:00:00.000+00:00
Duration: 9 min 59 s
Description: Watch this Scientific Journal Video about Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes at JoVE.com

이 연구는 국립 보건 연구원 (05A1-CSPP16-014, HJL)과 과학 기술부 (MOST 103-2320-B-400-015-MY3, HJL)의 연구비 지원에 의해 뒷받침되었다.

동물과 관련된 모든 연구는 동물 관리위원회 (Institutional Animal Care Committee)가 승인 한 의정서를 준수했습니다. 1. 세포 외 소포 / 엑소 좀 격리 및 검증 <ol><li> 문화 유방 상피 기저 세포, NAMECs, 500 mL의 MCDB 170, pH 7.4 + 500 mL의 중탄산 나트륨 (0.2438 %)을 함유 한 DMEM / F12로 제조 된 신선한 무 혈청 배지; EGF (5 ng / mL); hydrocoritisone (0.5 μg / mL); 인슐린 (5 μg / mL); 소 뇌하수체 추출물 (BPE; 35㎍ / mL); 및 GW627368X (1 μg / mL)를 15 cm 접시에 넣었다. </li><li> hemocytometer로 세포를 계산 후 4 일 동안 0 일째에 15cm dish 당 12mL의 배지에 1.2x10 6 세포를 사육한다. </li><li> 배양 4 일 후 탁상용 원심 분리기를 사용하여 300 xg에서 5 분간 원심 분리한다. 상등액을 원추형 튜브 ( 그림 1 )로 옮긴다. </li><li> 상등액을 원심 분리한다.탁상용 원심 분리기에서 2,000 xg에서 20 분간. 뜨는 원심 분리기 튜브에 뜨는를 전송하고 죽은 세포와 세포 잔해를 남겨주세요 ( 그림 1 ). </li><li> 4 ℃에서 30 분 동안 10,000 xg에서 상층 액을 원심 분리한다. 상등액을 새로운 초 원심 분리기 튜브 ( 그림 1 )로 옮긴다. </li><li> 4 ℃에서 60 분 동안 110,000 xg에서 상층 액을 원심 분리한다. 뜨는을 제거하고 PBS ( 그림 1 )에서 EV / exosome 펠렛을 resuspend. </li><li> 4 ℃에서 60 분 동안 110,000 xg에서 상층 액을 원심 분리한다. 뜨는을 제거합니다. PBS에 EV / exosome 펠렛을 Resuspend ( 그림 1 ). 100 μL에서 NAMEC - 조건 매체 240-480 ML에서 격리 된 펠렛을 Resuspend. </li><li> BCA 단백질 분석으로 EV 현탁액의 단백질 농도를 측정하십시오. 농도가 20-40 μg / 100 μL 정도인지 확인하십시오. -20 ℃에서 보관추가 분석. </li><li> 이전에 Gardiner 등이 설명한 것처럼 나노 입자 추적 분석 (NTA)을 통해 EVs / exosomes의 농도와 크기를 측정하십시오 . 11 . NTA 분석을 위해 PBS로 EVs / exosomes (20 μg / 100 μL)를 10,000 배로 희석합니다. 참고 : NTA 분석의 결과는 분석 된 소포의 수와 크기를 반영합니다. </li><li> 린 (Lin) 등 4 에 의해 이전에 기술 된 바와 같이, 투과 전자 현미경 (TEM)으로 EVs / 엑소 좀을 이미지화한다. </li></ol> 2. 밀도 구배를 이용한 Exosome 정제 <ol><li> PBS (2 mL) 중 40 % (w / v) iodixanol에서 1.7 단계의 110,000 g 펠렛을 재현 탁한다. Ultracentrifuge 튜브에 밀도 기울기를 형성하기 위해 PBS (각각 2 mL)에 30 %, 20 %, 10 % 및 5 % (w / v) iodixanol의 분량으로 혼합물을 중첩하십시오. </li><li> 4 ° C에서 8 시간 동안 200,000 xg에서 혼합물을 원심 분리하십시오. </li><li> 각 그래디언트 분율 (10도 프랙털ons; 1 mL / 분획)을 피펫으로 튜브의 꼭대기에서 꺼냈다. </li><li> SDS-PAGE 12 및 Western blot으로 각 분획에서 exosome marker ( 예 : CD81, CD9, CD63 및 Tsg101)의 존재를 분석합니다. 0.1 % (w / v) SDS를 포함하는 10 % 겔에 각 분획의 50 μL 현탁액을 적재하고 겔 전기 영동으로 분획으로 단백질을 분리한다. </li><li> 단백질을 겔에서 PVDF 막으로 옮기고 exosome 마커 ( 예 : CD81, CD9, CD63 및 Tsg101)에 대한 항체와 멤브레인을 4 ℃에서 하룻밤 동안 보관하십시오 (재료 표 ). 참고 : 결과는 exosomes를 포함하는 분획을 식별합니다. </li></ol> 3. 세포 외 소포 / 엑소 좀 표지 <ol><li> 엑소 솜 단백질 20 μg / 100 μL에서 10 μM carboxyfluorescein succinimidyl diacetate ester (CFSE)에 1.7 단계에서 얻은 EVs / exosomes를 정지시킨다. 병렬 샘플 공동 작성동일한 방식으로 처리 된 CFSE 및 PBS만이 나중에 EV / 엑소 좀 섭취 검정에 대한 음성 대조군으로 사용된다. 30 분 동안 37 ° C에서 혼합물을 둡니다. </li><li> EV 50 분의 PBS에서 EVs / exosomes를 일시 중지하고 4에서 60 분 동안 110,000 xg에서 현탁액을 원심 분리하십시오 ° C. 뜨는를 제거하고 PBS에 EV / exosome 펠렛을 resuspend. 한 번 단계 3.2를 반복하십시오. </li><li> exosomal 단백질 / 100 μL 20 μg의 농도에서 PBS에 EVs / exosomes을 일시 중지하고 세포에 EVs / exosomes를 추가하기 전에 0.22 μm의 세포막을 통해 EVs / exosomes를 필터링합니다. </li></ol> 4. 세포 외 소포 / 엑소 좀 흡수 분석법 <ol><li> 배양 배지를 만들기 위해 MCDB 170, pH 7.4 + 500 mL의 DMEM / F12를 중탄산 나트륨 (0.2438 %)과 혼합한다. EGF (5 ng / mL); hydrocoritisone (0.5 μg / mL); 인슐린 (5 μg / mL); 및 BPE (35 μg / mL) 4 . 6 잘 접시에 인간 mammary 상피 HMLE 세포를 플레이트 (1 × 10 6 &lt;/ sup&gt; 세포 / 웰) EV / 엑소 좀 처리 하루 전에. 2-6 H 조 HMLE 세포의 문화 매체에 단계 3.3에서 얻은 2 μg / ML CFSE 형광 라벨 EVS / exosome을 추가합니다. 단계 3.1에서 설명한 병렬 준비와 함께 부정적인 제어 그룹의 HMLE 세포를 처리합니다. </li><li> 2-6 시간 배양 후 실온에서 PBS 4 mL로 세포를 두 번 씻어 낸다. </li><li> 10 분 0.25 % 트립신으로 세포를 분리하고 0.2 % FBS를 포함하는 PBS에 세포를 다시 일시 중지합니다. 형광 셀 분석기 4 를 사용하여 세포의 형광 강도에서 EV / exosome 이해를 측정합니다. 공 촛점 현미경을 사용하여 EVs / exosomes 또는 부정적인 컨트롤로 치료 세포를 이미지. 참고 : 세포의 녹색 형광은 EV / exosome 섭취로 인해 발생합니다. 현미경 설정에 대해서는 그림 6 의 범례를 참조하십시오. </li></ol> 5. 일차 마우스 유방 상피 세포의 분리 <ol><li&gt; 10cm 접시에 0.1 % 젤라틴 용액 12ml를 넣어 젤라틴 코팅 요리를 준비하십시오. 30 분 동안 37 ° C 배양기에 접시를 놓습니다. 젤라틴 코팅이 건조 될 때까지 젤라틴 용액을 제거하고 4 시간 동안 층류 후드에서 뚜껑을 접시에서 내려 놓습니다. </li><li> 가위를 사용하여 12 주 된 처녀 암컷 C57BL / 6 마우스에서 2, 3, 4, 5 번 유방 땀샘 ( 그림 2 )을 해부하고 면도기를 사용하여 작은 조각 (2mm 2 )으로 땀샘을 자릅니다. </li><li> 추가로 0.2 % 콜라게나 제 타입 IV, 0.2 % 트립신, 5 % FBS, 5 μg / ML gentamycin을 포함하는 DMEM / F12 50 ML와 37 ° C, 120 rpm의 교반에서 60 분 (10 생쥐) 유방 땀샘의 해리 , 1x pen-strep. </li><li> 10 분 동안 350 XG에서 원심 분리하여 혼합물에서 상피 organoids를 펠렛. </li><li> 상온에서 5 분 동안 0.1 MG / ML DNase I와 DMEM / F12의 4 ML에 상피 organoids를 중단. 최종 6 ML의 DMEM / F12를 추가10 mL의 부피. </li><li> 현탁액을 실온에서 10 분 동안 400 xg에서 원심 분리하고 상등액을 버린다. </li><li> 10 ML의 DMEM / F12에 펠렛을 Resuspend. 서스펜션을 원심 분리하지만 속도가 400 x g에 도달하면 브레이크를 맞 춥니 다. 뜨는 물을 버린다. 참고 : 브레이크를 치면, 상피 organoids 신속하게 pelleted, fibroblasts, 하나의 세포로, 여전히 뜨는에 머물러 있습니다. </li><li> 5.6 단계와 5.7 단계를 5-7 회 반복하십시오. hemocytometer에 정지 현탁액을 추가하고 원심 분리 ( 그림 3 ) 각 라운드 후 현미경하에 organoid 혼합물에서 fibroblasts의 정리를 확인하십시오. </li><li> 1x ITS, 5 % FBS, 50㎍ / mL gentamycin, 10ng / mL EGF 및 1x 펜 - 스트렙토닌을 함유하는 DMEM / F12로 단계 5.1에서 얻은 젤라틴 코팅 된 접시에 유기체를 플레이트한다. </li><li> 48 시간 후, FBS없이 새로운 배지로 배지를 교체하여 배양 물에서 부유 세포를 제거한다1 x ITS, 50 μg / mL gentamycin, 10 ng / mL EGF 및 1x pen-strep를 함유하는 DMEM / F12). </li><li> 유방 상피 세포의 단일 층이 3 일 안에 부착 된 상피 기관 종에서 이동 및 성장한다는 것을 확인하십시오. </li></ol> 6. 일차 마우스 기초 / Luminal 유방 상피 세포의 분리 <ol><li> 3 일 배양 후, 단백질 분해 및 콜라겐 분해 효소 활성을 갖는 천연 효소 혼합물을 마우스 기본 유방 세포에서 20 분 동안 분리한다. PBS에서 5 % FBS로 효소 활성을 중성화하십시오. </li><li> 현탁액을 실온에서 10 분간 450 xg으로 원심 분리하고 상등액을 버린다. 20 분 동안 5 MG / ML dispase에 세포 펠렛을 Resuspend. PBS에서 5 % FBS로 효소 활성을 중성화하십시오. </li><li> 현탁액을 실온에서 10 분간 450 xg으로 원심 분리하고 상등액을 버린다. </li><li> 희석과 10 7 세포 / ML의 농도 0.2 % FBS와 100 μL PBS에 세포를 다시 정지항 -CD49f 및 항 -EpCAM 항체를 얼음에서 1 시간 동안 어둠 속에서 처리 하였다. </li><li> PBS로 세포를 씻어 낸 다음 어둠 속에서 30 분 동안 얼음 위에 형광 물질이 결합 된 2 차 항체로 세포를 부화시킵니다. </li><li> 0.2 % FBS로 PBS에서 세포를 세척하고 재현 탁한다. </li><li> EpCAM hi / CD49f lo luminal mammary 상피 세포를 세포 분류기 4로 분류하십시오. </li></ol> 7. 세포 외 소포 / 엑소 좀 처리 <ol><li> 젤라틴 코팅 6 잘 요리 (2 X 10 5 세포 / 음)에 단계 6.7에서 정렬 EpCAM 안녕 / CD49f lo luminal mammary 상피 세포 씨앗 후 PBS 또는 2 μg / ML NAMEC 파생 EVs / exosomes로 그들을 치료 1.7 단계에서 얻은 </li><li> 장기간의 배양 처리에서 EV / 엑소 좀의 생물학적 효능을 보장하기 위해 PBS 또는 2 μg / mL NAMEC 파생 EVs / 엑소 좀을 함유하는 새로운 배지로 세포 배양 배지를 교체하십시오. 마우스 prima를 분할하지 마라.10 일간의 치료 중 luminal cell을 관찰 하였다. </li></ol> 유방 상피 세포의 지방 패드 주입 <ol><li> 3 주된 여성 C57BL / 6 마우스를 isofluorane 2-3 % 흡입제로 마취하십시오. </li><li> 마취 된 마우스를 뒤쪽에 놓습니다. 중년 복부 면도기 / 모발 크림을 사용하여 모피를 제거하고 70 % 알콜 및 포비돈 요오드 3 회 교대로 수술 부위를 닦으십시오. </li><li> 가위로 복부 흉부 - 사타구니 부위를 따라 피부를 통해 1.5 cm 수직 절개를하고 오른쪽 및 왼쪽 4 번째 유방 지방 패드를 교대로 노출시킵니다. </li><li> 유선 실질을 가위로 제거하여 지방 패드를 모두 지 웁니다. 지방 패드에서 림프절을 찾은 다음 림프절 아래의 전체 샘 실질을 제거하십시오. 참고 : 지방 패드의 3 분의 2는 그대로 두어야합니다. </li><li> 단계 7.2에서 얻은 세포를 천연 효소 혼합물 ( 물질 표 참조)로 단백질 분해성 a콜라겐 분해 효소 활성 10 분. PBS에서 5 % FBS로 효소 활성을 중성화하십시오. </li><li> 현탁액을 실온에서 10 분간 450 xg으로 원심 분리하고 상등액을 버린다. hemocytometer로 세포를 세고 15 μL에 원하는 세포 복용량 (10 4 -10 2 세포 / 패드)이 포함 된 농도의 PBS에 세포를 정지시킵니다. </li><li> 27G 바늘에 부착 된 100 μL 유리 주사기를 사용하여 지방 패드에 유방 상피 세포 현탁액의 15 μL를 주입. </li><li> 다른 쪽의 지방 패드에 대해 8.4-8.7 단계를 반복하십시오. </li><li> 상처 클립으로 피부를 닫으십시오. </li></ol> 9. 유선 전체 마운트 <ol><li> 세포 주입 후 8 주째에 이산화탄소와 자궁 경부 탈구로 마우스를 안락사하십시오 (단계 8.7). </li><li> 가위를 사용하여 흉부 영역에서 사타구니 영역으로 피부 레이어를 통해 수직 절개를 만들고 오른쪽과 왼쪽 모두를 노출 4 분ammary 지방 패드. 네 번째 유방 땀샘을 제거하십시오 ( 그림 2 ). </li><li> 유리 현미경 슬라이드에 지방 패드를 퍼지하고 하룻밤 실온에서 Kahle의 고정액 (4 % 포름 알데히드, 30 % EtOH 및 2 % 빙초산)로 지방 패드를 고정합니다. 주의 : Kahle의 정착액은 자극제입니다. 화학 물질 후드에서이 단계를 수행하십시오. </li><li> 지방 패드를 50 분간 70 % EtOH 250 mL에 넣고 15 분 동안 씻은 다음 5 분 동안 250 mL의 dH 2 O에서 씻으십시오. 살찐 명반을 실온에서 하룻밤 동안 밤새 카민의 명반 (카민 1g과 황산 칼륨 2.5g을 dH 2 O 500mL에 넣음)으로 염색합니다. </li><li> 지방 패드를 15 분 동안 250 mL의 70 % EtOH, 15 분 동안 95 % EtOH 250 mL 및 15 분 동안 100 % EtOH 250 mL로 씻으십시오. </li><li> 뚱뚱한 패드가 투명하게 될 때 일 동안 자일 렌에있는 뚱뚱한 패드를 청소하고 자일 렌 부화를 멈추십시오. 주의 : 크실렌은 자극제입니다. 화학 물질 후드에서이 단계를 수행하십시오. </li><li> 슬라이드 마운트하기디지털 슬라이드 스캐너를 사용하여 지방 패드의 이미지 (2,400 dpi)를 촬영합니다. </li></ol>

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저자는 공개 할 것이 없습니다.

엑소 좀은 종종 그것들을 방출 한 세포의 특성을 가지고 있으며 방출 된 엑소 좀의 양은 자극 4 에 의해 유도 될 수있다. 세포의 배양 배지를 수집하여 EV / exosome 수집을위한 차동 초 원심 분리 (differential centrifuge)에 적용 할 수 있습니다 ( 그림 1 ). 현재 EVs / exosomes를 분리하는 이상적인 방법에 대한 일반적인 합의는 없다. 여기에서 사용 된 최적의 방법은 다?...

Exosomes는 막과 세포질 단백질, 지질 및 RNA를 세포간에 전달하여 세포 간 통신을 매개 할 수 있습니다 1 . Exosome-mediated communication은 많은 생리 학적 및 병리학 적 과정 ( 즉, 항원 제시, 내성 2 의 발달 및 종양 진행 3 )에 관여하는 것으로 입증되었습니다. 엑소 좀은 종종 그것들을 방출하는 원천 세포와 비슷한 내용을 가지고있다. 따라서, exosomes은 소스 세포에서 특정 세포 속성을 수행하고 4 그들을 섭취 세포 에이 특성을 전송할 수 있습니다. Exosomes는 50 ~ 150 nm의 이중층 멤브레인 베 시클이며 특정 마커 ( 예 : CD9, CD81, CD63, HSP70, Alix 및 TSG101)를 나타냅니다. 따라서, 엑소 좀은 다양한 측면에 대한 다양한 방법으로 특징 지어 져야한다. 투과 전자 현미경으로 막 소포를 시각화 할 수 있습니다.엑소 좀 4 , 5 와 같은 나노 입자 추적 분석 (NTA)과 동적 광산란 분석 (DLS)은 정제 된 엑소 좀의 크기와 수를 측정하는 데 사용됩니다 4 . 엑소 좀의 지질막 함량은 밀도 구배로 확인할 수 있습니다. CD9, CD81, CD63, HSP70, Alix 및 TSG101 6 , 7 같은 엑소 좀 마커는 웨스턴 블 랏팅으로 측정 할 수 있습니다. 유방 기저 세포는 유방 패드에 이식 될 때 유선을 생성 할 수있는 능력이 있지만 luminal 세포는 8 , 9 , 10 일 수 없습니다. 따라서, 유방 기초 세포는 유방 재 분산 단위라고도합니다. 유방 기저 세포 및 내강 세포의 모델을 사용하여 EVs / 엑소 좀이 다른 세포 집단간에 세포 특성을 전달하는 능력을 검사 할 수 있습니다. 이 일유방 기초 상피 세포에서 유래 한 EVs / exosomes를 이용하여 유방 기초 상피 세포에서 유방 내피 세포로 유선 형성능을 전달하는 방법을 보여줍니다. 근위 유방 상피 세포는 기저 세포에서 분비 된 EV / 엑소 좀을 섭취 한 후 기저 세포 특성을 획득하고 유방 땀 샘을 형성 할 수 있습니다 4 .

<table><tbody><tr><td>MCDB 170&amp;nbsp;</td><td>USBiological</td><td>M2162</td><td></td></tr><tr><td>DMEM/F12</td><td>Thermo</td><td>1250062</td><td></td></tr><tr><td>Optima L-100K ultracentrifuge</td><td>Beckman</td><td>393253</td><td></td></tr><tr><td>SW28 Ti Rotor</td><td>Beckman</td><td>342204</td><td></td></tr><tr><td>SW41 Rotor</td><td>Beckman</td><td>331306</td><td></td></tr><tr><td>NANOSIGHT LM10</td><td>Malvern</td><td>NANOSIGHT LM10</td><td>for nanoparticle tracking analysis (NTA)</td></tr><tr><td>Optiprep&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D1556</td><td>60% (w/v) solution of iodixanol in water (sterile).</td></tr><tr><td>CD81 antibody</td><td>GeneTex</td><td>GTX101766</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CD9 antibody</td><td>GeneTex</td><td>GTX100912</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CD63 antibody</td><td>Abcam</td><td>Ab59479</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>TSG101 antibody</td><td>GeneTex</td><td>GTX118736</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>GAPDH</td><td>GeneTex</td><td>GTX100118</td><td>1:6,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CFSE (carboxyfluorescein succinimidyl diacetate ester)</td><td>Thermo</td><td>V12883</td><td></td></tr><tr><td>FACSCalibur</td><td>BD Biosciences</td><td></td><td>fluorescence cell analyzer</td></tr><tr><td>collagenase Type IV&amp;nbsp;</td><td>Thermo</td><td>17104019</td><td></td></tr><tr><td>trypsin</td><td>Thermo</td><td>27250018</td><td></td></tr><tr><td>&amp;nbsp;ITS</td><td>Sigma-Aldrich</td><td>I3146</td><td>a mixture of recombinant human insulin, human transferrin, and sodium selenite</td></tr><tr><td>accutase</td><td>ebioscience</td><td>00-4555-56</td><td>a natural enzyme mixture with proteolytic and collagenolytic enzyme activity</td></tr><tr><td>dispase&amp;nbsp;</td><td>STEMCELL</td><td>7913</td><td>5 mg/mL = 5 U/mL</td></tr><tr><td>anti-CD49f antibody</td><td>Biolegend</td><td>313611</td><td>1:50</td></tr><tr><td>anti-EpCAM antibody</td><td>Biolegend</td><td>118213</td><td>1:200</td></tr><tr><td>FACSAria</td><td>BD Biosciences</td><td></td><td>cell sorter</td></tr><tr><td>carmine alum</td><td>Sigma-Aldrich</td><td>C1022</td><td></td></tr><tr><td>human mammary epithelial cells (HMLE cells, NAMECs)</td><td></td><td></td><td>gifts from Dr. Robert Weinberg</td></tr><tr><td>permount</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>SP15-500</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Zymeset&amp;nbsp;</td><td>BSB101</td><td></td></tr><tr><td>EGF</td><td>Peprotech</td><td>AF-100-015</td><td></td></tr><tr><td>Hydrocoritisone</td><td>Sigma-Aldrich</td><td>SI-H0888</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SI-I9278</td><td></td></tr><tr><td>BPE (bovine pituitary extract)</td><td>Hammod Cell Tech&amp;nbsp;</td><td>1078-NZ</td><td></td></tr><tr><td>GW627368X&amp;nbsp;</td><td>Cayman</td><td>10009162</td><td></td></tr><tr><td>15 cm culture dish</td><td>Falcon&amp;nbsp;</td><td>353025</td><td></td></tr><tr><td>table-top centrifuge</td><td>Eppendrof&amp;nbsp;</td><td>Centrifuge 3415R</td><td></td></tr><tr><td>ultracentrifuge tube</td><td>Beckman</td><td>344058</td><td></td></tr><tr><td>PBS (Phosphate-buffered saline)&amp;nbsp;</td><td>Corning</td><td>46-013-CM</td><td></td></tr><tr><td>BCA Protein Assay</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>23228</td><td></td></tr><tr><td>Transmission Electron Microscopy</td><td>Hitachi</td><td>HT7700</td><td></td></tr><tr><td>gelatin&amp;nbsp;</td><td>STEMCELL</td><td>7903</td><td></td></tr><tr><td>10 cm culture dish</td><td>Falcon&amp;nbsp;</td><td>353003</td><td></td></tr><tr><td>6-well culture dish</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>female C57BL/6 mice</td><td>NLAC (National Laboratory Animal Center</td><td></td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>BioWest&amp;nbsp;</td><td>S01520</td><td></td></tr><tr><td>gentamycin</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>15710072</td><td></td></tr><tr><td>Pen/Strep</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>DNase I</td><td>5PRIMER</td><td>2500120</td><td></td></tr><tr><td>isofluorane&amp;nbsp;</td><td>Halocarbon</td><td>NPC12164-002-25</td><td></td></tr><tr><td>formaldehyde</td><td>MACRON</td><td>H121-08</td><td></td></tr><tr><td>EtOH (Ethanol)</td><td>J.T. Baker</td><td>800605</td><td></td></tr><tr><td>glacial acetic acid</td><td>Panreac</td><td>131008.1611</td><td></td></tr><tr><td>aluminum potassium sulfate</td><td>Sigma-Aldrich</td><td>12625</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>Leica</td><td>3803665</td><td></td></tr><tr><td>0.22 &amp;mu;m membranes</td><td>Merck Millipore</td><td>Millex-GP</td><td></td></tr><tr><td>AUTOCLIP Wound Clips, 9 mm</td><td>BD Biosciences</td><td>427631</td><td></td></tr><tr><td>AUTOCLIP Wound Clip Applier</td><td>BD Biosciences</td><td>427630</td><td></td></tr><tr><td>CellMask&amp;trade; Deep Red</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>C10046</td><td>plasma membrane stain</td></tr></tbody></table>

transfer of mammary gland-forming ability between mammary basal epithelial cells and mammary luminal cells via extracellular vesicles/exosomes

세포는 단백질, 지질 및 핵산을 포함하는 ~ 100-nm 세포 외 소포 (EVs) 인 엑소 좀을 통해 전달할 수 있습니다. Non-adherent / mesenchymal mammary epithelial cell (NAMEC) 유래 세포 외 소포는 차별적 인 초 원심 분리를 통해 NAMEC 배지에서 분리 할 수 ​​있습니다. 밀도에 따라 EV는 110,000 x g에서 초 원심 분리를 통해 정제 할 수 있습니다. 초 원심 분리로 얻은 EV 준비물은 가용성 단백질에 의한 오염을 방지하기 위해 연속 밀도 구배를 사용하여 더욱 분리 할 수 ​​있습니다. 그런 다음 정화 된 EVs는 나노 입자 추적 분석을 사용하여 추가로 평가할 수 있으며, 이는 준비 과정에서 소포의 크기와 수를 측정합니다. 50 내지 150 nm 범위의 세포 외 소포가 엑소 좀이다. NAMEC에서 추출한 EVs / exosomes는 유선 상피 세포에서 섭취 할 수 있는데, 이는 유동 세포 계측법과 공 촛점 현미경으로 측정 할 수 있습니다. 일부 유방 줄기 세포 특성 ( 예 : 유선 형성 능력)은줄기 모양의 NAMEC에서 NAMEC 유래 EVs / 엑소 좀을 통해 유방 상피 세포로 전달 될 수있다. 격리 된 일차 성 EpCAM hi / CD49f lo 내시경 상피 ​​세포는 마우스 지방 패드에 이식 된 후 유방 땀샘을 형성 할 수 없으며, EpCAM lo / CD49f hi 기저 유방 상피 세포는 이식 후 유방 땀샘을 형성합니다. EpCAM hi / CD49f lo luminal mammary 상피 세포에 의한 NAMEC 유래 EVs / 엑소 좀의 섭취는 지방 패드에 이식 된 후 유방 땀샘을 생성하게한다. 줄기 모양의 유방 상피 세포로부터 유래 된 EVs / 엑소 좀은 유방 내 형성 능력을 EpCAM hi / CD49f lo 내강 유방 상피 세포로 전달한다.

PGE 2 / EP 4 신호 전달을 막음으로써 유방 기초 형 줄기 세포 4 에서 EV / 엑소 좀 방출을 유발한다는 것이 보여지기 때문에,이 연구는 유방 상피 기저 세포 (NAMEC) 배양으로부터 유도 된 EVs / 엑소 좀을 분리하는 방법을 제시한다. NAMEC은 혈청이없는 배지에서 배양되기 때문에 혈청에서 유래 된 EV / 엑소 좀은 존재하지 않는다. 혈청 함유 배지?...

Watch this Scientific Journal Video about Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes at JoVE.com

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

이 프로토콜은 non-adherent / mesenchymal 유방 상피 세포에서 세포 외 소포 (EVs) / 엑소 좀을 정화, 정량화 및 특성화하고 유선 형성능을 내유성 상피 세포로 전달하는 방법을 설명합니다. 줄기 모양의 유방 상피 세포로부터 유래 된 EVs / 엑소 좀은이 세포 특성을 EVs / 엑소 좀을 섭취하는 세포로 전달할 수있다.

extracellular vesicles / exosomes를 통한 유방 기초 상피 세포와 유방 Luminal 세포 사이 유선 형성 능력의 이전

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

transfer-mammary-gland-forming-ability-between-mammary-basal

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JoVE Journal

Developmental Biology

11.6K 조회수.  National Health Research Institutes.  이 프로토콜은 non-adherent / mesenchymal 유방 상피 세포에서 세포 외 소포 (EVs) / 엑소 좀을 정화, 정량화 및 특성화하고 유선 형성능을 내유성 상피 세포로 전달하는 방법을 설명합니다. 줄기 모양의 유방 상피 세포로부터 유래 된 EVs / 엑소 좀은이 세포 특성을 EVs / 엑소 좀을 섭취하는 세포로 전달할 수있다. 

비디오: Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

C57BL/6J 마우스의 Stromal 셀 및 암종 세포 이식 내유

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

연구

의학

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

쥐 유방 상피 세포 이식에 갑피 흰 지방 패드

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

암 연구학

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

마우스 내유 상피 세포는 Lactogenic 차별 동안 Mammospheres를 형성

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

생물학

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

유방암 연구를 위한 잠재적인 기능적인 시험관 내 모형으로 유방 상피 및 내피 세포 스페로이드

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

내유 상피 이식 절차

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

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

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

마우스 모델에서 유선 개발 및 기능 평가

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

비 효소, Mammospheres의 자연 발생에 대한 사전 침습 유방 병변의 혈청이없는 조직 문화

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

마우스 유선의 냉동 섹션에 간접 면역 형광

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

세포 외 기질 식 및 선 형태 분석을 그대로, 전체 마우스 유 방 땀 샘의 절연

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...