Name: primary culture of porcine retinal pigment epithelial cells
Uploaded: 2022-09-23T14:00:00
Description: Watch this Scientific Journal Video about Primary Culture of Porcine Retinal Pigment Epithelial Cells at JoVE.com

저자는이 연구에서 세포에 기여한 모든 동물에게 감사와 존경을 표하고 싶습니다. 이 연구는 중국 국가 핵심 R & D 프로그램 (2019YFA0111200, Yi Liao & Yuan Gao 및 Grant nos. 2018YFA0107301, Wei Li)의 보조금으로 부분적으로 지원되었습니다. 저자는 컨포칼 이미징에 대한 기술 지원에 대해 샤먼 대학교 의과대학 중앙 연구소의 Jingru Huang과 Xiang You에게 감사를 표합니다.

실험 동물의 사용은 시력 및 안과 연구 협회 (ARVO)의 규정을 준수했으며 샤먼 대학교 실험 동물 관리 윤리위원회의 승인을 받았습니다.
1. 실험용 수술기구, 조직소화효소, 세포배양액의 제조
<ol><li>안구 해부 전날 두 쌍의 안과 수술 가위와 집게를 세척 및 고압 멸균 한 다음 일반 프로토콜 오븐에서 65 ° C의 밤새 수술 장치로 상자를 건조시켜 실험 수술 장치를 ?...

<ol>
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여기에서는 RPE 세포의 시험관 내 특성화 및 RPE 관련 장애 연구를 위한 좋은 모델을 생성하는 돼지 안구에서 RPE 세포의 분리, 배양 및 특성화를 위한 상세하고 최적화된 프로토콜이 설명되었습니다. 인간, 마우스 및 래트 눈으로부터 RPE를 단리하기 위한 방법은 이전에23,24,25에 기술되었다. 그러나 일부 실험실에서는 사람...

망막 색소 상피(RPE)는 망막1 외층의 광수용체와 맥락모세혈관 사이에 위치하며, 혈액-망막 장벽 형성, 영양소 및 망막 대사산물 운반 및 교환, 정상적인 시각 주기를 유지하기 위한 비타민 A 재활용, 식균작용 및 흘린 광수용체 외부 분절(POS)의 제거를 포함한 여러기능을 수행합니다.2,3 . POS는 시력을 생성하기 위해 지속적인자가 갱신이 필요하기 때문에 RPE 세포는 망막 항상성을 유지하기 위해 분리 된 POS를 지속적으로 삼켜야합니다4. 따라서 RPE 기능 장애는 연령 관련 황반 변성(AMD)4, 색소성 망막염(RP)5, Leber 선천성 무균증6, 당뇨병성 망막증7 등과 같은 많은 실명 안과 질환을 유발합니다. 지금까지 이러한 질병의 정확한 발병 기전은 아직 파악하기 어렵습니다. 결과적으로 RPE 세포 생물학, 병리학적 변화 및 기본 메커니즘을 연구하기 위해 RPE 세포 배양이 확립되었습니다.
세포 생물학을 연구하는 가장 간단한 모델로서, RPE 세포의 배양은 이미 1920년대에 시작되었다8. ARPE-19가 RPE 세포로서 널리 사용되고 있지만, 색소 침착, 조약돌 형태, 특히 이 세포주의 장벽 기능의 손실은많은 우려를 불러일으킨다9. 이에 비해, 일차 인간 RPE 세포의 배양은 생리학적 및 병리학적 연구를 위한 보다 현실적인 시나리오를 제공한다9. 그러나 상대적으로 제한된 가용성으로 인해 사용이 제한되고 윤리적 문제가 항상 존재합니다. 또한 여러 그룹이 마우스 모델을 사용하여 RPE 세포를 배양했습니다. 그러나 마우스 눈의 크기가 작고 단일 배양에는 일반적으로 많은 마우스가 필요하므로 편리하지 않습니다9. 최근 과학자들은 인간 배아 줄기 세포 또는 유도 만능 줄기 세포를 사용하여 RPE 세포를 유도하는 새로운 방법을 개발했습니다. 비록 이 기술이 유전된 RPE 장애의 치료를 위한 특별한 잠재력을 갖지만, 이는 시간이 많이 걸리고 보통 성숙한 RPE 세포(10)를 생성하는데 수개월이 필요하다. 이러한 문제를 극복하기 위해 실험실에서 고순도 RPE 세포를 일상적으로 분리하고 배양하는 따라하기 쉬운 프로토콜을 소개합니다. 적절한 배양 조건 하에서, 이들 세포는 전형적인 RPE 기능을 나타내고 전형적인 RPE 형태를 나타낼 수 있다. 따라서이 배양 방법은 RPE 생리학을 이해하고, 세포 독성을 연구하고, 관련 안구 질환의 병리학 적 메커니즘을 조사하고, 약물 스크리닝을 수행하는 좋은 모델을 제공 할 수 있습니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ARPE-19 cells</td>
			<td>CCTCC</td>
			<td>GDC0323</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Yeasen</td>
			<td>36101ES60</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscopy</td>
			<td>Zeiss</td>
			<td>LSM 880 with Airyscan</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Touch</td>
			<td>Bio-Rad</td>
			<td>1708370</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Sangon</td>
			<td>F619301</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm culture dish</td>
			<td>NEST</td>
			<td>121621EH01</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well culture plate</td>
			<td>NEST</td>
			<td>29821075P</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F12 Medium</td>
			<td>Gibco</td>
			<td>C11330500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM basic Medium</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM2</td>
			<td>World Precision Instruments</td>
			<td>EVOM2</td>
			<td>For TER measurement</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ExCell Bio</td>
			<td>FSP500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Mouse IgG (H/L):HRP</td>
			<td>Bio-Rad</td>
			<td>0300-0108P</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Rabbit IgG (H/L):HRP</td>
			<td>Bio-Rad</td>
			<td>5196-2504</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrocortisone</td>
			<td>MCE</td>
			<td>HY-N0583/CS-2226</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342 solution (20 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 96 Instrument</td>
			<td>Roche</td>
			<td>5815916001</td>
			<td></td>
		</tr>
		<tr>
			<td>Liothyronine</td>
			<td>MCE</td>
			<td>HY-A0070A/CS-4141</td>
			<td></td>
		</tr>
		<tr>
			<td>laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM(1X)+GlutaMAX Medium</td>
			<td>Gibco</td>
			<td>10566-016</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAA(100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GP syringe filter unit</td>
			<td>Millipore</td>
			<td>SLGPR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>N1</td>
			<td>Sigma-Aldrich</td>
			<td>SLCF4683</td>
			<td></td>
		</tr>
		<tr>
			<td>NcmECL Ultra</td>
			<td>New Cell&#38;Molecular Biotech</td>
			<td>P10300</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat Powdered Milk</td>
			<td>Solarbio</td>
			<td>D8340</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>SparkJade</td>
			<td>SJ-MV0061</td>
			<td></td>
		</tr>
		<tr>
			<td>Na+-K+ ATPase antibody</td>
			<td>Abcam</td>
			<td>ab76020</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>PAGE Gel Fast Preparation Kit(10%)</td>
			<td>Epizyme</td>
			<td>PG112</td>
			<td></td>
		</tr>
		<tr>
			<td>primary Human RPE cells&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>Generous gift from Shoubi Wang lab&#160;</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad by Dotmatics</td>
			<td>version 8.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktails</td>
			<td>APExBIO</td>
			<td>K1024</td>
			<td></td>
		</tr>
		<tr>
			<td>PRE65 antibody</td>
			<td>Proteintech</td>
			<td>17939-1-AP</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>PEDF antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-390172</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>100 x penicillin/streptomycin&#160;</td>
			<td>Biological Industries</td>
			<td>03-031-1BCS</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>RARBIO</td>
			<td>RA-9005</td>
			<td></td>
		</tr>
		<tr>
			<td>ReverTra Ace qPCR RT Master Mix</td>
			<td>Toyobo</td>
			<td>FSQ-201</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile centrifuge tubes</td>
			<td>NEST</td>
			<td>601052</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tubes</td>
			<td>NEST</td>
			<td>602052</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Damas-beta</td>
			<td>107-35-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo-Fisher&#160;</td>
			<td>15596026</td>
			<td>RNA extraction solution</td>
		</tr>
		<tr>
			<td>TB Green Fast qPCR Mix</td>
			<td>Takara</td>
			<td>RR430A</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well transwell inserts</td>
			<td>Labselect</td>
			<td>14212</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF antibody</td>
			<td>Proteintech</td>
			<td>19003-1-AP</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>VEGF ELISA kit</td>
			<td>Novusbio</td>
			<td>VAL106</td>
			<td></td>
		</tr>
		<tr>
			<td>ZO-1 antibody</td>
			<td>ABclonal</td>
			<td>A0659</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
	</tbody>
</table>

primary culture of porcine retinal pigment epithelial cells

망막 색소 상피 (RPE)는 망막의 맥락막과 신경 망막 사이에 위치한 편광 색소 상피 세포의 단층입니다. 식균 작용, 영양소/대사 산물 수송, 비타민 A 대사 등을 포함한 여러 기능이 RPE에 의해 매일 수행됩니다. RPE 세포는 재생 능력이 거의 또는 전혀 없는 말기 분화된 상피 세포입니다. RPE 세포의 손실은 연령 관련 황반 변성과 같은 시각 장애로 이어지는 여러 안과 질환을 초래합니다. 따라서 세포주보다 RPE in vivo와 더 유사한 1차 RPE 세포의 시험관 내 배양 모델을 구축하는 것은 RPE 세포의 특성 및 기계론적 연구에 매우 중요합니다. 인간의 안구의 공급원이 제한적이라는 사실을 고려하여 우리는 1 차 돼지 RPE 세포를 배양하기위한 프로토콜을 만듭니다. 이 프로토콜을 사용하면 RPE 세포를 성인 돼지 안구에서 쉽게 분리 할 수 있습니다. 그 후, 이러한 해리된 세포는 배양 접시/삽입물에 부착되고, 증식하여 합류 단층을 형성하고, 2주 이내에 생체 내 상피 조직의 주요 특징을 빠르게 재설정합니다. qRT-PCR에 의해, 1차 돼지 RPE 세포는 천연 RPE 조직과 유사한 수준에서 여러 시그니처 유전자를 발현하는 반면, 이러한 유전자의 대부분은 인간 RPE 유사 세포인 ARPE-19에서 손실/고도로 감소한다는 것이 입증되었습니다. 또한 면역형광 염색은 배양된 일차 세포에서 긴밀한 접합, 조직 극성 및 세포골격 단백질의 분포와 비타민 A 대사에 중요한 이성질화효소인 RPE65의 존재를 보여줍니다. 전체적으로 우리는 고순도 및 천연 RPE 기능을 가진 1차 돼지 RPE 세포 배양에 대한 따라하기 쉬운 접근 방식을 개발했으며, 이는 RPE 생리학을 이해하고 세포 독성을 연구하며 약물 스크리닝을 용이하게 하는 좋은 모델이 될 수 있습니다.

1차 돼지 RPE(pRPE) 세포를 10% FBS가 있는 DMEM/Basic 배지에서 배양하고, 시딩 후 2일(그림 2A), 6일(그림 2B) 및 10일(그림 2C)에 광학 현미경으로 세포 형태를 촬영했습니다. 1주 후, 조약돌 형태를 갖는 착색된 pRPE 세포의 합류 단층이 관찰되었다.
1차 pRPE 세포의 특성을 더 잘 분석하기 위해, Passage 3(P3)18</su...

Watch this Scientific Journal Video about Primary Culture of Porcine Retinal Pigment Epithelial Cells at JoVE.com

Primary Culture of Porcine Retinal Pigment Epithelial Cells

여기서, 시험 관 내에서 일차 돼지 망막 색소 상피세포를 배양하는 방법을 제시한다.

돼지 망막 색소 상피 세포의 1 차 배양

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

RPE 관련 장애는 혼합 질환의 주요 부분으로 남아 있지만 RPE 관련 장애의 생리학적 및 병리학적 연구를 위한 좋은 모델로 작용하기 위해 1차 RPE 세포의 가상 배양에 효과적인 잔재가 없습니다. 이 프로토콜에서 우리는 1차 IPC를 배양하기 위해 따르기 쉬운 프로토콜을 제공하여 나중에 RP 특성을 신속하게 복원하고 유지할 수 있으며, 이 방법을 사용함으로써 사람들은 RPE 장애에 대한 새로운 치료법의 개발을 촉진할 수 있는 macistate 연구 및 보호 약물 스크리닝에서 RP 세포를 신속하게 생성할 수 있다고 믿습니다. 단계별 값 시연을 통해 RP 세포를 연구하려는 여러 실험실에서 이 방법을 훨씬 쉽게 복제할 수 있습니다.조직 소화 효소 말라쿼드를 해동하기 시작하고 1X PBS를 멸균한다. 2 % 페니실린 및 2 % 스트렙토 마이신으로 보충 된 0.22 마이크로 미터 주사기 필터 장치를 통해 용액을 여과합니다. 배양 플레이트와 트랜스웰 삽입물의 웰을 1X PBS로 세척합니다.피펫으로 웰 및 트랜스웰에서 PBS를 제거하고 하부 챔버에 1밀리리터의 신선한 1X PBS를 추가하고 상부 챔버에 밀리리터당 10마이크로그램의 라미닌 용액 600마이크로리터를 추가합니다. 플레이트와 트랜스웰 삽입물을 이 헬리컬처 인큐베이터에서 섭씨 37도 및 5%이산화탄소에서 밤새 배양합니다. 그 후, 상부 챔버에서 라미네이트 용액을 제거하고 셀을 안착하기 전에 차가운 1X PBS 1 밀리리터로 두 번 세척하십시오. 라미네이트 흐름 후드를 75 % 에탄올과 자외선으로 청소하십시오.약 25 밀리리터의 75 % 에탄올을 함유 한 3 개의 50 밀리리터 멸균 원심 분리 튜브, 약 25 밀리리터의 1X PBS를 포함하는 3 개의 50 밀리리터 멸균 원심 분리 튜브 및 약 15 밀리리터의 1X PBS를 포함하는 3 개의 10 센티미터 멸균 세포 배양 접시를 준비하십시오. 10 센티미터의 페트리 접시에 약 15 밀리리터의 75 % 에탄올에 4 개의 돼지 안구를 담그고 가위와 집게를 사용하여 모든 잔류 결합 조직과 근육을 잘라냅니다. 안구의 오염을 제거하기 위해 75 % 에탄올로 채워진 3 개의 50 밀리리터 멸균 원심 분리 튜브에 4 개의 안구를 담그고 순차적으로 씻으십시오.그런 다음 1X PBS로 채워진 3 개의 50 밀리리터 멸균 원심 분리 튜브에서 안구를 순차적으로 씻으십시오. 매분마다 튜브를 뒤집어 안구를 철저히 씻으십시오. 4개의 안구를 1X PBS가 포함된 10센티미터 멸균 세포 배양 접시에 옮깁니다.시신경과 작은 파편을 제거하기 위해 각 안구의 외부 표면을 다시 다듬습니다. 가위를 사용하여 윤부와 공막의 교차점에서 작은 절단을하십시오. 그런 다음 각막, 홍채, 수정체, 유리체 및 신경 망막을 제거합니다.RPE 맥락막 공막 복합체를 새로운 10cm 세포 배양 접시에 옮기고 아이 컵을 4 잎 클로버 모양으로 평평하게하기 위해 4 번 자릅니다. 4개의 RPE 맥락막 공막 복합체를 새로운 10cm 접시에 넣고 20ml의 신선한 트립신 EDTA 용액을 부어 RPE 맥락막 공막 복합체를 병합하여 EDTA 용액 분해를 수행합니다. 접시를 섭씨 37도의 세포 배양 인큐베이터에 거의 30분 동안 놓습니다.30 분 후, 인큐베이터에서 접시를 꺼내고 10 % FBS가 포함 된 예열 된 세포 배양 배지 20ml를 접시에 추가합니다. EDTA 용액에서 트립을 중화하려면 5밀리리터 전사 피펫을 사용하여 여러 번 부드럽게 피펫팅하여 RPE 세포를 해리합니다. 세포 현탁액을 15 밀리리터 원심분리 튜브에 모은 다음, FBS로 또 다른 10 밀리리터의 신선한 배양 배지를 부드럽게 피펫팅하여 접시에 있는 RPE 맥락막 공막 복합체를 세척합니다.실온에서 5분 동안 튜브를 300G으로 원심분리하여 RPE 세포를 수집합니다. 피펫을 사용하여 SuperAgent를 흡인하고 FBS로 배양 배지 5ml를 더 추가하여 세포를 다시 부유시킵니다. 300G에서 5 분 더 원심 분리합니다.SuperAgent를 디캔트하고 12ml의 배양 배지로 세포를 다시 부유시킵니다. 다음 시드 100, 000 내지 200, 000 세포를 웰 당 12 웰 배양 플레이트 또는 트랜스웰 삽입물에 넣는다. 2 일마다 배양 배지를 교체하고 세포가 삽입 당 웰 표면을 완전히 덮을 때 혈청 농도를 1 %로 줄입니다.세포가 수확 될 때까지 2 일마다 배양 배지를 교체하십시오. 2일차, 6일째 및 10일차의 1차 돼지 RPE 세포의 대표 이미지가 여기에 나와 있습니다. 일차 돼지 RPE 세포, 일차 인간 RPE 세포, ARPE 19 세포 및 돼지 RPE 맥락막 조직에서 시그니처 유전자의 mRNA 수준에 대한 QRTPCR 분석은 이 그래픽 이미지에 의해 검출됩니다.여기서 GAP DH는 RTPCR을 위한 하우스키핑 유전자로서 사용되었다. 4개의 생물학적 복제물이 1차 돼지 RPE 세포에 사용되었고, RPE 19 세포에는 3개의 생물학적 복제물이 1차 인간 RPE 세포 및 돼지 RPE 맥락막 조직에 사용되었습니다. ARPE 19 세포에서의 유전자 발현 수준을 대조군으로 설정하였다.ARPE 19 및 돼지 RPE 세포 및 일차 인간 RPE 세포 및 돼지 RPE 맥락막 조직에서 RPE 65 단백질의 웨스턴 혈액 분석이 이 그림에 나와 있습니다. Vinculin은 여기에서 로딩 컨트롤로 사용되었습니다. 이 그림은 1%FBS를 갖는 DMEM 기본 배지에서 돼지 RPE 세포의 2주 배양을 보여줍니다.라민과 함께 또는 라민없이 배양 된 세포를 RPE 65, 나트륨 칼륨 APTAs, Z01 및 Hex로 염색 하였다. 라미닌을 사용하거나 사용하지 않고 배양 된 세포 시트의 TR 측정이이 그림에 나와 있습니다. 일차 돼지 RPE 및 ARPE 19 세포에서 혈관 내피 성장 인자 또는 VEGF 및 색소 상피 유래 인자 또는 PEDF의 웨스턴 혈액 분석을 이 그림에 나타내었다.배양된 1차 돼지 RPE 및 ARPE 19 세포의 부문별 기능이 이 이미지에 나와 있습니다. 이 프로토콜의 가장 큰 부분은 RPE chide glial 복합체에서 RP 세포를 용해시키는 것입니다. 매번 신선한 트립과 용액을 사용하고 소화 시간을 적절하게 조절하여 8개의 RP 세포를 용해시키는 것이 좋습니다.

primary-culture-of-porcine-retinal-pigment-epithelial-cells

Research

JoVE Journal

Biology

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

기본 인간 난관 상피 세포의 전의 VIVO 문화

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

연구

생물학

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

인간 iPS 세포의 마이크로 RNA 발현 프로파일, IP의 유래 망막 색소 상피 세포, 태아의 망막 색소 상피

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

지리적 위축의 눈이 큰 모델에 인간 배아 줄기 세포 유래 망막 색소 상피 세포의 subretinal 이식

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

마우스 망막 안료 상피에서 1 차 셀 문화

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

비 바이러스 유전자 치료를 위한 포유류 1차 안료 상피 세포의 격리, 배양 및 유전 공학

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

1차 마우스 망막 색소 상피 세포의 효율적인 해부 및 배양

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

일차 마우스 망막 색소 상피 세포의 분리

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

고분해능 호흡 측정법을 이용한 일차 인간 망막 색소 상피 세포의 생체 에너지 실시간 분석

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

기니피그 눈에서 망막 색소 상피 세포의 분리

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

인간 조직에서 유방 상피 세포를 분리하기 위한 프로토콜

Isolation and Culture of Primary Human Mammary Epithelial Cells

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

저자 스포트라이트: 육아종성 유선염 치료를 위한 동양 의학과 서양 의학의 통합

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

Isolation and Culture of primary human mammary epithelial cells(원발성 인간 유방 상피세포의 분리 및 배양)

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...