이 연구는 한국과학기술연구원이 지원하는 '화학및 방사선측정기준 수립', 2020-GP2020-0003, '생체재료 및 의료융합측정기준 및 기술개발', '2020-GP2020-0004 프로그램 보조금'을 지원했다. 이 연구는 과학기술정보통신부(MSIT), 국립연구재단(NRF-2019M3A9F3065868), 보건복지부(MOHW), 한국보건산업진흥원(KHIDI, HI20C0558), 산업통상자원부(MOTIE), 산업통상자원부(MOTIE) 및 한국산업기술연구원(MOTIE)의 평가(MOTIE)20009350 지원했다. ORCID ID (유희민: 0000-0002-5951-2137; 강덕진: 0000-0002-5924-9674; 김세일: 0000-0003-3465-7118; 이주은: 0000-0002-2495-1439; 이진아: 0000-0002-3661-3701). 박창우에게 실험에 도움을 주신 것에 대해 감사드립니다.

1. 세균 세포 배양 및 유산 균 세포 없는 상체체의 준비 (LCFS)
참고: 단계 1.2 – 1.9 혐기성 챔버에서 실시됩니다.
<ol><li>L-시스테인이 들어 있는 MRS 한천 접시와 국물을 준비하고 오토클레이브를 통해 살균합니다.</li>
	<li>H2 혐기성 챔버에서 MRS 한천 플레이트를 20 ppm 산소로 37°C로 유지한 사전 배양한다.</li>
	<li>해동 유산균 세균육및 항마판을 세균배양으로 접종한다(도1A(i). </li>
	<li>단일 세균 식민지가 얻을 때까지 37 °C 및 20 ppm 산소에서 H2 혐기성 챔버에서 2 - 3 일 동안 박테리아를 배양한다.</li>
	<li>훈게이트 형 무성 배양 튜브를 세척하고 건조시십시오. 배양 튜브를 121°C에서 15분 동안 자동 클락.</li>
	<li>그런 다음 37°C 및 20 ppm 산소에서 H2 혐기성 챔버에서 튜브를 배양하여 산소를 제거합니다.</li>
	<li>튜브에 MRS 국물의 2 - 3 mL을 배치합니다. 부틸 고무 스토퍼로 튜브를 밀봉하고 캡을 나사.</li>
	<li>루프가 있는 단일 콜로니를 획득하여 1x PBS의 500 μL로 1.5mL 배양 튜브에 배치하십시오. (그림1A (ii)).</li>
	<li>1 mL 주사기를 사용하여 콜로니를 일시 중단(도 1A (iii) 이렇게 하면 튜브 뚜껑 중앙에 1mL 주사기의 바늘을 삽입하고 중단된 식민지를 흡입한 다음 MRS 국물 매체로 다시 다시 분리합니다. (그림1A (iv)).</li>
	<li>2 일 동안 셰이커 인큐베이터에서 MRS 국물 미디어를 배양 (37 °C, 5 % CO2,200 rpm).</li>
	<li>분광계를 사용하여 광학 밀도(OD)를 측정하여 OD620의 흡광도가 2.0에 도달할 때까지 세균 성장 곡선을 모니터링합니다.</li>
	<li>세균성 펠릿과 조건부 매체를 1,000 x g에서 15분 동안 원심분리하여 분리합니다. 수집된 세균성 펠릿을 1x PBS로 세척하고 10% 태아 소 혈청으로 보충된 RPMI 1640의 4mL에서 재연합니다. 배지에 항생제를 포함하지 마십시오.</li>
	<li>세균성 펠릿을 RPMI에서 유지하고 셰이커 인큐베이터에서 37°C에서 4시간 동안 5%CO2를 100rpm의 속도로 배양한다.</li>
	<li>프로바이오틱 상류제의 제조를 위해 원심분리를 통해 세균펠릿을 1000 x g에서15분 동안 제거하십시오. 멸균 필터는 0.22 μm 필터를 사용하여 회수 된 상체를 사용하고 사용전까지 -80 °C에 저장합니다.</li>
</ol>2. 스페로이드 생성
<ol><li>
대장암 세포주 준비
	<ol>
<li>DLD-1, HT-29 및 WiDr 세포주를 70-80%까지 단층층으로 성장시키고 5% CO2 인큐베이터에서 37°C에서 플레이트를 배양합니다(성장 배지: 10% 태아 소 혈청(FBS) 및 1% 페니실린-streptomycin)을 함유한 RPMI.</li>
		<li>100mm 페트리 접시로 자란 셀의 경우, 1x PBS의 4mL로 접시를 두 번 씻으시다. 0.25% 트립신-EDTA의 1mL을 추가하고 세포를 해리하기 위해 5% CO2 인큐베이터에서 37°C에서 2분 동안 페트리 접시를 배양한다.</li>
		<li>인큐베이션 후 현미경으로 세포 해리를 확인하고 5mL의 성장 배지로 트립신-EDTA를 중화시한다.</li>
		<li>해리된 세포를 15mL 원물 관및 원심분리기로 300 x g로 3분 간 이송한다.</li>
		<li>상체를 버리고 성장 매체의 3mL로 부드럽게 다시 중단하십시오.</li>
		<li>혈종계를 사용하여 실행 가능한 세포를 결정하기 위해 트라이팬 블루로 세포를 계산합니다. (그림1B (i))</li>
	</ol>
</li>
	<li>
스페로이드 형성
	<ol>
<li>15mL 원물 관에서 세포를 2.1.5로부터 희석하여 1- 2 x 105 세포/mL(도 1B(ii)를얻습니다.</li>
		<li>세포 현탁액에 0.6%의 메틸셀룰로오스의 최종 농도를 추가하고 희석된 세포를 멸균 저장소로 옮길 수 있습니다. 참고: 각 세포주에 대해 필요한 메틸셀룰로오스의 양은 적정화되고 그에 따라 결정되어야 합니다.</li>
		<li>멀티채널 파이펫을 사용하여 200 μL의 셀을 초저 부착 96웰 라운드 하단 마이크로플레이트의 각 웰에 분배합니다. (그림1B (iii))</li>
		<li>플레이트를 37°C에서 24-36h의 5% CO2 인큐베이터에서 배양한다.</li>
		<li>24 - 36 시간 후, 스페로이드 형성을 보장하기 위해 가벼운 현미경으로 플레이트를 관찰하십시오.</li>
	</ol>
</li>
</ol>3. LCFS로 3D 대장 암세포 치료
<ol><li>2단계와 3단계에서 설명된 바와 같이 스페로이드를 생성합니다.</li>
	<li>LCFS 치료를 수행하기 전에 실온(RT)에서 10-20분 동안 냉동 LCFS를 해동하십시오.</li>
	<li>LCFS 주식 솔루션을 성장 매체로 접종합니다. 연속적으로 25%, 12.5%, 성장 매체의 6%로 희석합니다(즉, 25% LCFS = 성장 매체의 150 μL + LCFS의 50 μL).</li>
	<li>인큐베이터에서 스페로이드를 함유한 세포 배양판을 꺼내 200μL 파이펫을 사용하여 각 우물에서 가능한 한 많은 성장 배지를 제거합니다.</li>
	<li>세포에 LCFS를 사용하여 성장 매체를 추가하고 24 - 48h에 대한 5 % CO2 인큐베이터에서 37 ° C에서 배양한다. 참고: 사용되는 부피는 다음과 같이 플레이트 크기에 따라 달라집니다: 6웰 세포 배양 판에 대한 2mL; 96웰 세포 배양판을 위한 200 μL.</li>
</ol>4. 스페로이드에 대한 세포 생존 가능성
<ol><li>8 -10 LCFS 처리 된 대장암 스페로이드를 불투명 벽다중 음판에서 준비하십시오 (세포 생존 성 소는 LCFS 치료 후 48 h로 수행됩니다).</li>
	<li>하룻밤 동안 세포 생존 능력 시약 (재료의 표참조)을 4 °C에서 해동하십시오.</li>
	<li>사용 전에 세포 생존능력 시약을 실온에 상량화합니다.</li>
	<li>분석작업을 수행하기 전에 스페로이드에서 성장 미디어의 50%를 제거합니다.</li>
	<li>각 웰에 셀 생존 능력 시약 100 μL을 추가합니다. 참고: 사용할 부피는 다음과 같이 플레이트 크기에 따라 달라집니다: 96웰 세포 배양 판에 대한 100 μL.</li>
	<li>시약을 5분 동안 격렬하게 섞어 세포 용해를 촉진합니다.</li>
	<li>37 °C에서 30 분 - 2 시간 동안 배양하십시오.</li>
	<li>발광을 기록합니다.</li>
</ol>5. 스페로이드에 대한 정량적 실시간 폴리머라제 연쇄 반응 분석
<ol><li>각 조건에 대해 2mL 튜브에 10 - 15 스페로이드를 준비하고 원심 분리기는 400 x g에서 3 분 동안 준비하십시오.</li>
	<li>상체를 버리고 얼음 차가운 1 x PBS의 1 mL에서 스페로이드를 두 번 씻으십시오. 참고: 원심분리를 피하고 스페로이드가 정착하게 하십시오.</li>
	<li>가능한 한 1x PBS의 많은 흡인및 시판 가능한 키트를 사용하여 RNA를 분리합니다.</li>
	<li>제조업체의 프로토콜에 따라 시판 되는 키트를 사용하여 RNA 1 μg에서 cDNA를 합성합니다.</li>
	<li>마스터 믹스를 준비하여 트리플리케이트로 모든 샘플을 실행합니다(표 1 및 표 2참조).</li>
	<li>템플릿 마스터 믹스의 20 μL에서 각 qPCR 플레이트에 증폭을 잘 수행합니다.</li>
	<li>반응을 잘 혼합하고 필요한 경우 회전합니다.</li>
	<li>계측기제조업체(표 3)의권장 사항에 따라 샘플을 실행합니다.</li>
</ol>6. 스페로이드에서 서양 블로팅
참고: 스페로이드를 수집할 때 200 μL 파이펫을 사용하고 팁 끝을 잘라 구조가 방해하지 않도록 하십시오.
<ol><li>각 조건에 대해 2mL 튜브에서 30 -40 스페로이드를 준비하십시오.</li>
	<li>튜브를 얼음 위에 놓고 스페로이드가 2mL 튜브의 바닥으로 정착하게 하십시오.</li>
	<li>상체를 버리고 1 mL 얼음 차가운 1 x PBS에서 스페로이드를 두 번 씻으십시오. 참고: 원심분리를 피하고 스페로이드가 정착하게 하십시오.</li>
	<li>가능한 한 1x PBS를 흡인하고 프로테아제 억제제 칵테일 (10 스페로이드 = RIPA 버퍼의 30 μL)와 RIPA 버퍼를 추가합니다.</li>
	<li>위아래로 파이펫팅하여 세포를 lyse하고 10 사이클 동안 얼음에 30 초 의 휴식과 함께 30 s에 대한 초음파 처리를 수행합니다.</li>
	<li>원심분리단백질은 4°C에서 15분 동안 15000 x g에서 15분 동안 리자트합니다.</li>
	<li>각 세포 용액에 대한 단백질 농도를 결정합니다.</li>
	<li>로드하기 전에 각 셀을 100°C에서 10분 동안 샘플 버퍼로 끓입니다.</li>
	<li>SDS-PAGE 젤의 우물에 동일한 양의 단백질을 적재하고 100 V에서 1 - 2 시간 동안 젤을 실행합니다.</li>
	<li>젤에서 PVDF 막으로 단백질을 전달합니다.</li>
	<li>이송 후, 블로킹 버퍼(0.05% Tween-20)를 사용하여 실온에서 1h의 멤브레인을 차단하십시오.</li>
	<li>1차항체(표 4)의1:1,000 희석제로 멤브레인을 배양하여 1x TBST에서 5% BSA 버퍼를 하룻밤 사이에 4°C로 배양한다.</li>
	<li>TBST로 멤브레인을 세 번, 세척할 때마다 15분 세척합니다.</li>
	<li>2 시간 동안 실온에서 차단 버퍼에서 이차 항체의 1:2,500 희석으로 멤브레인을 배양하십시오 (재료표참조).</li>
	<li>TBST로 멤브레인을 세 번, 세척할 때마다 15분 세척합니다.</li>
	<li>화학 발광 기판으로 HRP 검출을 위한 막을 준비합니다.</li>
	<li>화학 발광 이미지를 획득합니다.</li>
</ol>7. 스페로이드의 프로피듐 이오디드 (PI) 염색
<ol><li>4.1 단계에서 설명된 바와 같이 5-10 스페로이드를 준비하고 37°C 및 5% CO2의인큐베이터에 스페로이드를 배치한다.</li>
	<li>1x PBS에서 PI 1:100의 1 mg/mL 재고를 희석시하십시오.</li>
	<li>96웰 플레이트의 각 웰에서 배지의 50%를 제거합니다.</li>
	<li>각 우물에 PI 용액의 100 μL을 추가하고 37 °C에서 인큐베이터에 우물을 넣고 10 - 15 분 동안 5 % CO2를 배치하십시오.</li>
	<li>1x PBS로 PI 용액을 씻어내라.</li>
	<li>성장 배지의 200 μL을 추가하고 형광 현미경을 사용하여 이미지를 가져 가라. 이미지 J를 사용하여 형광 강도를 분석하여 스페로이드의 생존 가능성을 가져옵니다.</li>
</ol>8. 스페로이드의 FACS 분석
<ol><li>이전에 설명한 대로 스페로이드를 생성합니다.</li>
	<li>각 조건에 대해 FACS 튜브에 30-40 스페로이드를 준비하고 원심분리기는 400 x g및 RT에서 3분 동안 준비합니다.</li>
	<li>슈퍼나탄을 흡기하고 1x PBS의 3mL로 스페로이드를 세척한 다음 4°C에서 3분 동안 400 x g에서 원심분리기를 세척합니다.</li>
	<li>수퍼네티드를 흡인하고 0.25% 트립신-EDTA의 200 μL을 추가한 다음 RT에서 2-3분 동안 배양합니다. 참고: 잠복기 시간은 스페로이드 크기와 셀 유형에 따라 다릅니다.</li>
	<li>FACS 버퍼 1mL을 추가하고 200 μL 파이펫을 사용하여 스페로이드를 부드럽게 분리합니다.</li>
	<li>3 분 동안 400 x g 및 4 °C에서 해리 된 세포를 원심 분리합니다. 참고: FACS 버퍼 = 1x PBS + 2.5% FBS, 0.22 μm 상단 필터를 사용하여 필터링.</li>
	<li>상체를 폐기하고 7-AAD/부속서 V 시약(7AAD(5μL), 부속서 V(5 μL)/시료를 추가합니다.</li>
	<li>부드럽게 세포를 소용돌이와 어둠 속에서 RT에서 13 - 30 분 동안 배양.</li>
	<li>FACS 버퍼 500 μL을 추가하고 원물 폴리스티렌 테스트 튜브를 사용하여 세포를 필터링하여 집계 셀을 제거합니다.</li>
	<li>원심분리기 400 x g및 4°C에서 3분 동안.</li>
	<li>각 튜브에 500 μL의 부속서 V 바인딩 버퍼를 추가하고 다시 일시 중지합니다.</li>
	<li>유동 세포계를 사용하여 분석합니다.</li>
</ol>

<ol>
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저자는 관련 재무 공시가 없습니다.

인접한 세포및 세포외 매트릭스(ECM)를 포함하는 조직 미세환경은 조직 생성에 근본적이며 세포 성장 및 조직개발(13)의제어에 결정적인 역할을 한다. 그러나, 2D 배양은 세포 상호 작용의 중단과 같은 몇몇 단점이 있습니다, 세포 형태에 있는 변경, 세포 외 환경 및 분열 의 접근14. 3D 세포 배양 시스템은 생체 내 효과를 더 잘 재현하기 위해 엄격하게 연구되었으?...

프로바이오틱스는 면역 항상성을 개선하고 에너지 대사를 주최하는 장내 가장 유리한 미생물입니다1. 유산균과 비피도박테리움의 프로바이오틱스는장2,3에서발견되는 가장 진보된 프로바이오틱스입니다. 이전 조사는 유산 균이 여러 암에 억제 및 항증식 효과가 있음을 보여 주었다, 대장암을 포함4. 또한, 프로바이오틱스는 염증성 장질환, 크론병, 궤양성 대장염5,6을예방한다. 그러나, probiotics와 대부분의 연구 2 차원에서 수행 되었다 (2D) 단층 고체 표면에 성장.
인공 배양 시스템은 암세포에 자연적이지 않은 환경 적 특징이 부족합니다. 이러한 한계를 극복하기 위해 3차원(3D) 배양 시스템이7,8을개발하였다. 3D내암세포는 세포 생존가능성, 증식, 형태학, 세포통신, 약물 민감도, 생체내 관련성9,10과 같은 기본적인 생물학적 메커니즘의 측면에서 개선을 보여준다. 더욱이, 스페로이드는 대장암의 다세포형으로 만들어지며 세포 세포 상호작용 및 세포외 매트릭스(ECM)11에의존한다. 우리의 이전 연구는 유산 균 발효를 사용 하 여 생산 하는 probiotic 세포 무료 supernatant (CFS) 대장암의 3D 배양에 항 암 효과 보여 보고 (CRC) 세포12. 우리는 CFS가 3D 스페로이드12에프로바이오틱 효과를 테스트하기위한 적절한 대체 전략이라고 제안했다.
여기서, 우리는 몇몇 3D 대장암 모방 시스템에 probiotic 세포 없는 supernatant (CFS)의 치료 효과의 분석을 위해 3D 대장암의 다세포 모형을 수용할 수 있는 접근법을 제시합니다. 이 방법은 체외에서 관련 프로바이오틱 및 항암 효과의 분석을 위한 수단을 제공한다.

<table>
	<tbody>
		<tr>
			<td> 
			10% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 &#181;l</td>
			<td>Biorad</td>
			<td>4561036</td>
			<td>Pkg of 10</td>
		</tr>
		<tr>
			<td>Applied Biosystems MicroAmp Optical Adhesive Film</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311971</td>
			<td>100 covers</td>
		</tr>
		<tr>
			<td>10x transfer buffer</td>
			<td>Intron</td>
			<td>IBS-BT031A</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>10X Tris-Glycine (W/SDS)</td>
			<td>Intron</td>
			<td>IBS-BT014</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Axygen 2.0 mL MaxyClear Snaplock Microcentrifuge Tube, Polypropylene, Clear, Nonsterile, 500 Tubes/Pack, 10 Packs/Case</td>
			<td>Corning</td>
			<td>SCT-200-C</td>
			<td>500 Tubes/Pack, 10 Packs/Case</td>
		</tr>
		<tr>
			<td>BD Difco Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>BD Difco Lactobacilli MRS Broth</td>
			<td>BD</td>
			<td>DF0881-17-5</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Cell viability assay</td>
			<td>Promega</td>
			<td>G9681</td>
			<td>100&#956;l/assay in 96-well plates</td>
		</tr>
		<tr>
			<td>Complete Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11697498001</td>
			<td>vial of 20 tablets</td>
		</tr>
		<tr>
			<td>Corning Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 &#177; 0.1</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>EMD Millipore Immobilon-P PVDF Transfer Membranes</td>
			<td>fisher Scientific</td>
			<td>IPVH00010</td>
			<td>26.5cm x 3.75m roll; Pore Size: 0.45um</td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>25/Pack, 500/Case</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, US origin</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000044</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit, 25 x 20 &#181;l rxns #1708890</td>
			<td>Biorad</td>
			<td>1708890</td>
			<td>25 x 20 &#181;L rxns</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>Biorad</td>
			<td>1725121</td>
			<td>5 x 1 mL</td>
		</tr>
		<tr>
			<td>Lactobacillus fermentum</td>
			<td>Korean Collection for Type Cultures</td>
			<td>KCTC 3112</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine hydrochloride monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C6852-25G</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>Methyl Cellulose (3500-5600mPa&#183;s, 2% in Water at 20&#176;C)</td>
			<td>TCI</td>
			<td>M0185</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL</td>
			<td>Applied Biosystems</td>
			<td>4346906</td>
			<td>20 plates</td>
		</tr>
		<tr>
			<td>Millex-GS Syringe Filter Unit, 0.22 &#181;m, mixed cellulose esters, 33 mm, ethylene oxide sterilized</td>
			<td>Millipore</td>
			<td>SLGS033SB</td>
			<td>250</td>
		</tr>
		<tr>
			<td>PE Annexin V Apoptosis Detection Kit with 7-AAD</td>
			<td>Biolegend</td>
			<td>640934</td>
			<td>100 tests</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Introgen</td>
			<td>P1304MP</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>89901</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit (250)</td>
			<td>Qiagen</td>
			<td>74106</td>
			<td>250</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Name of Materials/Equipment/Software</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>anti - p-I&#954;B&#945; (B-9)</td>
			<td>Santa cruze</td>
			<td>sc-8404</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>anti-BclxL (H-5)</td>
			<td>Santa cruze</td>
			<td>sc-8392</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>anti-PARP 1 (C2-10)</td>
			<td>Santa cruze</td>
			<td>sc-53643</td>
			<td>50 &#181;l ascites</td>
		</tr>
		<tr>
			<td>anti-&#946;-actin (C4)</td>
			<td>Santa cruze</td>
			<td>sc-47778</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>BD FACSVerse</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>San Diego, CA, USA</td>
		</tr>
		<tr>
			<td>Synergy HTX Multi-Mode Microplate Reader</td>
			<td>BioT</td>
			<td>S1LFA</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo fisher</td>
			<td>HERAcell 150i</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube 15 ml</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube 50 ml</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Ultra-Low Attachment Multiple Well Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Ultra-Low Attachment Multiple Well Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 50 mL Reagent Reservoirs, 5/Bag, Sterile</td>
			<td>Costar</td>
			<td>4870</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Thermofisher</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>invitrogen</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>EnSpire Multimode Reader</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Enspire 2300</td>
		</tr>
		<tr>
			<td>Eppendorf Research Plus Multi Channel Pipette, 8-channel</td>
			<td>Eppendorf</td>
			<td>3122000051</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo software</td>
			<td>TreeStar</td>
			<td></td>
			<td>Ashland, OR, USA</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson immunoresearch</td>
			<td>115-035-062</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jackson immunoresearch</td>
			<td>111-035-144</td>
			<td>2.0 mL</td>
		</tr>
		<tr>
			<td>GraphPad Prism 5</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Inc., San Diego, CA, USA</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageQuant LAS 4000 mini</td>
			<td>Fujifilm</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Incubated shaker</td>
			<td>Lab companion</td>
			<td>SIF-6000R</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi Gauge Ver. 3.0,</td>
			<td>Fujifilm</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>Optical density (OD)LAMBDA UV/Vis Spectrophotometers</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Waltham, MA, USA</td>
		</tr>
		<tr>
			<td>Phase-contrast microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr>
			<td>SPL microcentrifuge tube 1.5mL</td>
			<td>SPL</td>
			<td>60015</td>
			<td></td>
		</tr>
		<tr>
			<td>SPL Multi Channel Reservoirs, 12-Chs, PS, Sterile</td>
			<td>SPL</td>
			<td>21012</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus Real-Time PCR system</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Waltham, MA, USA</td>
		</tr>
		<tr>
			<td>Vibra-Cell Ultrasonic Liquid Processors</td>
			<td>SONICS-vibra cell</td>
			<td>VC 505</td>
			<td>500 Watt ultrasonic processor</td>
		</tr>
		<tr>
			<td>Vinyl Anaerobic Chamber</td>
			<td>COY LAB PRODUCTS</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating cell death using cell-free supernatant of probiotics in three-dimensional spheroid cultures of colorectal cancer cells

이 원고는 프로바이오틱스배양으로 간주되는 유산균 발효 세포 배양에서 수퍼나티를 사용하여 암세포의 다세포 유형의 3차원(3D) 스페로이드에서 암세포 사망을 평가하는 프로토콜을 설명합니다. 유산균 세포 없는 상퍼나탄(LCFS)을 테스트하기 위해 3D 배양을 사용하는 것은 특히 L. 발효가 장 내항암 효과를 생성할 수 있기 때문에 2D 단층에서 테스트하는 것보다 더 나은 옵션입니다. L. 발효 상체는 3D 배양 조건에서 여러 대장암(CRC) 세포에 대한 증식 효과를 보유한 것으로 확인되었다. 흥미롭게도, 이러한 효과는 암세포 사망을 유도하는 L. 발효의 주목할 만한 능력을 입증하는 배양 모델과 강하게 관련이 있었다. 안정스페로이드는 아래에 제시된 프로토콜을 사용하여 다양한 CRC(대장암세포)로부터 생성되었다. 3D 스페로이드를 생성하는 이 프로토콜은 시간 절약및 비용 효율적입니다. 이 시스템은 여러 유형의 CRC 스페로이드에서 LCFS의 항암 효과를 쉽게 조사하기 위해 개발되었습니다. 예상대로, 실험 중 LCFS로 치료된 CRC 스페로이드는 qRT-PCR, 서부 블로팅 및 FACS 분석에 의해 분석된 특정 세포사멸 분자 마커를 발현하였다. 따라서, 이 방법은 세포 생존가능성을 탐구하고 항암제의 효능을 평가하는 데 유용하다.

우리는 다양한 대장암 세포주로부터 스페로이드를 얻는 프로토콜을 설명합니다. 메틸셀룰로오스를 보충하셔야 스페로이드를 생성해야 했습니다. 우리는 또한 LCFS 준비의 방법을 제시하고 프로바이오틱스와 대장암 사이의 상관 관계를 연구하는 모델을 제시한다. 스페로이드 형성 및 LCFS 준비 프로토콜은 도 1A,B에괄호로 도시된다. 도 2A에?...

Watch this Scientific Journal Video about Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells at JoVE.com

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

여기에서 방법은 유산균 세포 없는 상체 (LCFS)의 항암 효과를 이해하기 위하여 제시됩니다. 대장암 세포선은 3D 배양에서 LCFS로 치료할 때 세포 사망을 보여줍니다. 스퍼로이드를 생성하는 과정은 스캐폴드와 제시된 분석 방법에 따라 최적화될 수 있으며 관련 시그널링 경로를 평가하는 데 유용하다.

대장암세포의 3차원 스페로이드 배양에서 프로바이오틱스의 세포없는 슈퍼네탄을 사용하여 세포 사멸 평가

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

evaluating-cell-death-using-cell-free-supernatant-probiotics-three

Research

JoVE Journal

Cancer Research

5.4K 조회수.  Korea Research Institute of Standards and Science (KRISS). 여기에서 방법은 유산균 세포 없는 상체 (LCFS)의 항암 효과를 이해하기 위하여 제시됩니다. 대장암 세포선은 3D 배양에서 LCFS로 치료할 때 세포 사망을 보여줍니다. 스퍼로이드를 생성하는 과정은 스캐폴드와 제시된 분석 방법에 따라 최적화될 수 있으며 관련 시그널링 경로를 평가하는 데 유용하다.

비디오: Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

기능 유전체학에게 검사를 활용하여 암 세포 회전 타원체 문화에 잠재적으로 소설 약 표적을 식별하는

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

연구

암 연구학

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

준비 및 췌장암 세포 및 섬유 아 세포의 3 차원 회전 타원 체 공동 문화의 변화 분석 결과

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

위쪽으로 이동: 간단 하 고 유연한 생체 외에서 3 차원 침공 분석 실험 프로토콜

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

3 차원 세 포스 페 로이드 침입 분석에서 완전 한 인간 종양 기반 매트릭스

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

암세포의 3D 스페로이드 배양에서 세포 생존력과 사망 평가

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

3차원 스페로이드 배양에서 줄기와 같은 세포의 분리

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

암 환자의 혈장 샘플에서 세포없는 DNA 의 검출

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

대장암 줄기세포를 연구하는 스페로이드의 3차원 모델

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

콜라겐 기반 스캐폴드를 사용한 신경모세포종의 3차원 체외 생체모방 모델

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

간세포 암에서 종양-기질 상호 작용을 조사하는 3차원 스페로이드 모델

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Methods Article

대장암세포의 3차원 스페로이드 배양에서 프로바이오틱스의 세포없는 슈퍼네탄을 사용하여 세포 사멸 평가