Name: evaluating cell death using cell-free supernatant of probiotics in three-dimensional spheroid cultures of colorectal cancer cells
Uploaded: 2020-06-13T14:45:00
Description: 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

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 세균배양으?...

<ol>
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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 ...

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

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 ...

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암 연구학

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

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

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

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

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

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. ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

유기체 배양을 사용하여 마우스 전립선 상피 세포의 분화 능력 평가

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

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

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

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

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

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

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 ...