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

本研究は、韓国規格研究所が出資する「化学・放射線測定基準の確立」、助成番号KRISS-2020-GP2020-0003、「生物材料と医療収束のための測定基準と技術の開発」によって支援されました。また、科学・ICT省、韓国国立研究財団(NRF-2019M3A9F3065868)、厚生省(MOHW)、韓国保健産業開発研究所(KHIDI、HI20C0558)、貿易産業エネルギー省(MOTIE)、韓国工業技術研究所(KEIT)、韓国評価研究所20009350も支援しました。ORCID ID(ヒーミンユー:0000-0002-5951-2137;ドゥッジンカン:0000-0002-5924-9674;Seilキム: 0000-0003-3465-7118;ジュウン・リー: 0000-0002-2495-1439;ジナ・リー:0000-0002-3661-3701)。チャン・ウー・パークの実験支援に感謝します。

1. 細菌細胞培養と ラクトバチルス 細胞フリー上清(LCFS)の調製
注:ステップ1.2 - 1.9は嫌気性の部屋で行われます。
<ol><li>L-システインを含むMRS寒天プレートとブロスを準備し、オートクレーブで殺菌します。</li>
	<li>20 ppm酸素で37 °Cで維持されたH2 嫌気性チャンバーにMRS寒天プレートをプレインキュベートします。</li>
	<li>乳酸菌の細菌ストックを解?...

<ol>
	<li>Bron, P. A., Van Baarlen, P., Kleerebezem, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+molecular+insights+into+the+interaction+between+probiotics+and+the+host+intestinal+mucosa.">Emerging molecular insights into the interaction between probiotics and the host intestinal mucosa.</a> Nature Reviews Microbiology. 10 (1), 66-78 (2012).</li><li>Ruiz, L., Delgado, S., Ruas-Madiedo, P., S&#225;nchez, B., Margolles, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bifidobacteria+and+their+molecular+communication+with+the+immune+system.">Bifidobacteria and their molecular communication with the immune system.</a> Frontiers in Microbiology. 8, 1-9 (2017).</li><li>Sanders, M. E., Merenstein, D. J., Reid, G., Gibson, G. R., Rastall, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotics+and+prebiotics+in+intestinal+health+and+disease:+from+biology+to+the+clinic.">Probiotics and prebiotics in intestinal health and disease: from biology to the clinic.</a> Nature Reviews Gastroenterology and Hepatology. 16 (10), 605-616 (2019).</li><li>Pandey, K. R., Naik, S. R., Vakil, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotics,+prebiotics+and+synbiotics-+a+review.">Probiotics, prebiotics and synbiotics- a review.</a> Journal of Food Science and Technology. 52 (12), 7577-7587 (2015).</li><li>Harish, K., Varghese, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotics+in+humans-evidence+based+review.">Probiotics in humans-evidence based review.</a> Calicut Medical Journal. 4 (4), 3 (2006).</li><li>Routy, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gut+microbiota+influences+anticancer+immunosurveillance+and+general+health.">The gut microbiota influences anticancer immunosurveillance and general health.</a> Nature Reviews Clinical Oncology. 15 (6), 382-396 (2018).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+of+the+cell-based+data-harvesting+efforts+that+drive+the+integration+of+cell+biology.">Most of the cell-based data-harvesting efforts that drive the integration of cell biology.</a> Nature Reviews Molecular Cell Biology. 8 (10), 839-845 (2007).</li><li>Shamir, E. R., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+organotypic+culture:+Experimental+models+of+mammalian+biology+and+disease.">Three-dimensional organotypic culture: Experimental models of mammalian biology and disease.</a> Nature Reviews Molecular Cell Biology. 15 (10), 647-664 (2014).</li><li>Jong, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+tissue+culture+models+in+cancer+biology.">Three-dimensional tissue culture models in cancer biology.</a> Seminars in Cancer Biology. 15 (5), 365-377 (2005).</li><li>Zanoni, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tumor+spheroid+models+for+in+vitro+therapeutic+screening:+a+systematic+approach+to+enhance+the+biological+relevance+of+data+obtained.">3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained.</a> Scientific Reports. 6, 19103 (2016).</li><li>Anton, D., Burckel, H., Josset, E., Noel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture:+A+breakthrough+in+vivo.">Three-dimensional cell culture: A breakthrough in vivo.</a> International Journal of Molecular Sciences. 16 (3), 5517-5527 (2015).</li><li>Lee, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Anti-Cancer+Activity+of+the+Probiotic+Bacterium+Lactobacillus+fermentum+Using+2D+vs.+3D+Culture+in+Colorectal+Cancer+Cells.">Characterization of the Anti-Cancer Activity of the Probiotic Bacterium Lactobacillus fermentum Using 2D vs. 3D Culture in Colorectal Cancer Cells.</a> Biomolecules. 9 (10), 557 (2019).</li><li>Koledova, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+cell+culture:+An+introduction.">3D cell culture: An introduction.</a> Methods in Molecular Biology. 1612, (2017).</li><li>Kapa&#322;czy&#324;ska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2D+and+3D+cell+cultures+-+a+comparison+of+different+types+of+cancer+cell+cultures.">2D and 3D cell cultures - a comparison of different types of cancer cell cultures.</a> Archives of Medical Science. 14 (4), 910-919 (2018).</li><li>Langhans, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+in+vitro+cell+culture+models+in+drug+discovery+and+drug+repositioning.">Three-dimensional in vitro cell culture models in drug discovery and drug repositioning.</a> Frontiers in Pharmacology. 9, 1-14 (2018).</li><li>Breslin, S., O'Driscoll, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture:+The+missing+link+in+drug+discovery.">Three-dimensional cell culture: The missing link in drug discovery.</a> Drug Discovery Today. 18 (5-6), 240-249 (2013).</li><li>Mazzocchi, A. R., Rajan, S. A. P., Votanopoulos, K. I., Hall, A. R., Skardal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+patient-derived+3D+mesothelioma+tumor+organoids+facilitate+patient-centric+therapeutic+screening.">In vitro patient-derived 3D mesothelioma tumor organoids facilitate patient-centric therapeutic screening.</a> Scientific Reports. 8, 2886 (2018).</li><li>Lv, D., Hu, Z., Lu, L., Lu, H., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture:+A+powerful+tool+in+tumor+research+and+drug+discovery.">Three-dimensional cell culture: A powerful tool in tumor research and drug discovery.</a> Oncology Letters. 14 (6), 6999-7010 (2017).</li><li>Thirumala, S., Gimble, J., Devireddy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylcellulose+Based+Thermally+Reversible+Hydrogel+System+for+Tissue+Engineering+Applications.">Methylcellulose Based Thermally Reversible Hydrogel System for Tissue Engineering Applications.</a> Cells. 2 (3), 460-475 (2013).</li><li>Chandrashekran, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylcellulose+as+a+scaffold+in+the+culture+of+liver-organoids+for+the+potential+of+treating+acute+liver+failure.">Methylcellulose as a scaffold in the culture of liver-organoids for the potential of treating acute liver failure.</a> Cell and Gene Therapy Insights. 4 (11), 1087-1103 (2018).</li><li>Lee, W., Park, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+patterned+stem+cell+differentiation+using+thermo-responsive+methylcellulose+hydrogel+molds.">3D patterned stem cell differentiation using thermo-responsive methylcellulose hydrogel molds.</a> Scientific Reports. 6, 1-11 (2016).</li><li>Fan, H., Demirci, U., Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+organoid+models:+Leaping+forward+in+cancer+research.">Emerging organoid models: Leaping forward in cancer research.</a> Journal of Hematology and Oncology. 12 (1), 1-10 (2019).</li><li>Drost, J., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+in+cancer+research.">Organoids in cancer research.</a> Nature Reviews Cancer. 18 (7), 407-418 (2018).</li><li>Liou, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Metabolic+Pathway+for+Activation+of+Dietary+Glucosinolates+by+a+Human+Gut+Symbiont.">A Metabolic Pathway for Activation of Dietary Glucosinolates by a Human Gut Symbiont.</a> Cell. 180 (4), 717-728 (2020).</li><li>Sherwin, E., Bordenstein, S. R., Quinn, J. L., Dinan, T. G., Cryan, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiota+and+the+social+brain.">Microbiota and the social brain.</a> Science. 366 (6465), 2016 (2019).</li><li>Honda, K., Littman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbiota+in+adaptive+immune+homeostasis+and+disease.">The microbiota in adaptive immune homeostasis and disease.</a> Nature. 535 (7610), 75-84 (2016).</li><li>B&#225;rcena, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Healthspan+and+lifespan+extension+by+fecal+microbiota+transplantation+into+progeroid+mice.">Healthspan and lifespan extension by fecal microbiota transplantation into progeroid mice.</a> Nature Medicine. 25 (8), 1234-1242 (2019).</li><li>Michalovich, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+and+disease+severity+magnify+disturbed+microbiome-immune+interactions+in+asthma+patients.">Obesity and disease severity magnify disturbed microbiome-immune interactions in asthma patients.</a> Nature Communications. 10, 5711 (2019).</li><li>Ansaldo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akkermansia+muciniphila+induces+intestinal+adaptive+immune+responses+during+homeostasis.">Akkermansia muciniphila induces intestinal adaptive immune responses during homeostasis.</a> Science. 364 (6446), 1179-1184 (2019).</li></ol>

隣接する細胞および細胞外マトリックス(ECM)を含む組織微小環境は、組織発生の基本であり、細胞増殖および組織発達13の制御において極めて重要である。しかし、2D培養物には、細胞間相互作用の中断、細胞形態の変化、細胞外環境、および分割14のアプローチなど、いくつかの欠点がある。3D細胞培養システムは、生体内の効果をより良く再現するために...

プロバイオティクスは、免疫恒常性および宿主エネルギー代謝を改善する腸内で最も有利な微生物である1.乳酸菌およびビフィズス菌からのプロバイオティクスは腸内で見つかったその種の最も先進的な 2,3.これまでの調査では、ラクトバチルスは大腸癌を含むいくつかの癌に対して阻害および抗増殖効果を有することが示されている。また、プロバイオティクスは炎症性腸疾患、クローン病、潰瘍性大腸炎5,6を予防する。しかし, プロバイオティクスを用いたほとんどの研究は、2 次元で行われました (2D) 固体表面上で成長している単層.
人工培養システムは、がん細胞にとって自然ではない環境特性を欠いています。この制限を克服するために、3次元(3D)培養システムが7,8に開発されている。3Dにおける癌細胞は、細胞生存率、増殖、形態、細胞細胞通信、薬物感受性、および生体内関連性9,10などの基本的な生物学的機構の点で改善を示す。さらに、スフェロイドは、多細胞型の大腸癌から作製され、細胞間相互作用および細胞外マトリックス(ECM)11に依存する。我々の以前の研究は、ラクトバチルス発酵を用いて産生されたプロバイオティクス無細胞上清(CFS)が大腸癌(CRC)細胞12の3D培養に抗癌効果を示したことを報告した。我々は、CFSが3Dスフェロイド12に対するプロバイオティクス効果をテストするための適切な代替戦略であることを提案した。
ここでは、プロバイオティクス無細胞上清(CFS)の治療効果を複数の3D大腸癌模倣システムに対する分析のために、多細胞型の3D大腸癌に対応できるアプローチを提示する。この方法は、インビトロで関連プロバイオティクスと抗癌効果の分析のための手段を提供します。.

<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

本稿は、 乳酸菌 発酵細胞培養物 の上清を用いた多細胞型癌細胞の三次元(3D)スフェロイドにおける癌細胞死亡を評価するプロトコルを、プロバイオティクス培養物と考える。 ラクトバチルス 細胞遊離上清(LCFS)をテストするための3D培養物の使用は、特に L.発酵 が腸内で抗癌効果を生み出すことができるため、2D単層での試験よりも優れた選択肢です。 L.発酵 物上清は、3D培養条件で複数の大腸癌(CRC)細胞に対する抗増殖効果の増加を有することを同定した。興味深いことに、これらの効果は培養モデルと強く関連し 、L.発酵 が癌細胞死を誘導する顕著な能力を示した。安定したスフェロイドは、以下に示すプロトコルを用いて多様なCRC(大腸癌細胞)から生成された。3Dスフェロイドを生成するこのプロトコルは、時間の節約とコスト効率が高いです。このシステムは、複数のタイプのCRCスフェロイドにおけるLCFSの抗癌効果を容易に調べる開発された。予想通り、LCFSで処理されたCRCスフェロイドは、実験中に細胞死を強く誘導し、qRT-PCR、ウェスタンブロッティング、およびFACS分析によって分析された特定のアポトーシス分子マーカーを発現した。したがって、この方法は、細胞の生存率を探索し、抗癌薬の有効性を評価するために有用である。

多様な大腸癌細胞株からスフェロイドを得るプロトコルについて述べています。メチルセルロースによる補充は、スフェロイドを生成するために必要とされた。また、LCFS調製の方法を提示し、プロバイオティクスと大腸癌の相関関係を研究するモデルを提示する。スフェロイド形成およびLCFS調製プロトコルは、図1A,Bに概略的に示される。?...

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

ここでは、ラクトバチルス細胞遊離上清(LCFS)の抗癌効果を理解する方法が提示されている。大腸癌細胞株は、3D培養でLCFSで治療した場合の細胞死を示す。スキャフォールドの生成過程はスキャフォールドに応じて最適化でき、提示される解析方法は、関係するシグナル伝達経路を評価するのに有用である。

大腸癌細胞の3次元スフェロイド培養におけるプロバイオティクスの細胞遊離上清を用いた細胞死の評価

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

Research

JoVE Journal

Cancer Research

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

癌細胞スフェロイド培養における潜在的に新規薬剤標的を同定するためのスクリーニング機能ゲノミクスを利用

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

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

準備と膵臓癌細胞と線維芽細胞の共培養を 3 次元回転楕円体の代謝アッセイ

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

上に移動: シンプルかつ柔軟なIn Vitro三次元侵略試金のプロトコル

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

三次元スフェロイド浸潤アッセイにおける完全ヒト腫瘍ベースマトリクス

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

癌細胞の3Dスフェロイド培養における細胞生存率と死の評価

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

オルガノイド培養を用いたマウス前立腺上皮細胞の分化能力の評価

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

3次元スフェロイド培養からの幹様細胞の分離

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

がん患者の血漿サンプルにおける無細胞DNAの検出

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

大腸癌幹細胞を研究するスフェロイドの3次元モデル

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

コラーゲンベースの足場を用いた神経芽細胞腫の3次元 in vitro 生体模倣モデル

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