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

Bu araştırma, Kore Standartlar ve Bilim Araştırma Enstitüsü tarafından finanse edilen "Kimya ve Radyasyon için ölçüm standartlarının oluşturulması", KRISS-2020-GP2020-0003 hibe numarası ve "Biyomalzemeler ve Tıbbi Yakınsama için Ölçüm Standartlarının ve Teknolojisinin Geliştirilmesi", KRISS-2020-GP2020-0004 programlarının hibe numarası ile desteklenmiştir. Bu araştırma bilim ve Bİt Bakanlığı (MSIT), Kore Ulusal Araştırma Vakfı (NRF-2019M3A9F3065868), Sağlık ve Refah Bakanlığı (MOHW), Kore Sağlık Endüstrisi Geliştirme Enstitüsü (KHIDI, HI20C0558), Ticaret, Sanayi ve Enerji Bakanlığı (MOTIE) ve Kore Sanayi Teknolojisi Değerlendirme Enstitüsü (KEIT, 20009350) tarafından da desteklendi. ORCID Kimliği (Hee Min Yoo: 0000-0002-5951-2137; Dukjin Kang: 0000-0002-5924-9674; Seil Kim: 0000-0003-3465-7118; Joo-Eun Lee: 0000-0002-2495-1439; Jina Lee: 0000-0002-3661-3701). Deneylere yardım için Chang Woo Park'a teşekkür ederiz.

1. Bakteriyel hücre kültürleri ve Lactobacillus hücresiz süpernatant hazırlanması (LCFS)
NOT: Adım 1.2 – 1.9 anaerobik bir odada gerçekleştirilir.
<ol><li>L-sistein içeren bir MRS agar tabağı ve et suyu hazırlayın ve otoklavlayarak sterilize edin.</li>
	<li>37 °C'de tutulan H2 anaerobik haznedeki MRS agar plakasını 20 ppm oksijenle önceden kuluçkaya yatırın.</li>
	<li>Lactobacillus bakteri stoğunu çözün ve agar plakasını bakteri kül...

<ol>
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Komşu hücreler ve hücre dışı matris (ECM) de dahil olmak üzere doku mikroçevrasyonu doku üretimi için temeldir ve hücre büyümesi ve doku gelişiminin kontrolünde çok önemlidir13. Bununla birlikte, 2D kültürlerin hücresel etkileşimlerin bozulması, hücre morfolojisindeki değişiklikler, hücre dışı ortamlar ve bölüm14yaklaşımı gibi çeşitli dezavantajları vardır. 3D hücre kültürü sistemleri in vivo etkileri daha iyi üretmek için titizli...

Probiyotikler, bağışıklık homeostazını geliştiren ve enerji metabolizmasını barındıran bağırsaktaki en avantajlı mikroorganizmalardır1. Lactobacillus ve Bifidobacterium probiyotikleri bağırsakta bulunan türünün en gelişmişidir2,3. Önceki araştırmalar Lactobacillus'un kolorektal kanser de dahil olmak üzere çeşitli kanserler üzerinde inhibitör ve antiproliferatif etkileri olduğunu göstermiştir4. Ayrıca, probiyotikler enflamatuar bağırsak hastalıklarını, Crohn hastalığını ve ülseratif koliti önler5,6. Bununla birlikte, probiyotiklerle yapılan çalışmaların çoğu katı yüzeylerde yetişen iki boyutlu (2D) monolayerlerde gerçekleştirildi.
Yapay kültür sistemleri, kanser hücreleri için doğal olmayan çevresel özelliklerden yoksundur. Bu sınırlamanın üstesinden gelmek için üç boyutlu (3D) kültür sistemleri geliştirilmiştir7,8. 3D kanser hücreleri hücre canlılığı, çoğalma, morfoloji, hücre-hücre iletişimi, ilaç duyarlılığı ve in vivo alaka düzeyi9,10gibi temel biyolojik mekanizmalar açısından iyileşmeler göstermektedir. Ayrıca, sferoidler çok hücreli kolorektal kanser türlerinden yapılır ve hücre-hücre etkileşimlerine ve hücre dışı matrise (ECM)bağlıdır 11. Önceki çalışmamız, Lactobacillus fermentum kullanılarak üretilen probiyotik hücresiz süpernatantın (CFS) kolorektal kanser (CRC) hücrelerinin 3D kültürleri üzerinde kanser önleyici etkiler gösterdiğini bildirmiştir12. CFS'nin probiyotik etkilerini 3D sferoidler üzerinde test etmek için uygun bir alternatif strateji olduğunu önerdik12.
Burada, probiyotik hücresiz süpernatantın (CFS) çeşitli 3D kolorektal kanser taklit sistemleri üzerindeki terapötik etkilerinin analizi için çok hücreli 3D kolorektal kanser türlerini barındırabilecek bir yaklaşım sunuyoruz. Bu yöntem, ilgili probiyotik ve anti-kanser etkilerinin in vitro analizi için bir araç sağlar.

<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

Bu makale, probiyotikkültürü olarak kabul edilen Lactobacillus fermentum hücre kültüründen süpernatantlar kullanarak çok hücreli kanser hücrelerinin üç boyutlu (3D) sferoidlerinde kanser hücresi ölümlerini değerlendirmek için bir protokol açıklanmaktadır. Lactobacillus hücresiz süpernatant (LCFS) test etmek için 3D kültürlerin kullanımı, özellikle L. fermentum bağırsak içinde kanser önleyici etkiler üretebileceğinden, 2D monolayerlerde test etmekten daha iyi bir seçenektir. L. fermentum supernatant'ın 3D kültür koşullarında çeşitli kolorektal kanser (CRC) hücrelerine karşı artan anti-proliferatif etkilere sahip olduğu belirlendi. İlginçtir ki, bu etkiler L. fermentum'un kanser hücre ölümünü teşvik etmedeki önemli yeteneğini gösteren kültür modeliyle güçlü bir şekilde ilişkiliydi. Aşağıda sunulan protokol kullanılarak çeşitli CRC'lerden (kolorektal kanser hücreleri) stabil sferoidler üretilmiştir. 3D küresel oluşturmanın bu protokolü zaman tasarrufu sağlar ve uygun maliyetlidir. Bu sistem, LCFS'nin birden fazla CRC sferoid türündeki kanser önleyici etkilerini kolayca araştırmak için geliştirilmiştir. Beklendiği gibi, deney sırasında LCFS ile tedavi edilen CRC sferoidleri hücre ölümünü güçlü bir şekilde indükledi ve qRT-PCR, batı şişkinliği ve FACS analizi ile analiz edildiği gibi spesifik apoptoz moleküler belirteçlerini ifade etti. Bu nedenle, bu yöntem hücre canlılığını keşfetmek ve anti-kanser ilaçlarının etkinliğini değerlendirmek için değerlidir.

Çeşitli kolorektal kanser hücre hatlarından sferoid elde etme protokolünü açıklıyoruz. Sferoid üretmek için metilselüloz ile takviye gerekiyordu. Ayrıca LCFS hazırlama yöntemini sunuyoruz ve probiyotikler ile kolorektal kanser arasındaki korelasyonu incelemek için bir model sunuyoruz. Küresel formasyon ve LCFS hazırlama protokolleri Şekil 1A,B'deşematik olarak gösterilmiştir. Şekil 2A'dagösterildiği gibi, %0,6'luk metil...

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

Burada Lactobacillus hücresiz süpernatantın (LCFS) kanser önleyici etkilerini anlamak için yöntemler sunulmaktadır. Kolorektal kanser hücre hatları 3D kültürlerde LCFS ile tedavi edildiğinde hücre ölümlerini göstermektedir. Sferoid üretme süreci iskeleye bağlı olarak optimize edilebilir ve sunulan analiz yöntemleri ilgili sinyal yollarını değerlendirmek için yararlıdır.

Kolorektal Kanser Hücrelerinin Üç Boyutlu Küresel Kültürlerinde Probiyotiklerin Hücresiz Süpernatantı Kullanılarak Hücre Ölümünün Değerlendirilmesi

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

İşlevsel Genomik Tarama kullanılması Kanser Hücre Sfero Kültürleri Potansiyel Yeni İlaç Hedefleri Belirlemek İçin

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

Hazırlık ve pankreas kanseri hücreleri ve fibroblastlar 3 boyutlu küresel ortak kültürlerin metabolik tahlil

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

Yukarı doğru hareket: Bir basit ve esnek Vitro üç boyutlu işgali tahlil iletişim kuralı

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

Üç boyutlu Küroid Istilası tahlil tam ınsan tümör tabanlı matris

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

Hücre viability ve ölüm kanser hücrelerinin 3D Küroid kültürlerde değerlendirilmesi

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

Organoid Kültürü Kullanarak Fare Prostat Epitel Hücrelerinin Ayırt Etme Kapasitesinin Değerlendirilmesi

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 Boyutlu Spheroid Kültürlerden Kök Benzeri Hücrelerin İzolasyon

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

Kanser Hastalarının Kan Plazma Örneklerinde Hücresiz DNA Tespiti

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

Kolon Kanseri Kök Hücrelerini İncelemek İçin Üç Boyutlu Bir Küresel Model

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

Kollajen Bazlı İskeleler Kullanılarak Nöroblastomun Üç Boyutlu İn Vitro Biyomimetik Modeli

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