Evaluering cell død ved hjælp af cellefri supernatant af probiotika i tre-dimensionelle sfæroid kulturer colorectal cancerceller
Summary
Her metoder præsenteres for at forstå anti-cancer virkninger af Lactobacillus celle-fri supernatant (LCFS). Kolorektal cancer cellelinjer viser celledødsfald, når de behandles med LCFS i 3D kulturer. Processen med at generere sfæroider kan optimeres afhængigt af stilladset, og de fremlagte analysemetoder er nyttige til evaluering af de involverede signalveje.
Abstract
Dette manuskript beskriver en protokol til at evaluere kræftcelledødsfald i tredimensionelle (3D) sfæroider af multicellulære typer kræftceller ved hjælp af supernatants fra Lactobacillus fermentumcellekultur , betragtes som probiotikakulturer. Brugen af 3D-kulturer til at teste Lactobacillus cellefri supernatant (LCFS) er en bedre mulighed end test i 2D monolayers, især da L. fermentum kan producere anti-cancer effekter i tarmen. L. fermentum supernatant blev identificeret til at besidde øget anti-proliferative virkninger mod flere kolorektal cancer (CRC) celler i 3D-kultur betingelser. Interessant nok var disse virkninger stærkt relateret til kulturmodellen, hvilket demonstrerede L. fermentums bemærkelsesværdige evne til at fremkalde kræftcelledød. Stabile sfæroider blev genereret fra forskellige CRCs (kolorektal cancerceller) ved hjælp af protokollen præsenteret nedenfor. Denne protokol til generering af 3D-sfæroid er tidsbesparende og omkostningseffektiv. Dette system blev udviklet til nemt at undersøge anti-cancer virkninger af LCFS i flere typer af CRC sfæroider. Som forventet, CRC sfæroider behandlet med LCFS stærkt induceret celledød under forsøget og udtrykt specifikke apoptose molekylære markører som analyseret af qRT-PCR, vestlige blotting, og FACS analyse. Derfor er denne metode værdifuld til at udforske celle levedygtighed og evaluere effekten af anti-cancer medicin.
Introduction
Probiotika er de mest fordelagtige mikroorganismer i tarmen, der forbedrer immunhomøostase og vært for energimetabolisme1. Probiotika fra Lactobacillus og Bifidobacterium er de mest avancerede af sin art, der findes i tarmen2,3. Tidligere undersøgelser har vist, at Lactobacillus har hæmmende og antiproliferative virkninger på flere kræftformer, herunder kolorektal cancer4. Desuden forhindrer probiotika inflammatoriske tarmsygdomme, Crohns sygdom og colitis ulcerosa5,6. Men de fleste undersøgelser med probiotika blev udført i todimensionelle (2D) monolag, der dyrkes på faste overflader.
Kunstige kultursystemer mangler miljømæssige træk, hvilket ikke er naturligt for kræftceller. For at overvinde denne begrænsning er der udviklet tredimensionelle (3D) kultursystemer7,8. Kræftceller i 3D viser forbedringer med hensyn til grundlæggende biologiske mekanismer, såsom celle levedygtighed, spredning, morfologi, cellecellekommunikation, lægemiddelfølsomhed og in vivo relevans9,10. Desuden er sfæroider fremstillet af flercellede typer af kolorektal cancer og er afhængige af cellecelleinteraktioner og den ekstracellulære matrix (ECM)11. Vores tidligere undersøgelse har rapporteret, at probiotiske celle-fri supernatant (CFS) produceret ved hjælp af Lactobacillus fermentum viste anti-cancer virkninger på 3D kulturer af kolorektal cancer (CRC) celler12. Vi foreslog, at CFS er en passende alternativ strategi til test af probiotiske virkninger på 3D-sfæroider12.
Her præsenterer vi en tilgang, der kan rumme flercellede typer af 3D-kolorektal cancer til analyse af terapeutiske virkninger af probiotisk cellefri supernatant (CFS) på flere 3D-kolorektal cancer-efterligningssystemer. Denne metode giver et middel til analyse af relaterede probiotiske og anti-cancer effekter in vitro.
Protocol
Representative Results
Discussion
Vævsmikromiljøet, herunder naboceller og den ekstracellulære matrix (ECM), er grundlæggende for vævsgenerering og afgørende for kontrollen med cellevækst og vævsudvikling13. 2D-kulturer har dog flere ulemper, såsom afbrydelse af cellulære interaktioner samt ændringer i cellemorfologi, ekstracellulære miljøer og tilgangen til division14. 3D-cellekultursystemer er blevet grundigt undersøgt for bedre at reproducere in vivo-effekter og er blevet bevist som mere pr…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Denne forskning blev støttet af “Etablering af målestandarder for kemi og stråling”, tilskudsnummer KRISS-2020-GP2020-0003 og “Udvikling af målestandarder og teknologi til biomaterialer og medicinsk konvergens”, tilskudsnummer KRISS-2020-GP2020-0004-programmer, finansieret af Korea Research Institute of Standards and Science. Denne forskning blev også støttet af Ministeriet for Videnskab og IKT (MSIT), National Research Foundation of Korea (NRF-2019M3A9F3065868), Ministeriet for Sundhed og Velfærd (MOHW), Korea Health Industry Development Institute (KHIDI, HI20C0558), Ministeriet for Handel, Industri & Energi (MOTIE) og Korea Evaluation Institute of Industrial Technology (KEIT, 20009350). ORCID ID (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). Vi takker Chang Woo Park for hjælp med eksperimenter.
Materials
10% Mini-PROTEAN TGX Precast Protein Gels, 15-well, 15 µl |
Biorad | 4561036 | Pkg of 10 |
| Applied Biosystems MicroAmp Optical Adhesive Film | Thermo Fisher Scientific | 4311971 | 100 covers |
| 10x transfer buffer | Intron | IBS-BT031A | 1 L |
| 10X Tris-Glycine (W/SDS) | Intron | IBS-BT014 | 1 L |
| Axygen 2.0 mL MaxyClear Snaplock Microcentrifuge Tube, Polypropylene, Clear, Nonsterile, 500 Tubes/Pack, 10 Packs/Case | Corning | SCT-200-C | 500 Tubes/Pack, 10 Packs/Case |
| BD Difco Bacto Agar | BD | 214010 | 500 g |
| BD Difco Lactobacilli MRS Broth | BD | DF0881-17-5 | 500 g |
| CellTiter-Glo 3D Cell viability assay | Promega | G9681 | 100μl/assay in 96-well plates |
| Complete Protease Inhibitor Cocktail | Sigma-Aldrich | 11697498001 | vial of 20 tablets |
| Corning Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1 | Corning | 21-040-CV | 500 mL |
| EMD Millipore Immobilon-P PVDF Transfer Membranes | fisher Scientific | IPVH00010 | 26.5cm x 3.75m roll; Pore Size: 0.45um |
| Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap | Corning | 352235 | 25/Pack, 500/Case |
| Fetal Bovine Serum, certified, US origin | Thermo Fisher Scientific | 16000044 | 500 mL |
| iScript cDNA Synthesis Kit, 25 x 20 µl rxns #1708890 | Biorad | 1708890 | 25 x 20 µL rxns |
| iTaq Universal SYBR Green Supermix | Biorad | 1725121 | 5 x 1 mL |
| Lactobacillus fermentum | Korean Collection for Type Cultures | KCTC 3112 | |
| L-Cysteine hydrochloride monohydrate | Sigma-Aldrich | C6852-25G | 25 g |
| Methyl Cellulose (3500-5600mPa·s, 2% in Water at 20°C) | TCI | M0185 | 500 g |
| MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL | Applied Biosystems | 4346906 | 20 plates |
| Millex-GS Syringe Filter Unit, 0.22 µm, mixed cellulose esters, 33 mm, ethylene oxide sterilized | Millipore | SLGS033SB | 250 |
| PE Annexin V Apoptosis Detection Kit with 7-AAD | Biolegend | 640934 | 100 tests |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140122 | 100 mL |
| Propidium Iodide | Introgen | P1304MP | 100 mg |
| RIPA Lysis and Extraction Buffer | Thermo Fisher Scientific | 89901 | 250 mL |
| RNeasy Mini Kit (250) | Qiagen | 74106 | 250 |
| RPMI-1640 | Gibco | 11875-119 | 500 mL |
| Trypsin-EDTA (0.25%), phenol red | Thermo Fisher Scientific | 25200056 | 100 mL |
| Name of Materials/Equipment/Software | Company | Catalog Number | Comments/Description |
| anti – p-IκBα (B-9) | Santa cruze | sc-8404 | 200 µg/mL |
| anti-BclxL (H-5) | Santa cruze | sc-8392 | 200 µg/mL |
| anti-PARP 1 (C2-10) | Santa cruze | sc-53643 | 50 µl ascites |
| anti-β-actin (C4) | Santa cruze | sc-47778 | 200 µg/mL |
| BD FACSVerse | BD Biosciences | San Diego, CA, USA | |
| Synergy HTX Multi-Mode Microplate Reader | BioT | S1LFA | |
| CO2 incubator | Thermo fisher | HERAcell 150i | |
| Conical tube 15 ml | SPL | 50015 | |
| Conical tube 50 ml | SPL | 50050 | |
| Corning Costar Ultra-Low Attachment Multiple Well Plate | Sigma-Aldrich | CLS7007 | |
| Corning Costar Ultra-Low Attachment Multiple Well Plate | Sigma-Aldrich | CLS3471 | |
| Costar 50 mL Reagent Reservoirs, 5/Bag, Sterile | Costar | 4870 | |
| Countess Cell Counting Chamber Slides | Thermofisher | C10228 | |
| Countess II FL Automated Cell Counter | invitrogen | AMQAF1000 | |
| EnSpire Multimode Reader | Perkin Elmer | Enspire 2300 | |
| Eppendorf Research Plus Multi Channel Pipette, 8-channel | Eppendorf | 3122000051 | |
| FlowJo software | TreeStar | Ashland, OR, USA | |
| Goat Anti-Mouse IgG (H+L) | Jackson immunoresearch | 115-035-062 | 1.5 mL |
| Goat Anti-Rabbit IgG (H+L) | Jackson immunoresearch | 111-035-144 | 2.0 mL |
| GraphPad Prism 5 | GraphPad Software | Inc., San Diego, CA, USA | |
| ImageJ | NIH | ||
| ImageQuant LAS 4000 mini | Fujifilm | Tokyo, Japan | |
| Incubated shaker | Lab companion | SIF-6000R | |
| Multi Gauge Ver. 3.0, | Fujifilm | Tokyo, Japan | |
| Optical density (OD)LAMBDA UV/Vis Spectrophotometers | Perkin Elmer | Waltham, MA, USA | |
| Phase-contrast microscope | Olympus | Tokyo, Japan | |
| SPL microcentrifuge tube 1.5mL | SPL | 60015 | |
| SPL Multi Channel Reservoirs, 12-Chs, PS, Sterile | SPL | 21012 | |
| StepOnePlus Real-Time PCR system | Thermo Fisher Scientific | Waltham, MA, USA | |
| Vibra-Cell Ultrasonic Liquid Processors | SONICS-vibra cell | VC 505 | 500 Watt ultrasonic processor |
| Vinyl Anaerobic Chamber | COY LAB PRODUCTS |
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