Tredimensionel kultur af vaskulariseret termogenisk fedtvæv fra mikrovaskulære fragmenter
Summary
Her præsenterer vi en detaljeret protokol, der skitserer brugen af mikrovaskulære fragmenter isoleret fra gnaver- eller humant fedtvæv som en ligetil tilgang til at konstruere funktionelt, vaskulariseret beige fedtvæv.
Abstract
Engineering termogeniske fedtvæv (fx beige eller brune fedtvæv) er blevet undersøgt som en potentiel terapi for metaboliske sygdomme eller for design af personlige mikrovæv til sundhedsscreening og lægemiddeltestning. Nuværende strategier er ofte ret komplekse og undlader nøjagtigt fuldt ud at skildre de multicellulære og funktionelle egenskaber af termogeniske fedtvæv. Mikrovaskulære fragmenter, små intakte mikrokar bestående af arteriol, venuler og kapillærer isoleret fra fedtvæv, tjener som en enkelt autolog kilde til celler, der muliggør vaskularisering og dannelse af fedtvæv. Denne artikel beskriver metoder til optimering af dyrkningsbetingelser for at muliggøre generering af tredimensionelle, vaskulariserede og funktionelle termogeniske fedtvæv fra mikrovaskulære fragmenter, herunder protokoller til isolering af mikrovaskulære fragmenter fra fedtvæv og dyrkningsbetingelser. Derudover diskuteres bedste praksis, ligesom teknikker til karakterisering af de konstruerede væv, og prøveresultater fra både gnavere og humane mikrovaskulære fragmenter tilvejebringes. Denne tilgang har potentiale til at blive brugt til forståelse og udvikling af behandlinger for fedme og metabolisk sygdom.
Introduction
Målet med denne protokol er at beskrive en tilgang til udvikling af vaskulariseret beige fedtvæv fra en enkelt, potentielt autolog kilde, mikrovaskulært fragment (MVF). Brunt og beige fedtvæv har vist sig at vise gavnlige egenskaber relateret til metabolisk regulering; Den lille mængde af disse fedtvævsdepoter hos voksne begrænser imidlertid den potentielle indvirkning på systemisk metabolisme, især ved syge tilstande som fedme eller type 2-diabetes 1,2,3,4,5,6,7. Der er betydelig interesse for brunt / beige fedt som et terapeutisk mål for forebyggelse af de skadelige metaboliske virkninger forbundet med fedme og dens comorbiditeter 8,9,10,11,12.
MVF’er er karstrukturer, der kan isoleres direkte fra fedtvæv, dyrkes og vedligeholdes i en tredimensionel konfiguration i længere perioder13,14,15. Tidligere arbejde fra vores gruppe og andre er begyndt at udnytte MVF’ernes multicellulære og multipotente kapacitet, specifikt da det vedrører dannelse af fedtvæv16,17,18. Som en opbygning af dette arbejde demonstrerede vi for nylig, at MVF’er afledt af gnavermodeller af sund og type 2-diabetes19 og fra mennesker (voksne over 50 år)20 indeholdt celler, der kunne induceres til dannelse af termogent eller beige fedtvæv.
Heri er en innovativ tilgang, hvorfra en enkelt kilde MVF anvendes, ikke kun i stand til at skabe beige fedtvæv, men også dets tilknyttede og kritiske vaskulære komponent21. Brugen af denne teknik kunne være af stor værdi for undersøgelser på udkig efter en ligetil væv-manipuleret tilgang til termogeniske fedtvæv dannelse. I modsætning til andre metoder, der stræber efter at konstruere beige fedtvæv 22,23,24,25,26,27,28, kræver processen beskrevet i denne undersøgelse ikke anvendelse af flere celletyper eller komplekse induktionsregimer. Vaskulariserede beige og hvide fedtmodeller kan oprettes med MVF’er, der stammer fra gnavere og menneskelige kilder, hvilket viser et stort oversættelsespotentiale. Slutproduktet af denne protokol er en konstrueret beige termogeniske fedtvæv med en struktur og metabolisk funktion kan sammenlignes med brunt fedtvæv. Samlet set præsenterer denne protokol ideen om, at en let tilgængelig og muligvis autolog kilde MVF kan være en værdifuld terapeutisk intervention og værktøj til at studere metaboliske lidelser.
Protocol
Representative Results
Discussion
Området brun / beige fedtvævsteknik er stort set umoden 22,23,24,25,26,27,28, hvor hovedparten af fedtmodeller udvikles til hvidt fedtvæv 8,22,31. Konstrueret brunt / beige mikrovæv består typi…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dr. Acosta støttes af National Institutes of Health tilskud CA148724 og TL1TR002647. Dr. Gonzalez Porras støttes af National Institute of Diabetes og fordøjelses- og nyresygdomme ved National Institutes of Health under tildelingsnummer F32-0DK122754. Dette arbejde blev delvist støttet af National Institutes of Health (5SC1DK122578) og University of Texas i San Antonio Department of Biomedical Engineering. Indholdet er udelukkende forfatternes ansvar og repræsenterer ikke nødvendigvis de officielle synspunkter fra National Institutes of Health. Figurer blev delvist oprettet med Biorender.com.
Materials
| Aminocaproic Acid | Sigma Aldrich | A2504-100G | Added in DMEM at the concentration of 1 mg/mL |
| Blunt-Tipped Scissors | Fisher scientific | 12-000-172 | Sterilize in autoclave |
| Bovin Serum Albumin (BSA) | Millipore | 126575-10GM | Diluted in PBS to 4 mg/mL and 1 mg/mL |
| Collagenase Type 1 | Fisher scientific | NC9633623 | Diluted to 6 mg/mL in BSA 4 mg/mL, Digestion of minced fat |
| Dexamethasone | Thermo Scientific | AC230302500 | Diluted in ethanol at a 2 mg/ml stock concentration |
| Disposable underpads | Fisher scientific | 23-666-062 | For fluid absorption during surgery |
| Dissecting Scissors | Fisher scientific | 08-951-5 | Sterilize in autoclave |
| Dulbecco′s Modified Eagle′s Medium (DMEM) | Fisher scientific | 11885092 | |
| Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham (DMEM/F12) | Sigma Aldrich | D8062 | |
| Fetal Bovine Serum | Fisher scientific | 16140089 | Added in DMEM to 20% v/v. |
| Fibrinogen | Sigma Aldrich | F8630-25G | Solubilized in DMEM at the concentration of 20 mg/mL, Protein found in blood plasma and main component of hydrogel |
| Flask, 250 mL | Fisher scientific | FB500250 | Allows for digestion of fat using a large surface area |
| Forceps | Fisher scientific | 50-264-21 | Sterilize in autoclave, For handling of tissue and filters |
| Forskolin | Sigma Aldrich | F6886 | Diluted in ethanol at a 10 mM stock concentration |
| Human MVF | Advanced Solutions Life Scienes, LLC | https://www.advancedsolutions.com/microvessels | Human MVFs (hMVFs) isolated from three different patients (52-, 54-, and 56-year old females) were used in the current study. |
| Indomethacine | Sigma Aldrich | I7378 | Diluted in ethanol at a 12.5 mM stock concentration |
| Insulin from porcine pancreas | Sigma Aldrich | I5523 | Diluted in 0.01 N HCl at a 5 mg/ml stock concentration |
| MycoZap | Fisher scientific | NC9023832 | Added in DMEM to 0.2% w/v, Mycoplasma Prophylactic |
| Pennycilin/Streptomycin (10,000 U/mL) | Fisher scientific | 15140122 | Added in DMEM to 1% v/v. |
| Petri dishes, polystyrene (100 mm x 15 mm). | Fisher scientific | 351029 | 3 for removal of blood vessels and mincing, 8 (lid) for presoaking of screens & 8 (dish) for use when filtering with 500 or 37 µM screens |
| Petri dishes, polystyrene (35 mm x 10 mm). | Fisher scientific | 50-202-036 | For counting fragments |
| Phosphate Buffer Saline (PBS) | Fisher scientific | 14-190-250 | Diluted to 1x with sterile deionized water. |
| Rat Clippers (Andwin Mini Arco Pet Trimmer) | Fisher scientific | NC0854141 | |
| Rosiglitazone | Fisher scientific | R0106200MG | Diluted in DMSO at a 10 mM stock concentration |
| Scissors | Fine Science Tools | 14059-11 | 1 for initial incision, 1 for epididymal incision, 1 for tip clipping |
| Screen 37 µM | Carolina Biological Supply Company | 652222R | Cut into 3" rounded squares and sterilized in ethylene oxide, Fragment entrapment and removal of very small fragments/single cells and debris |
| Screen 500 µM | Carolina Biological Supply Company | 652222F | Cut into 3" rounded squares and sterilized in ethylene oxide, Removes larger fragments/debris |
| Serrated Hemostat | Fisher scientific | 12-000-171 | Sterilize in autoclave, For clamping of skin before incision |
| Steriflip Filter 0.22 μm | Millipore | SE1M179M6 | |
| Thrombin | Fisher scientific | 6051601KU | Diluted in deionzed water to 10 U/mL, Used as a clotting agent turning fibrinogen to fibrin |
| Thyroid hormone (T3) | Sigma Aldrich | T2877 | Diluted in 1N NaOH at a 0.02 mM stock concentration |
| Zucker diabetic fatty (ZDF) rats – obese (FA/FA) or lean (FA/+) male | Charles River | https://www.criver.com/products-services/find-model/zdf-rat-lean-fa?region=3611 https://www.criver.com/products-services/find-model/zdf-rat-obese?region=3611 |
Obtained from Charles River (Wilmington, MA). Rats were acquired at 4 weeks of age and fed Purina 5008 until euthanasia (15-19 weeks of age). Glucose levels (blood from the lateral saphenous vein) were greater than 300 mg/dL in all FA/FA rats used in the study. All animals were housed in a temperature-controlled environment with a 12-h light-dark cycle and fed ad libitum. |
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