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Abstract
Introduction
Protocol
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Biologia

Etablering af 3D endometrieorganoider fra musens livmoder

Published: January 06, 2023
doi:
10.3791/64448
, , , , ,
1Department of Pathology & Immunology,Baylor College of Medicine, 2Center for Drug Discovery,Baylor College of Medicine, 3Departments of Molecular Virology and Microbiology and Medicine,Baylor College of Medicine

Summary

Denne protokol beskriver metoder til etablering af museendometrieepitelorganoider til genekspression og histologiske analyser.

Abstract

Endometrievæv linjer livmoderens indre hulrum og er under cyklisk kontrol af østrogen og progesteron. Det er et væv, der består af luminalt og kirtelepitel, et stromalt rum, et vaskulært netværk og en kompleks immuncellepopulation. Musemodeller har været et kraftfuldt værktøj til at studere endometrium og afsløre kritiske mekanismer, der styrer implantation, placentation og kræft. Den seneste udvikling af 3D endometrieorganoidkulturer præsenterer en state-of-the-art model til at dissekere de signalveje, der ligger til grund for endometriebiologi. Etablering af endometrieorganoider fra genetisk manipulerede musemodeller, analyse af deres transkriptomer og visualisering af deres morfologi ved en enkeltcelleopløsning er afgørende værktøjer til undersøgelse af endometriesygdomme. Dette papir skitserer metoder til at etablere 3D-kulturer af endometrieepitel fra mus og beskriver teknikker til kvantificering af genekspression og analyse af organoidernes histologi. Målet er at tilvejebringe en ressource, der kan bruges til at etablere, dyrke og studere genekspression og morfologiske egenskaber ved endometrieepitelorganoider.

Introduction

Endometrium – det indre slimhindevæv i livmoderhulen – er et unikt og meget dynamisk væv, der spiller kritiske roller i en kvindes reproduktive sundhed. I løbet af den reproduktive levetid har endometrium potentialet til at gennemgå hundredvis af cyklusser med spredning, differentiering og nedbrydning, koordineret af den samordnede virkning af ovariehormonerne – østrogen og progesteron. Undersøgelser af genetisk manipulerede mus har afdækket grundlæggende biologiske mekanismer, der understøtter endometrieresponset på hormoner og kontrol af embryoimplantation, stromalcelledecidualisering og graviditet1. In vitro-studier har imidlertid været begrænsede på grund af vanskeligheder med at opretholde ikke-transformeret primært museendometrievæv i traditionelle 2D-cellekulturer 2,3. Nylige fremskridt inden for kulturen af endometrievæv som 3D-organsystemer eller organoider giver en ny mulighed for at undersøge biologiske veje, der styrer endometriecellegenerering og differentiering. Mus og humane endometrieorganoidsystemer er blevet udviklet fra rent endometrieepitel indkapslet i forskellige matricer4,5, mens humant endometrium er blevet dyrket som stilladsfri epitel/stromale co-kulturer 6,7 og for nylig som kollagenindkapslede epitel-/stromale samlinger8 . Væksten og det regenerative potentiale af epitelorganoidkulturer understøttes af en defineret cocktail af vækstfaktorer og små molekylehæmmere, der er blevet empirisk bestemt til at maksimere vækst og regenerering af organoiderne 4,5,9. Desuden tillader evnen til at fryse og optø endometrieorganoider langsigtet bankning af endometrieorganoider fra mus og mennesker til fremtidige undersøgelser.

Gensplejsede mus har afsløret de komplekse signalveje, der styrer tidlig graviditet og decidualisering, og er blevet brugt som modeller for graviditetstab, endometriecancer og endometriose. Disse genetiske undersøgelser er stort set opnået med cellespecifik deletion af loxP-flankerede alleler (“floxed”) ved hjælp af cre-rekombinaser, der er specifikt aktive i kvindeligt reproduktionsvæv. Disse musemodeller omfatter den meget anvendte progesteronreceptor-cre 10, som har stærk rekombinaseaktivitet i endometrieepitel- og stromalvævet, lactoferrin i-cre, som inducerer endometrieepitelrekombination hos voksne mus11 eller Wnt7a-cre, som udløser epitelspecifik deletion i Müllerian-afledte væv12 . Dyrkning af endometrievæv fra genetisk manipulerede musemodeller som 3D-organoider har givet en glimrende mulighed for at undersøge endometriebiologi og lette identifikationen af vækstfaktorer og signalveje, der styrer endometriecellefornyelse og differentiering13,14. Metoder til isolering og dyrkning af museendometrievæv er beskrevet i litteraturen og rapporterer brugen af forskellige enzymatiske strategier til isolering af livmoderepitel til efterfølgende dyrkning af endometrieepitelorganoider4. Mens tidligere litteratur giver en kritisk ramme for endometrieepitelorganoidkulturprotokoller 4,5,6, giver dette papir en klar, omfattende metode til generering, vedligeholdelse, behandling og analyse af disse organoider. Standardisering af disse teknikker er vigtig for at fremskynde fremskridt inden for kvinders reproduktive biologi. Her rapporterer vi en detaljeret metode til enzymatisk og mekanisk oprensning af museendometrieepitelvæv til den efterfølgende kultur af endometrieorganoider i et gelmatrixstillads. Vi beskriver også metoderne til downstream histologiske og molekylære analyser af gelmatrix-indkapslede museendometrieepitelorganoider.

Protocol

Musehåndtering og eksperimentelle undersøgelser blev udført i henhold til protokoller godkendt af Institutional Animal Care and Use Committee (IACUC) ved Baylor College of Medicine og retningslinjer fastlagt af NIH Guide for pleje og brug af forsøgsdyr. 1. Isolering af livmoderepitel fra mus ved hjælp af enzymatiske og mekaniske metoder BEMÆRK: Dette afsnit beskriver de trin, der kræves for at etablere, passage, fryse og optø epitelendometrieo…

Representative Results

Fasekontrastbilleder af museendometrieorganoiderVi etablerede organoider fra WT-museendometrieepitel, som beskrevet i vedlagte protokol (se diagram i figur 1). Efter enzymatisk dissociation af museendometriepitelet blev epitelarkene mekanisk adskilt fra livmoderstromale celler og yderligere dissocieret med kollagenase for at generere en enkeltcellesuspension. Hvis denne metode til epitel- og stromalcelleseparation udføres korrekt, bør den give prøver med en kontaminer…

Discussion

Her beskriver vi metoder til at generere endometrieepitelorganoider fra museendometrium og de protokoller, der rutinemæssigt anvendes til deres nedstrømsanalyse. Endometrieorganoider er et kraftfuldt værktøj til at studere de mekanismer, der styrer endometrierelaterede sygdomme, såsom endometriose, endometriecancer og implantationsfejl. Skelsættende undersøgelser offentliggjort i 2017 rapporterede betingelserne for dyrkning af langsigtede og vedvarende kulturer af endometrieorganoider fra mus og humant epitel<sup …

Declarações

The authors have nothing to disclose.

Acknowledgements

Vi takker Dr. Stephanie Pangas og Dr. Martin M. Matzuk (M.M.M.) for kritisk læsning og redigering af vores manuskript. Undersøgelser blev støttet af Eunice Kennedy Shriver National Institute of Child Health and Human Development tilskud R00-HD096057 (D.M.), R01-HD105800 (D.M.), R01-HD032067 (M.M.M.) og R01-HD110038 (M.M.M.) og af NCI- P30 Cancer Center Support Grant (NCI-CA125123). Diana Monsivais, Ph.D. har en Next Gen Pregnancy Award fra Burroughs Wellcome Fund.

Materials

Organoid Media Formulation
Name Company Catalog Number Final concentration
Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-free Corning 354230 100%
Trypsin from Bovine Pancreas Sigma Aldrich T1426-1G 1%
Advanced DMEM/F12 Life Technologies 12634010 1X
N2 supplement Life Technologies 17502048 1X
B-27™ Supplement (50X), minus vitamin A Life Technologies 12587010 1X
Primocin Invivogen ant-pm-1 100 µg/mL
N-Acetyl-L-cysteine Sigma Aldrich A9165-5G 1.25 mM
L-glutamine Life Technologies 25030024 2 mM
Nicotinamide Sigma Aldrich N0636-100G 10 nM
ALK-4, -5, -7 inhibitor, A83-01 Tocris 2939 500 nM
Recombinant human EGF Peprotech AF-100-15 50 ng/mL
Recombinant human Noggin Peprotech 120-10C 100 ng/mL
Recombinant human Rspondin-1 Peprotech 120-38 500 ng/mL
Recombinant human FGF-10 Peprotech 100-26 100 ng/mL
Recombinant human HGF Peprotech 100-39 50 ng/mL
WNT3a R&D systems 5036-WN 200 ng/mL
Other supplies and reagents
Name Company Catalog Number Final concentration
Collagenase from Clostridium histolyticum Sigma Aldrich C0130-1G 5 mg/mL
Deoxyribonuclease I from bovine pancreas Sigma Aldrich DN25-100MG 2 mg/mL
DPBS, no calcium, no magnesium ThermoFisher 14190-250 1X
HBSS, no calcium, no magnesium ThermoFisher 14170112 1X
Falcon Polystyrene Microplates (24-Well) Fisher Scientific #08-772-51
Falcon Polystyrene Microplates (12-Well) Fisher Scientific #0877229
Falcon Cell Strainers, 40 µm Fisher Scientific #08-771-1
Direct-zol RNA MiniPrep (50 µg) Genesee Scientific 11-331
Trizol reagent Invitrogen 15596026
DMEM/F-12, HEPES, no phenol red ThermoFisher 11039021
Fetal Bovine Serum, Charcoal stripped Sigma Aldrich F6765-500ML 2%
Estratiol (E2) Sigma Aldrich E1024-1G 10 nM
Formaldehyde 16% in aqueous solution, EM Grade VWR 15710 4%
Epredia Cassette 1 Slotted Tissue Cassettes Fisher Scientific 1000961
Epredia Stainless-Steel Embedding Base Molds Fisher Scientific 64-010-15 
Ethanol, 200 proof (100%) Fisher Scientific 22-032-601 
Histoclear Fisher Scientific 50-899-90147
Permount Mounting Medium Fisher Scientific 50-277-97
Epredia Nylon Biopsy Bags Fisher Scientific 6774010
HistoGel Specimen Processing Gel VWR 83009-992
Hematoxylin solution Premium VWR 95057-844
Eosin Y (yellowish) solution Premium VWR 95057-848
TBS Buffer, 20X, pH 7.4 GenDEPORT T8054 1X
TBST (10X), pH 7.4 GenDEPORT T8056 1X
Citric acid  Sigma Aldrich C0759-1KG
Sodium citrate tribasic dihydrate Sigma Aldrich S4641-500G
Tween20 Fisher Scientific BP337-500 
Bovine Serum Albumin (BSA) Sigma Aldrich A2153-100G 3%
DAPI Solution (1 mg/mL) ThermoFisher 62248 1:1000 dilution
VECTASHIELD Antifade Mounting Medium Vector Labs H-1000-10
Clear Nail Polish Fisher Scientific NC1849418
Fisherbrand Superfrost Plus Microscope Slides Fisher Scientific 22037246
VWR Micro Cover Glasses VWR 48393-106
SuperScript VILO Master Mix ThermoFisher 11755050
SYBR Green PCR Master Mix ThermoFisher 4364346
Krt8 Antibody (TROMA-I)  DSHB TROMA-I  1:50 dilution
Vimentin Antobody Cell Signaling 5741S 1:200 dilution
Donkey anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary
Antibody, Alexa Fluor 594		 ThermoFisher A-21209 1:250 dilution
Donkey anti-Rabbin IgG (H+L) Highly Cross-Adsorbed Secondary
Antibody, Alexa Fluor 488		 ThermoFisher A-21206 1:250 dilution
ZEISS Stemi 508 Stereo Microscope ZEISS
ZEISS Axio Vert.A1 Inverted Routine Microscope with digital camera ZEISS
Primer Sequence Forward (5'-3') Reverse (5'-3') _
Lipocalin 2 (Lcn2) GCAGGTGGTACGTTGTGGG CTCTTGTAGCTCATAGATGGTGC
Lactoferrin (Ltf) TGAGGCCCTTGGACTCTGT ACCCACTTTTCTCATCTCGTTC
Progesterone (Pgr) CCCACAGGAGTTTGTCAAGCTC TAACTTCAGACATCATTTCCGG
Glyceraldehyde 3 phosphate dehydrogenase (Gapdh) CAATGTGTCCGTCGTGGATCT GCCTGCTTCACCACCTTCTT
DOWNLOAD MATERIALS LIST

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Tags

3D Endometrial OrganoidsMouse UterusUterine EpitheliumEnzymatic DissociationMechanical DissociationCell CultureOrganoid EstablishmentCell PelletCollagenaseDNA Solution
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Tang, S., Parks, S. E., Liao, Z., Cope, D. I., Blutt, S. E., Monsivais, D. Establishing 3D Endometrial Organoids from the Mouse Uterus. J. Vis. Exp. (191), e64448, doi:10.3791/64448 (2023).
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