Generation af svin testikel Organoider med testis specifik arkitektur ved hjælp af Microwell Culture
Summary
Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.
Abstract
Organoider er tre dimensionelle strukturer bestående af flere celletyper, der er i stand til at rekapitulere væv arkitektur og funktioner af organer in vivo. Dannelsen af organoider har åbnet forskellige veje for grundlæggende og Translationel forskning. I de seneste år har testikel organoider høstet interesse i området for mandlig reproduktiv biologi. Testikel organoider giver mulighed for undersøgelse af celle-celle interaktioner, vævs udvikling, og kimcelle niche mikromiljø og lette høj gennemstrømning stof og toksicitet screening. En metode er nødvendig for pålideligt og reproducerbart generere testikel organoider med testikel specifikke væv arkitektur. Mikrobrøndens kultur system indeholder et tæt udvalg af pyramideformede mikrobrønde. Testikel celler afledt af præ-pubertale testiklerne centrifugeres ind i disse mikrobrønde og dyrkes for at generere testikel organoider med testis-specifikke væv arkitektur og celle foreninger. Tusindvis af homogene organoider kan genereres via denne proces. Den protokol, der rapporteres her, vil være af bred interesse for forskere, som studerer mandlig reproduktion.
Introduction
I de seneste år har der været en genopblussen af interesse i tredimensionale (3D) organoider. Forskellige organer såsom tarm1, mave2, bugspytkirtlen3,4, leveren5, og hjernen6 er blevet med succes afledt i 3D organoid systemer. Disse organoider har arkitektoniske og funktionelle ligheder med organerne in vivo og er mere biologisk relevante for undersøgelse af vævs mikromiljø end enkeltlags kultur systemer7. Som et resultat, testikel organoider er begyndt at samle interesse samt8,9,10,11,12. De fleste af metoder rapporteret indtil videre er komplekse, ikke-høj gennemløb10 og kræver tilsætning af ECM proteiner8,10. Denne kompleksitet fører også til problemer med reproducerbarhed. En enkel og reproducerbar metode er nødvendig, der giver mulighed for generering af testikel organoider med celle-foreninger, der er ligesom testikel in vivo.
Vi har for nylig rapporteret et system til at løse disse krav12. Ved hjælp af grisen som model anvendte vi en centrifugal tvungen aggregerings tilgang i strips med mikrobrønde-systemet. I strips med mikrobrønde-systemet indeholder hver brønd et stort antal identiske mindre mikrobrønde13. Dette giver mulighed for generering af talrige sfæroider af ensartet størrelse. Strips med mikrobrønde-systemet aktiverede generering af et stort antal ensartede organoider med en testis-specifik arkitektur. Systemet er simpelt og kræver ikke tilsætning af ECM-proteiner.
Protocol
Representative Results
Discussion
We have established a simple method that allows the consistent, repeatable generation of large numbers of testicular organoids with tissue architecture that is similar to testis in vivo12. While the approach was developed using porcine testis cells, it is more broadly applicable also to mouse, non-human primate and human testis12. A number of different methods have been reported for producing testicular organoids8,9…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH/NICHD HD091068-01 to Dr. Ina Dobrinski.
Materials
| 100 mm ultra low attachment tissue culture dish | Corning | #CLS3262 | |
| 100 mm tissue culture dish | Corning | #353803 | |
| Aggrwell 400 | Stemcell Technologies | #34411 | |
| Anti-Adherence Rinsing Solution | Stemcell Technologies | #07010 | |
| Collagenase type IV from Clostridium histolyticum | Sigma-Aldrich | #C5138 | referred as Collagenase IV S |
| Collagenase type IV Worthington | Worthington-Biochem | #LS004189 | referred as Collagenase IV W |
| Deoxyribonuclease I from bovine pancreas | Sigma-Aldrich | #DN25 | |
| Dulbecco’s Modified Eagle’s Medium/F12 | Gibco | #11330-032 | |
| Dulbecco’s Modified Eagle’s Medium – high glucose | Sigma-Aldrich | #D6429 | |
| Dulbecco’s Phosphate Buffered Saline | Sigma-Aldrich | #D8537 | |
| Epidermal Growth Factor | R&D Systems | #236-EG | |
| Falcon Cell Strainers 70 µm | FisherScientific | #352350 | |
| Falcon Cell Strainers 40 µm | FisherScientific | #352340 | |
| Fetal Bovine Serum | ThermoFisher Scientific | #12483-020 | |
| Insulin-Transferrin-Selenium | Gibco | #41400-045 | |
| Penicillin-Streptomycin | Sigma-Aldrich | #P4333 | |
| Porcine testicular tissue | Sunterra Farms Ltd (Alberta, Canada) | ||
| Steriflip-GP Sterile Centrifuge Tube Top Filter Unit | Millipore | #SCGP00525 | |
| Trypsin-EDTA | Sigma | #T4049 |
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