A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System
Summary
A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.
Abstract
The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .
Protocol
Discussion
The presented ovarian follicle encapsulation method in a FA-IPN allows follicle culture in a 3D environment in vitro. A FA-IPN is a dynamic, cell-responsive matrix in which the initial mechanical properties are determined by the combination of both fibrin and alginate. During the culture, the encapsulated follicle activates proteases that degrade only one component of the IPN, the fibrin, which results in a gradually decreasing gel rigidity that is contributed solely by the remaining alginate at the end of the c…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was funded by NIH (U54HD41857 and PL1EB008542, a P30 Biomaterials Core within the Oncofertility Consortium Roadmap grant).
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Fetuin | Sigma-Aldrich, St. Louis, MO | F3385 | ||
| FBS | Invitrogen, Gibco | 10082-139 | ||
| Aprotinin | Roche | 10236624001 | ||
| CaCl2 | Wako | 039-00475 | 40 mM | |
| EGF | Sigma | A412 | ||
| rFSH | A.F. Parlow, National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases | |||
| hCG | Sigma | CG-5 | ||
| Hyaluronidase | Sigma | A1603 | ||
| ITS | Sigma | I1884-1VL | ||
| L-15 | Gibco | 11415 | ||
| αMEM+Gluta MAX | Gibco | 32561 | ||
| Pen-Strep | Cellgro | 30-002-CI | ||
| TBS | Pierce | 28379 | ||
| Tisseel Fibrin kit | Baxter | 921030 | ||
| Sodium Alginate | FMC BioPolymers | LF200DL | Mw 418kDa |
Riferimenti
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