1Institute for BioNanotechnology in Advanced Medicine, Northwestern University, 2Department of Obstetrics and Gynecology, Northwestern University, Feinberg School of Medicine, 3Center for Reproductive Research, Northwestern University, 4The Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 5Department of Chemical and Biological Engineering, Northwestern University
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Shikanov, A., Xu, M., Woodruff, T. K., Shea, L. D. A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System. J. Vis. Exp. (49), e2695, doi:10.3791/2695 (2011).
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 .
1. Follicle Isolation
Experiments on animals were performed in accordance with the guidelines and regulations set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the established Institutional Animal Use and Care protocol at Northwestern University.
For optimal results, all dissections are carried out in L15 media for pH control at ambient levels of CO2, on 37°C heated stages for temperature control, and on a clean bench to minimize bacterial contamination. The dissection media (DM) is prepared with L15 media supplemented with 50 IU/mL penicillin and 50 μL/mL streptomycin and 1% FBS. The maintenance media (MM) is prepared with αMEM media supplemented with 50 IU/mL penicillin and 50 μL/mL streptomycin and 1% FBS.
2. Follicle Encapsulation, Method 1 - "The Drop Method"
3. Follicle Encapsulation, Method 2 - "The Parafilm Method"
4. Follicle Imaging and Media Change
5. Follicle Recovery from the 3D Matrix and in vitro Oocyte Maturation (IVM)
6. Representative Results:
We described a novel encapsulation method of ovarian follicles in a FA-IPN for in vitro culture (Figure 1). Ovarian follicles consist of an oocyte surrounded by several layers of somatic cells. Communication between the multiple cellular compartments is essential for healthy follicle development and oocyte maturation. Follicle encapsulation in a 3D hydrogel supports follicle expansion while maintaining the architecture of the follicle 9,11,12. As follicles develop, their volume expands exponentially and the encapsulating biomaterial should allow this expansion without the development of an inhibiting compressive force. FA-IPN is a network built of two natural materials, where fibrin is proteolytically degradable by plasmin activated by the follicle and alginate is biologically inert 13(Figure 2). During follicle growth, fibrin degradation begins locally near the follicle, and continues until the fibrin is cleared from the hydrogel. The non-degradable alginate component, which remains intact throughout the culture period, supports the 3D structure of the hydrogel.
Follicles were isolated at the secondary stage of development (150-180 μm diameter) and expanded to 400 μm at the antral stage of development in the FA-IPN gels. With hCG stimulation, the cultured follicles can undergo cumulus cell expansion, and oocytes can resume meiosis and progress to metaphase II for fertilization. These results suggest that the encapsulation method and the encapsulating material allowed follicle culture and successful maturation in vitro (Figure 3).
Fibrin degradation around the encapsulated follicles starts on the first day of culture and is completed by day 6. Aprotinin, a soluble plasmin inhibitor, can be used to alter fibrin degradation and to extend mechanical gradient in the encapsulating material (Figure 4). If follicles are cultured with 0.01 TIU/mL aprotinin, the fibrin is degraded slower for the first 4 days. Nonetheless, the follicles can still develop to the antral stage and oocytes remain competent to resume meiosis to metaphase II after aprotinin is removed.A high concentration of aprotinin (0.1 TIU/mL) significantly inhibits fibrin degradation, matrix stiffness prevents follicle expansion and somatic cell invasion into the matrix is observed.
Figure 1. Flowchart of follicle development. The ovarian follicle consists of a centrally located oocyte surrounded by one or more layers of somatic cells, which support oocyte development. As follicles develop from secondary to the preovulatory antral stage, the somatic cells surrounding the oocyte proliferate and differentiate, and the oocyte increases in size. in vitro maturation (IVM) is the final step for the follicle culture, when the somatic cells adjacent to the oocyte, termed cumulus cells, expand after hCG stimulation, and the oocyte resumes meiosis and progresses to a metaphase II (MII) stage.
Figure 2a. Alginate and fibrin-alginate for 3D follicle culture in vitro . (a)Alginate is a natural biomaterial that is suitable for in vitro follicle culture due to its gentle gelation and biochemical characteristics, such as mesh size, controllable rigidity and biological inertness. Alginate is a linear polysaccharide copolymer of α-L-guluronic acid (G) and β-D-mannuronic acid (M). Areas with repeating G monomers, termed "G-blocks", are cross-linked to form a hydrogel in the presence of divalent calcium ions.
Figure 2b. (b-i) Small encapsulated follicles experience low compressive force in alginate at the beginning of the culture. However, as the follicle expands the displaced volume in the bead is increasing, which results in greater compressive force in the opposite direction of follicle expansion (b-ii).
Figure 2c. The chains of individual polymers are completely entangled in the fibrin-alginate solution prior to cross-linking. Both components of FA-IPN start to cross-link immediately as they are exposed to the mixture of thrombin and calcium.
Figure 3a. Flowchart for follicle isolation and encapsulation in a FA-IPN. Secondary follicles are isolated from a 16-day old mouse (A-i). The reproductive organs are dissected (A-ii), and isolated follicles are transferred to a dish with maintenance media (A-iii).
Figure 3b. The drop method for follicle encapsulation in fibrinogen alginate solution (B:i-iv). Two drops of fibrinogen-alginate solution, the rinsing drop (10 μL) and the encapsulation drop (90 μL) are pipetted into the dish (B-i). Next 3-5 follicles are transferred to a rinsing drop (B-ii). After rinsing and media removal, follicles are transferred to the encapsulation drop (B-iii). Each follicle is aspirated with 5 μL fibrinogen-alginate solution with a 10 μL pipette tip and then expelled into thrombin/calcium solution (B-iv).
Figure 3c. The bead crosslinks for 5 minutes. The parafilm method for follicle encapsulation in fibrinogen alginate solution (C:i-iii). Fibrinogen-alginate solution (7.5 μL) is pipetted on the parafilm coated glass slide and follicles are transferred individually to each drop after rinsing (C-i, ii). Thrombin solution (7.5 μL) is added to each drop (C-iii).
Figure 3d - e. The drops are covered with a second parafilm coated glass slide and the FA-IPN is crosslinked in the incubator for 5 minutes. (D) The encapsulated follicles are transferred to growth media in a 96-well plate. (E) An image of an encapsulated follicle in the FA-IPN (white arrow).
Figure 4. Fibrin degradation by the growing follicle. Follicles degrade the fibrin component of the FA-IPN during the culture period, which is demonstrated by a clear circle around the follicle. The follicle's 3D architecture is supported by the remaining alginate. On day 0 (D0) the fibrin is still intact and the matrix around the follicle is cloudy; after 1 day in culture (D1) the clear ring around the follicle appears (white arrow) and the fibrin degradation front continues to radially expand on day 2 (D2) and day 4 (D4) of culture.
Figure 5. Images of follicle growth. Representative images ofsecondary follicle growth in the FA-IPN on day 0 (A), 4 (B), 6 (C) and 8 (D) of culture. After 8 days, antral follicles were matured in vitro , and the resulting MII stage oocytes are shown (E, the polar body is shown with black arrow).
Figure 6. Fibrin degradation was inhibited by aprotinin. Growing follicles on day 2, 4, 10 and 12 are shown in the first row. Aprotinin at concentrations 0.01 TIU/mL (second row) and 0.1 TIU/mL was added to the culture media on days 0, 2, and 4. Only follicle cultures with 0.01 TIU/mL aprotinin degraded the fibrin after aprotinin removal and reached antral stage. Follicles cultured in 0.1 TIU/mL aprotinin did not grow in the FA-IPN.
|CO2 incubator||37°C incubator with 5% CO2|
|COC||Cumulus oocyte complex|
|EGF||Epidermal growth factor|
|FA-IPN||Fibrin-alginate interpenetrating network|
|FBS||Fetal Bovine Serum|
|hCG||Human chorionic gonadotropin|
|ITS||Insulin Transferrin Selenium supplement|
|IVF dish||Center well 60 mm in vitro fertilization dish|
|MII stage oocyte||Metaphase II stage oocyte|
|rFSH||Recombinant Follicular Stimulating Hormone|
|TBS||Tris buffered saline|
|TIU||Trypsin inhibitory units|
Table 1. Abbreviations
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 culture. The dynamic mechanical properties obtained with a FA-IPN have been proposed to be consistent with the natural environment of the developing follicles and contributed to the improved rate of oocyte meiotic maturation comparing to alginate alone.
The FA-IPN demonstrates mild and fast gelation, with each component of the system cross-linking by an independent mechanism. We have described elsewhere 5 that the speed of the gel formation can be controlled by fibrinogen and thrombin concentrations. The degradation rate can be adjusted by aprotinin inhibition of fibrin proteolysis. Murine secondary follicles are usually cultured for 8-12 days and follicles from other species require longer culture periods. Thus, delayed fibrin degradation by aprotinin could potentially provide an extended dynamic environment for longer cultures.
The described encapsulation methods can be applied to other systems, such as the encapsulation and culture of micro-tissues or embryoid bodies, in which cell-cell contact can be retained yet the aggregate can partially degrade the matrix and create a space for expansion. In conclusion, the FA-IPN encapsulation method presents a sterile hydrogel culture system with dynamic, cell-responsive mechanical properties and a controllable degradation rate.
No conflicts of interest declared.
This work was funded by NIH (U54HD41857 and PL1EB008542, a P30 Biomaterials Core within the Oncofertility Consortium Roadmap grant).
|CaCl2||Wako Pure Chemical Industries, Ltd.||039-00475||40 mM|
|rFSH||National Institute of Diabetes and Digestive and Kidney Diseases|
|L-15||GIBCO, by Life Technologies||11415|
|αMEM+Gluta MAX||GIBCO, by Life Technologies||32561|
|TBS||Pierce, Thermo Scientific||28379|
|Tisseel Fibrin kit||Baxter Internationl Inc.||921030|
|Sodium Alginate||FMC BioPolymers||LF200DL||Mw 418kDa|