Name: Application of a Novel Hyaluronan Hydrogel for Three-Dimensional Follicle Culture and Methodology for Mouse Ovarian Follicle Cryopreservation (Video) | JoVE Journal
Uploaded: 2025-05-09T13:40:00.000+00:00
Duration: 10 min 31 s
Description: Learn a novel 3D follicle culture method using hyaluronan hydrogel for mouse oocyte maturation. This technique preserves native architecture and includes vitrification protocols for effective cryopreservation.

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5 minutes

We want to acknowledge the entire embryology team at Cleveland Clinic for their assistance as well as the REI department and especially Dr. Falcone for support. This project was funded through a research fund at the Cleveland Clinic.

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</ol>

No conflicts of interest or disclosures.

The ability to control the mechanical properties and biodegradability of the tyramine-linked HA hydrogel offers many advantages for tissue engineering applications. Our laboratory is the first to apply this specific HA gel for ovarian follicle growth. This patented tyramine-substituted sodium hyaluronate gel (TS-NAHY) is a novel hyaluronan-based hydrogel system developed at the Cleveland Clinic. Crosslinking of the gel is driven by exposure of the peroxidase in the activated gel mix to an oxidizing agent. This can be done both in vitro or in vivo. Formed TS-NAHY hydrogels display a wide spectrum of properties from weak gel, a paste to a fragile solid, depending on the concentration of gel40.
The disappointing progress with ovarian follicle culture highlights the need to design new culture models. Creation of a culture system based exclusively on native extracellular matrix components may be a more advantageous approach. The HA culture model described is easy to use in a physiological manner with no requirement for additional ECM components. The gel is transparent, allowing detailed visualization of follicles. The viscoelastic properties of the HA gel facilitate the adjustment of rigidity as well as moldability. This feature increases the versatility of this biomatrix. Rigidity of a biomatrix can impact granulosa cell proliferation and antrum formation30,41,42. Follicles derived from culture environments permissive to antrum formation have been reported to have different gene expression profiles than those in a non-supportive culture system43. Primate follicles appear to require a more rigid matrix44. Tailoring the biomatrix to meet the requirements of different animal species, including humans, will likely be important for successful in vitro maturation.
For mouse preantral follicle growth, we have tested gel concentrations ranging from 2-5 mg/mL36. Oocyte maturation rates ranged from 44% to 58%. Higher concentrations of the HA gel allowed more moldability and retention of a 3-D structure, but follicle expansion was affected. Lower HA concentrations permitted more radial expansion of the follicle but increased the risk of the follicle being spontaneously extruded before the end of IVC. The 3-3.5 mg/mL concentration of HA worked best for mouse preantral follicles. Keeping the gel drop small was essential for forming HA gel beads with sufficient depth to keep follicles in a 3-D configuration throughout IVC. Larger drops flattened, resulting in the descent of follicles through the gel and attachment to the plate surface. One limitation of the use of this biomaterial for embedding is the extremely fast gelation time. It makes seeding more than two HA gel beads at a time difficult. Increasing the efficiency of follicle seeding into the HA gel is one area we are trying to improve on.
The culture of follicle clusters in a 3-D environment using HA has great potential. Normal ovarian architecture is retained, with different-sized follicles in contact with each other and the supporting stroma. LH may help the growth of smaller preantral follicles in the FL-C by inducing changes in the early differentiating thecal cells, so it was included in the FCM medium45. Continuing the culture of FL-C past 12 days to see if a new wave of growth can be initiated in any of the smaller follicles still embedded after the hCG trigger needs further study. Further optimization of the culture milieu may be a prerequisite for the cultivation of follicles of different size ranges in a tissue fragment. An advantage of this 3-D HA culture model with FL-C is that it allows close mimicking of in vivo follicular arrangement and interactions. Another important attribute is that unlike calcium alginate and other polymer systems, ovulation and maturation can be induced without physically removing the follicles from either the tissue or the gel matrix.
The time needed for in vitro follicle culture and obtaining mature oocytes for freezing is long, especially in large mammals. The ability to cryopreserve harvested follicles or follicle clusters presents a way to delay this step until a later and possibly more favorable time. If such technology can someday be applied to human follicles during the ovarian harvest, it may be beneficial. Whole ovary cryopreservation for fertility preservation is, at present, the only option for patients. But whether it is, in fact, the best method remains to be determined. In this paper, we present a vitrification methodology for preantral follicle cryopreservation that gives excellent post-warming survival and maturation rates after 3-D culture.
In conclusion, we have described a new 3-D culture model using hyaluronan, a component of native ECM. The HA encapsulation technique allows the retention of trophic factors in close proximity to the developing follicles. The methodology for encapsulation in the biomatrix is simple and can accommodate isolated follicles as well as follicle clusters. The latter may open new avenues of research and provide insight into the fundamental biology of folliculogenesis and its regulation. Oocytes from HA-encapsulated follicles have been shown to be functionally competent37. These oocytes can be fertilized, form blastocysts in vitro, and implant upon transfer to pseudo-pregnant mice. These data validate the use of tyramine-linked hyaluronan as a biomatrix for 3-D follicle culture and in vitro oocyte maturation. This protocol could potentially be applied to ovarian follicle culture in other animal models, including humans. Other possible applications for this 3-D HA gel system might be embryoid body and organoid culture.

Human in vitro folliculogenesis remain a challenge even four decades after the first birth from in vitro fertilization. To date, methodology for human ovarian follicle culture that supports the production of a viable embryo resulting in a healthy baby is still lacking1. The optimal physical properties necessary for human in vitro follicle growth remain to be determined. The intact ovary is populated with thousands of follicles at various stages of development, and regulation of their growth is a complex process (Figure 1)2. Germinal vesicle stage (GV) oocytes from human preantral follicles take as long as 30 days in culture to become meiotically mature and reach the metaphase II stage3. Bidirectional communication between the oocyte and surrounding granulosa cells through gap junctions is critical for cytoplasmic and nuclear maturation4,5,6.
Conventional 2-D culture systems are not ideal for follicle culture, especially in larger mammalian models that require extended time in culture. Follicles attach to the dish, and the link between granulosa cells and the oocyte becomes more tenuous as granulosa cells migrate away. Three-dimensional (3-D) culture systems for follicles have therefore emerged as a means to more closely mimic in vivo physiology7,8.
Encapsulation of follicles within a matrix to promote 3-D growth has been one approach for preserving follicular architecture during in vitro culture (IVC). Biomatrices from natural polymers (such as collagen, agarose, fibrin, alginate, and hyaluronic acid), as well as synthetic polymers (such as polyethylene glycol, polyvinyl alcohol, and polyglycolic acid), have been tested7,9,10,11,12,13. The mechanical properties of a biomatrix have been shown to influence nutrient diffusion, thecal cell differentiation, antrum formation as well hormonal secretion14. Collagen, as a part of the cell&#39;s natural extracellular matrix (ECM), is one of the earliest matrices tested and was initially promising15,16,17. However, the logistics of standardizing preparations of collagen, poor mechanical properties, and stability have limited its use18. Agarose has been tested on cumulus-oocyte complexes (COCs) released from antral follicles as well as primordial follicles19,20. More recently, a printed 3-D agarose mold has shown promise for scaffold-free follicle culture21. Calcium alginate encapsulation, first reported in 2003, has, to date, been the most widely studied system for IVC22. It has been tested on mice, bovine, monkey&#160;as well as human follicles23,24,25,26,27. With calcium alginate, follicles are singly loaded into micro drops of the polymer and exposed to calcium chloride to generate a gel bead. Extraction of follicles from the bead requires treatment with a chelating agent. However, this matrix has some drawbacks. Alginate is a polysaccharide isolated from algae, and while it provides support, it is not part of the follicle&#39;s natural extracellular matrix. Data suggest a higher incidence of spindle defects after IVC in alginate28. Later modifications of the system by combining alginate with fibrin or other extracellular matrix components (ECM) have helped make the calcium alginate system more effective29,30.
Growing evidence points to the extracellular matrix as a key modulator in cell growth10,31,32 . It not only provides support but plays a critical role in cell attachment, function, growth, and communication. One of the major components of ECM is hyaluronan, a naturally occurring glycosaminoglycan. In the ovarian follicle, hyaluronan is produced by granulosa cells and contributes to the structural integrity and function of the developing follicle33,34. Integration of hyaluronan into a follicle culture model may therefore aid in creating a more physiological environment and enhance production of functionally competent oocytes.
This work describes the novel application of a tyramine-linked hyaluronan as a biomatrix for fresh and frozen ovarian follicle cultivation and in vitro maturation of oocytes (IVM). We also detail techniques for follicle cryopreservation by vitrification on two types of devices. One method involves direct immersion in liquid nitrogen whereas in the second method follicles are enclosed in a straw before immersion. The primary aim is to show that despite differences, both methodologies and devices can be reliably used for follicle cryopreservation.

<table><tbody><tr><td>Anti-alpha tubulin-FITC labelled</td><td>Sigma-Aldrich</td><td>F2168</td><td></td></tr><tr><td>Anti-beta tubulin-FITC labelled</td><td>Sigma-Aldrich</td><td>F2043</td><td></td></tr><tr><td>BZ-X700</td><td>Keyence</td><td></td><td></td></tr><tr><td>Center well dish</td><td>Fisher Scientific</td><td>08-772-12</td><td></td></tr><tr><td>Collagenase Type I</td><td>Worthington Biochemical Corporation</td><td>LS004196</td><td></td></tr><tr><td>Crycap vial-vented</td><td>Hampton Research</td><td>HR4-904</td><td></td></tr><tr><td>Cryoloop</td><td>Hampton Research</td><td>HR4-974</td><td></td></tr><tr><td>Crystal cap</td><td>Hampton Research</td><td>HR4-733</td><td></td></tr><tr><td>Culture dish 60mm</td><td>Fisher Scientific&amp;nbsp;</td><td>08-772B</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Epidermal growth factor (EGF)</td><td>R &amp;amp;D Systems</td><td>236-EG</td><td></td></tr><tr><td>Ethylene Glycol (EG)</td><td>Sigma-Aldrich</td><td>293237</td><td></td></tr><tr><td>Fetal Bovine Serum-Heat Inactivated</td><td>ThermoFisher Scientific</td><td>10082-147</td><td></td></tr><tr><td>Follicle Stimulating Hormone (FSH)</td><td>Sigma-Aldrich</td><td>F4021</td><td></td></tr><tr><td>Global-Hepes&amp;nbsp; medium</td><td>CooperSurgical</td><td>LGGH-100</td><td></td></tr><tr><td>Hoechst 33342</td><td>Sigma-Aldrich</td><td>B2261</td><td></td></tr><tr><td>Human chorionic gonadotrophin (hCG)</td><td>Sigma-Aldrich</td><td>CG10</td><td></td></tr><tr><td>Human serum albumin</td><td>CooperSurgical</td><td>GHSA-125</td><td></td></tr><tr><td>Hyaluronidase</td><td>CooperSurgical</td><td>ART-4007-A</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>CVS Pharmacy Inc.</td><td>372441</td><td></td></tr><tr><td>Insulin-transferrin-selenium (ITS)</td><td>ThermoFisher Scientific</td><td>41400-045</td><td></td></tr><tr><td>Leibovitz medium (L-15)</td><td>ThermoFisher Scientific</td><td>11415-064</td><td></td></tr><tr><td>Luteinizing hormone</td><td>Sigma-Aldrich</td><td>L9773</td><td></td></tr><tr><td>Magnetic wand</td><td>Hampton Research</td><td>HR4-729</td><td></td></tr><tr><td>Micropipettes (1000 &amp;micro;m)</td><td>Minitube</td><td>19025/0050</td><td></td></tr><tr><td>Micropipettes (175 , 200, and 275&amp;micro;m)</td><td>CooperSurgical</td><td>MXL3-175, MXL3-200, MXL3-275</td><td></td></tr><tr><td>Millex GV filter 0.22 &amp;micro;m</td><td>Millipore</td><td>SLGU033RS</td><td></td></tr><tr><td>Mineral oil</td><td>CooperSurgical</td><td>LGOL-500</td><td></td></tr><tr><td>Minimum Essential Medium alpha (MEM)</td><td>ThermoFisher Scientific</td><td>32561-037</td><td></td></tr><tr><td>Oocyte Imaging System-Spindleview</td><td>Hamilton Thorne</td><td></td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>ThermoFisher Scientific</td><td>10010-023</td><td></td></tr><tr><td>Propidium iodide</td><td>Sigma-Aldrich</td><td>P4170</td><td></td></tr><tr><td>Rapid i</td><td>VitroLife</td><td>14406</td><td></td></tr><tr><td>SmartBox</td><td>VitroLife</td><td>14423</td><td></td></tr><tr><td>Synthetic Protein Substitute (SPS)</td><td>CooperSurgical</td><td>ART-3011</td><td></td></tr><tr><td>Tyramine -linked Hyaluronan Biohydrogel Kit&amp;nbsp;</td><td>LifeCore</td><td>ENG-00151</td><td></td></tr><tr><td>Ultrasonic sealer</td><td>VitroLife</td><td>14415</td><td></td></tr><tr><td>Universal GPS Culture dish 8x 100 &amp;micro;l wells</td><td>CooperSurgical</td><td>UGPS-010</td><td></td></tr></tbody></table>

application of a novel hyaluronan hydrogel for three-dimensional follicle culture and methodology for mouse ovarian follicle cryopreservation

Application of a Novel Hyaluronan Hydrogel for Three-Dimensional Follicle Culture and Methodology for Mouse Ovarian Follicle Cryopreservation

The 3-D architecture of the ovarian follicle and the complex interactions between somatic cell components and the oocyte that are necessary for cytoplasmic and nuclear maturation are difficult to maintain in conventional two-dimensional (2-D) culture systems. We describe a novel 3-D culture model using a tyramine-linked hyaluronan hydrogel for encapsulation and culture of mouse ovarian follicles. The hyaluronan encapsulation technique allows 3-D growth of follicles and retention of trophic factors in close proximity to the developing follicles. This hydrogel is highly versatile and can be applied to isolated follicles as well as ovarian tissue fragments. The viscoelastic properties of the HA gel enable adjustment of rigidity as well as moldability based on gel concentration. Preantral follicles developing in this culture model are able to complete meiotic maturation within 10-12 days of culture and ovulate a metaphase II oocyte upon triggering with hCG. This paper also details two approaches to ovarian follicle cryopreservation by vitrification.

This paper details methodology for using a novel tyramine linked hyaluronan gel for in vitro culture of mouse preantral follicles36,37. Figure 6 illustrates the differences between preantral follicle growth when placed in a conventional 2-D culture system versus a single follicle encapsulated in HA gel for 3-D culture. The native follicle architecture is maintained during the 12 days of culture with an antrum clearly visible on the last day of growth.
The HA gel is very versatile, allowing the growth of isolated follicles singly or in groups and also ovarian tissue is mechanically broken into small clusters of follicles. The gel is transparent making it possible to visualize follicles even if at different depths. Encapsulated follicles and FL-C exhibit radial expansion from continued granulosa cell proliferation (Figure 7). Initial follicle diameters average 139.8 ± 28 µm with the GV oocyte diameter measuring 63.5 ± 4.6 µm. In singly cultured follicles, final diameters measure at about 385.6 ± 36.7 µm, a roughly 3-fold increase in size. Ovulated metaphase II oocytes measure around 84.8 ± 3.8 µm. Within cultured FL-clusters, follicle size is fairly diverse (Figure 5, Figure 7). Ovulated oocytes after hCG trigger are found near the follicle (Figure 8). The majority of metaphase II oocytes will be retrieved from the ovulated COCs. Follicles still embedded after trigger usually contain GV and metaphase I oocytes.
Table 1 contrasts maturation rates between isolated follicles and FL-clusters from fresh or frozen ovaries. FL-C from cryopreserved ovaries had significantly lower maturation rates. Microscopic observations showed them to frequently have broken basal lamina, making them quite susceptible to premature oocyte extrusion. The fragile nature of follicle clusters was somewhat countered by encapsulation. Collagenase treatment of cryopreserved ovaries was avoided as it was especially damaging with low survival and low yield of intact follicles.
Cryopreservation of isolated follicles is much more effective than whole ovary preservation. High maturation rates can be achieved with both vitrification methods examined (Table 2). Despite large differences in cooling rates, oocyte maturation after IVC did not differ. The CL open carrier does allow for more efficiency as up to ten follicles can be loaded on a single CL open carrier. This also shortens the overall time for recovering multiple cryopreserved follicles. However, for any eventual clinical application of vitrification for human follicles the closed sealed system may be preferable.
Chromatin arrangement around the nucleolus of the GV oocyte can be used to identify oocytes most likely to fertilize after ovulation and develop to blastocysts38. Figure 9 illustrates live staining of oocytes to visualize the chromatin distribution pattern.
<img alt="Folliculogenesis stages diagram; primary to mature follicles, microscopic cell structure details." class="xfigimg" src="/files/ftp_upload/67786/67786fig01.jpg" data-altupdatedat="1783396841000" data-alt="Figure 1"> 
Figure 1: Schematic of follicle growth. This diagram illustrates the different stages of follicle development from a primary follicle to the secondary preantral stage and finally to a fully mature tertiary follicle ready for ovulation. A microscopic image of a typical preantral follicle is also shown with its different morphologic features. <a href="https://www.jove.com/files/ftp_upload/67786/67786fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Hyaluronan hydrogel preparation diagram; mixing process and experiment setup for cell encapsulation." class="xfigimg" src="/files/ftp_upload/67786/67786fig02.jpg" data-altupdatedat="1783396841000" data-alt="Figure 2"> 
Figure 2: Schematic of HA encapsulation method. Structure of the hyaluronan gel and the different steps for follicle embedding are illustrated in this diagram. <a href="https://www.jove.com/files/ftp_upload/67786/67786fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopic image of pollen grain sizes; diagrams A-E show measurements and cellular structures." class="xfigimg" src="/files/ftp_upload/67786/67786fig03.jpg" data-altupdatedat="1783396841000" data-alt="Figure 3"> 
Figure 3: Follicle isolation and encapsulation. (A, B) Preantral follicle selected for embedding at magnification 40x and 200x. (C) Apoptotic follicle is shown with healthy preantral follicle with oocyte not quite central. (D) Image of HA gel bead seeded with follicles and (E) with two FL-C. Images taken with a stereomicroscope to show the entire gel bead. <a href="https://www.jove.com/files/ftp_upload/67786/67786fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cryopreservation systems: open-loop LN setup, closed plastic straw assembly; process apparatus shown." class="xfigimg" src="/files/ftp_upload/67786/67786fig04.jpg" data-altupdatedat="1783396841000" data-alt="Figure 4"> 
Figure 4: Vitrification devices for cryopreservation of isolated follicles. With the CL open carrier device, the vitrification step is conducted by direct immersion of follicles into liquid nitrogen. The rate of cooling is, therefore, extremely high, over -20,000 °C/min. In contrast, with the RI closed carrier, follicles are loaded on the inner plastic stick and dropped into an outer straw immersed in liquid nitrogen. This closed vitrification method avoids direct contact with liquid nitrogen. However, the cooling rate is significantly lower at -1220 °C/min. Loading and recovery of follicles from either carrier is easy. The CL open carrier accommodated loading up to ten follicles per device as compared to just two with the RI closed carrier. This figure has been modified from35. <a href="https://www.jove.com/files/ftp_upload/67786/67786fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell aggregation process, microscope images of cell clusters at various stages, 50-200μm scale." class="xfigimg" src="/files/ftp_upload/67786/67786fig05.jpg" data-altupdatedat="1783396841000" data-alt="Figure 5"> 
Figure 5: Representative images of problems encountered. (A) Follicle cluster with oocyte being extruded. (B) Isolated follicles with broken basal lamina membrane and one with an extruded oocyte. (C) Embedded follicle under a bubble in the gel. (D) Follicle cluster that remained in gel (left) compared to cluster embedded too deep that eventually attached to the dish. The wide range of follicle sizes in FL-C is clearly visible. <a href="https://www.jove.com/files/ftp_upload/67786/67786fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Follicle development process diagram; 2D vs 3D culture, granulosa cell growth, oocyte maturation." class="xfigimg" src="/files/ftp_upload/67786/67786fig06.jpg" data-altupdatedat="1783396841000" data-alt="Figure 6"> 
Figure 6: Comparison of follicle growth in conventions 2-D versus 3-D culture in HA. With 2-D growth, flattening of the follicle and attachment of granulosa cells to the tissue culture dish was observed by Day 4, leaving the oocyte vulnerable to granulosa cell migration, disruption of gap junctions, and premature oocyte extrusion. The HA-encapsulated follicle remained unattached throughout the culture interval. Granulosa cell expansion occurred in all directions, encasing the oocyte and maintaining 3-D architecture. This figure has been modified from36. <a href="https://www.jove.com/files/ftp_upload/67786/67786fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell development stages; microscope images of organoid growth; scale bars; antrum formation focus." class="xfigimg" src="/files/ftp_upload/67786/67786fig07.jpg" data-altupdatedat="1783396841000" data-alt="Figure 7"> 
Figure 7: Representative images of follicles encapsulated in tyramine-linked hyaluronan gel. (A) Preantral follicle collected after collagenase digestion of fresh ovary on Day 1. (B) Gel drop seeded with four preantral follicles imaged on Day 1 and (C) Day 4 of culture (D) Follicle cluster from fresh ovary on day 2 (E) on Day 6 and (F) on Day 9 of culture. (G) Follicle cluster mechanically dissected from vitrified whole ovary shown on Day 2 and (H) on Day 6 of culture. (I) Follicle with antrum formation is clearly visible on Day 9 of culture. <a href="https://www.jove.com/files/ftp_upload/67786/67786fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Embryo cell stages, microscopy image sequence, developmental biology, cell division process." class="xfigimg" src="/files/ftp_upload/67786/67786fig08.jpg" data-altupdatedat="1783396841000" data-alt="Figure 8"> 
Figure 8: Oocyte ovulation. (A, B) Ovulated cumulus-oocyte complex (COC) shown next HA-gel bead. (C) Oocytes were imaged after enzymatic treatment of COCs with hyaluronidase to remove surrounding cumulus cells. Numerous metaphase II oocytes. (D) Metaphase II oocyte with prominent polar body. Magnification 400x. (E) Live imaging of metaphase II oocyte using polarized light and an imaging system to visualize meiotic spindle and assess organization, done as described in37. Magnification 400x. Normal birefringent spindle visible. (F) Metaphase II oocyte fixed and stained with anti-alpha/beta-tubulin and propidium iodide to visualize meiotic spindle organization. <a href="https://www.jove.com/files/ftp_upload/67786/67786fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy, cell division diagram, mitosis observation, experimental result, bioimaging." class="xfigimg" src="/files/ftp_upload/67786/67786fig09.jpg" data-altupdatedat="1783396841000" data-alt="Figure 9"> 
Figure 9: Chromatin reorganization in GV oocytes. Chromatin arrangement in GV oocytes after antrum formation was examined by staining of DNA with Hoechst 33342 (50 ng/mL). Representative follicles were harvested from HA beads by gentle pipetting. Granulosa cells were removed using hyaluronidase. GV oocytes were then stained for 15 min. (See protocol by Monti et al.38). Images were taken at 40x magnification. (A) GV oocyte shown on Day 1 at culture initiation exhibiting the non-surrounded chromatin (NSN) staining pattern. (B) GV oocyte from growing follicle with antrum shown on the day of hCG trigger. Chromatin condensed and formed a perinuclear ring around the nucleolus. <a href="https://www.jove.com/files/ftp_upload/67786/67786fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td colspan="2">Fresh Ovary</td>
			<td>Frozen Ovary</td>
		</tr>
		<tr>
			<td></td>
			<td>FL-Isolated</td>
			<td>FL-Cluster</td>
			<td>FL-Cluster</td>
		</tr>
		<tr>
			<td>Follicles Observed During IVC</td>
			<td>130</td>
			<td>154</td>
			<td>69</td>
		</tr>
		<tr>
			<td rowspan="2">Ovulation after HCG  (%)</td>
			<td>71%</td>
			<td>66%</td>
			<td>93%</td>
		</tr>
		<tr>
			<td> (92/130)</td>
			<td> (101/154)</td>
			<td>(64/69)</td>
		</tr>
		<tr>
			<td rowspan="2"> GVBD  (%)</td>
			<td>30%</td>
			<td>28%</td>
			<td>52%</td>
		</tr>
		<tr>
			<td> (28/92)</td>
			<td> (28/101)</td>
			<td>(33/64)</td>
		</tr>
		<tr>
			<td>MII oocyte formation (%)</td>
			<td>59%</td>
			<td>55%</td>
			<td>*34%</td>
		</tr>
		<tr>
			<td></td>
			<td>(54//92)</td>
			<td>(56/101)</td>
			<td>(22/64)</td>
		</tr>
	</tbody>
</table>
Table 1: Outcomes with HA-embedded follicles from fresh and vitrified ovaries. Follicles and FL-C from fresh ovaries were matured in vitro after encapsulation in HA gel. HA gel was also tested on follicles from ovaries vitrified using an EG/DMSO protocol39. With fresh ovaries, both individual follicles (FL) and follicle clusters (FL-C) were collected after collagenase digestion. For vitrified ovaries, exposure to collagenase was, in fact, damaging to follicles. The best approach with cryopreserved ovaries was to isolate FL-clusters rather than individual follicles and to only use mechanical dissection with needles. The table contrasts outcomes between HA beads seeded with follicles in groups of 4-6 versus beads with a single FL-C containing 6-10 follicles. *The maturation rate with FL-C from cryopreserved ovaries was significantly lower (p = 0.008; Chi-Square analysis to test for significance).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Carrier</td>
			<td>RI</td>
			<td>CL</td>
		</tr>
		<tr>
			<td>(Closed)</td>
			<td>(Open)</td>
		</tr>
		<tr>
			<td>Survival (%)</td>
			<td>100% (24/24)</td>
			<td>100% (41/41)</td>
		</tr>
		<tr>
			<td>Antrum formation* (%)</td>
			<td>25.0% (6/24)</td>
			<td>75.6% (31/41)*</td>
		</tr>
		<tr>
			<td>Ovulation rate (%)</td>
			<td>66.7% (16/24)</td>
			<td>87.8% (36/41)</td>
		</tr>
		<tr>
			<td>Maturation rate (% MIIs)</td>
			<td>81.3% (13/16)</td>
			<td>69.4% (25/36)</td>
		</tr>
	</tbody>
</table>
Table 2: Outcomes after cryopreservation of isolated follicles on two different vitrification devices. High maturation rates were achieved with both the open CL device as well as the closed RI carrier, with its lower cooling rate. Antrum formation was the only outcome measure observed to be significant but did not impact the overall maturation rate (p &lt; 0.05; Chi-square analysis to test for significance).

This protocol describes a novel three-dimensional (3-D) culture model using a tyramine-linked hyaluronan hydrogel to encapsulate and culture preantral follicles from the mouse ovary. We also detail two approaches to ovarian follicle cryopreservation by vitrification.

The 3-D architecture of the ovarian follicle and the complex interactions between somatic cell components and the oocyte that are necessary for ...

This research has been focused on developing a 3D culture system for in vitro follicle growth and oocyte maturation. Creating such a system using components of a cell's native extracellular matrix will enhance follicle development. The biggest challenge is applying the knowledge from animal models to human follicle culture.Access to human ovarian tissue is limited. And human follicles require a significantly longer time in culture to achieve oocyte maturation. The 3D culture model developed in this study may be especially useful for culturing human ovarian follicles, allowing retention of 3D architecture during the prolonged time needed in culture for oocyte maturation.To begin, obtain ovaries from 10 to 14 day old B6D2F1 mouse pups for the procedure. Pipette 6 milliliters of pre-equilibrated follicle culture medium, or FCM, into two 60 millimeter dishes and overlay with mineral oil, and incubate. Pipette 1 milliliter of collagenase to the center well of the dish, and 3 milliliters of ovarian tissue, or OT media, in the outer well.Move the ovaries to the collagenase solution. At the end of the collagenase incubation, move the collagenase treated ovaries to the outer well to rinse. Change micropipettes and transfer the ovaries to a fresh dish filled with OT medium.To harvest the follicles, repeatedly aspirate and expel enzyme treated ovarian tissue through pipette tips cut to different sizes. Mechanically tease tissue using 27 gauge needles to eight release of follicles. Examine under a dissecting microscope at 40x magnification and pick secondary preantral follicles with a centrally located oocyte enclosed within an intact basement membrane to a fresh OT dish.Place the FCM2 dish in the incubator for 60 minutes before starting the embedding process. Make two 25 microliter drops of HA gel without hydrogen peroxide on the lid for rinsing. Move follicles or follicle clusters to be embedded from the FCM dish to a drop of the hyaluronan, or HA gel, to rinse free of culture medium.Place the FCM dish back under the bubbler to gas. For embedding follicles, place 1 microliter of 0.03%hydrogen peroxide drop onto the lid of a 60 millimeter dish. Now, add 25 microliters of 3 milligrams per milliliter HA gel and mix.Pipette approximately eight to 10 microliters of the HA hydrogen peroxide mixture into two separate wells of the eight well culture dish. And quickly transfer the follicles, follicle clusters, or ovarian tissue fragments into the center of each drop. Ensure there are no bubbles within the drop.After three minutes, add 100 microliters of preequilibrated follicle culture medium to each well. Overlay with warm preequilibrated mineral oil and incubate the dish. Prepare the equipment and the solutions required for the vitrification process.Position the RI carrier into the slot in the cryo box containing liquid nitrogen. Place two drops of prewarmed vitrification solution 1, or VS1, side by side on the lid of a 60 millimeter dish near the top. Now, place two follicles in the first drop, rinse, and quickly move them to the second drop of VS1 for a five minute incubation.While the follicles incubate, make three VS2 drops just below the VS1 drops. Then quickly move the follicles sequentially through the three VS2 drops within 60 seconds. For vitrification, pick up the follicles from VS2.View the RI carrier under the dissecting scope. And adjust the focus to visualize the tiny hole in the plastic stick. Deposit follicles with minimal fluid into the hole.Drop the plastic stick into the precooled outer straw. And use an ultrasonic sealer to close the straw. Place the straw in a goblet attached to the cryo cane.Cover the cane with a plastic protective sleeve and plunge it into liquid nitrogen. For open carrier vitrification, prepare the carrier by inserting the metal CL stem into the magnetized vial cap, ensuring a firm hold. Place a rack into the cryo box to hold cryo vials, ensuring liquid nitrogen is below the top of the vials.Fill a magnetized invented cryo vial with liquid nitrogen and place in the rack. Grasp the CL open carrier by its attached magnetic cap using a magnetic wand. Dip the CL carrier in a separate drop of VS2 to create a cryoprotectant film.Pick up all follicles or follicle clusters and place them on the film with minimal fluid. Immediately immerse the CL into the cryo vial filled with liquid nitrogen to vitify the sample. Place the cryo vial on the cryo cane, and then cover with a plastic sleeve.Plunge the cane into a liquid nitrogen storage tank. Prepare a center well dish with 3 milliliters of preequilibrated FCM in the outer well and 1 milliliter in the center well. After overlaying the medium with oil, place the dish under the bubbler to gas.Place 0.5 milliliters of warming solutions WS1 and WS2 preequilibrated to 37 degrees Celsius into two labeled center well dishes. Move the cryo canes with samples from the storage tank into a cryo box filled with liquid nitrogen. First, remove the plastic cane cover.Then keeping the straw immersed, remove it from the goblet and slide it into the holding slot in the cryo box. Cut the outer straw just above the black dot. Lift the inner plastic stick out of the outer straw and quickly immerse the stick into WS1, gently swirling to unload the follicles within 10 seconds.After two minutes in WS1, use a micro pipette to transfer follicles or follicle clusters to WS2 for a three minute incubation. Then transfer them to an FCM dish and incubate the follicles for one to two hours in the center well before embedding, For open carrier CL warming, repeat the FCM and WS dish preparation. Transfer the cryo cane into a cryo box and remove the plastic cane cover.Use a magnetic wand to lift the cryo vial cap until the metal stem of the CL is visible. Grasp the cap with the attached CL carrier using the magnetic wand. And quickly immerse the CL into WS1, swirling to unload follicles within 10 seconds.Preantral follicle growth was maintained in the three-dimensional hyaluronan gel culture, preserving native follicle architecture with an antrum visible by day 12. Encapsulated follicles in hyaluronan gel exhibited radial expansion due to granulosis cell proliferation with follicle diameters increasing from around 139.8 micrometers to 385.6 micrometers over the culture period. Ovulated metaphase two oocytes were found near the follicle after human chorionic gonadotropin trigger.The oocytes measured approximately 84.8 micrometers in diameter. With fresh ovaries, both individual follicles and follicle clusters exhibited good maturation rates. Follicle clusters from cryopreserved ovaries exhibited lower maturation rates leading to premature oocyte extrusion.Isolated follicle cryopreservation was more effective than whole ovary preservation. Follicle vitrification on both carriers achieved high maturation rates, regardless of cooling rate differences. The CL open carrier system allowed for efficient cryo-preservation of multiple follicles, reducing processing time.

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Research

JoVE Journal

Biology

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.

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

Here, we describe a protocol outlining how to isolate human ovarian follicles from frozen-thawed cortical tissue to perform gene expression analyses.

Gene Expression Analyses in Human Follicles

We describe methods to culture ovarian follicles in a novel scaffold-free agarose mold that complement in vitro gametogenesis.

Production and Use of Customizable Agarose Molds for Scaffold-Free Mouse Ovarian Follicle Culture

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Learn a novel 3D follicle culture method using hyaluronan hydrogel for mouse oocyte maturation. This technique preserves native architecture and includes vitrification protocols for effective cryopreservation.

Application of a Novel Hyaluronan Hydrogel for Three-Dimensional Follicle Culture and Methodology for Mouse Ovarian Follicle Cryopreservation (Video) | JoVE Journal

Application of a Novel Hyaluronan Hydrogel for Three-Dimensional Follicle Culture and Methodology for Mouse Ovarian Follicle Cryopreservation

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