Name: extraction, labeling, and purification of lineage-specific cells from human antral follicles
Uploaded: 2022-11-30T13:40:00
Description: Watch this Scientific Journal Video about Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles at JoVE.com

The authors acknowledge support from the Queenie Victorina Neri Research Scholar Award (D.J.) and the Hung-Ching Liu Research Scholar Award (L.M.). N.L.G is supported by the NYSTEM Stem Cell and Regenerative Medicine postdoctoral training grant.

All procedures involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) at Weill Cornell Medicine. All xenotransplantation experiments using ovarian tissue were performed in accordance with relevant guidelines and regulations. Both ovaries were isolated from a 14-year-old brain-dead organ donor with no history of radio/chemotherapy and no documented history of endocrine or reproductive conditions. The institutional review board (IRB) Committee of Weill Cornell Medicine approved the collecti...

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	<li>Aghadavod, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+granulosa+cells+from+follicular+fluid;+Applications+in+biomedical+and+molecular+biology+experiments.">Isolation of granulosa cells from follicular fluid; Applications in biomedical and molecular biology experiments.</a> Advanced Biomedical Research. 4, 250 (2015).</li><li>Man, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+antral+follicles+of+xenograft+versus+ovarian+origin+reveals+disparate+molecular+signatures.">Comparison of human antral follicles of xenograft versus ovarian origin reveals disparate molecular signatures.</a> Cell Reports. 32 (6), 108027 (2020).</li><li>Schmidt, K. L., Ernst, E., Byskov, A. G., Nyboe Andersen, A., Yding Andersen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+primordial+follicles+following+prolonged+transportation+of+ovarian+tissue+prior+to+cryopreservation.">Survival of primordial follicles following prolonged transportation of ovarian tissue prior to cryopreservation.</a> Human Reproduction. 18 (12), 2654-2659 (2003).</li><li>Jensen, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outcomes+of+transplantations+of+cryopreserved+ovarian+tissue+to+41+women+in+Denmark.">Outcomes of transplantations of cryopreserved ovarian tissue to 41 women in Denmark.</a> Human Reproduction. 30 (12), 2838-2845 (2015).</li><li>Newton, H., Aubard, Y., Rutherford, A., Sharma, V., Gosden, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+temperature+storage+and+grafting+of+human+ovarian+tissue.">Low temperature storage and grafting of human ovarian tissue.</a> Human Reproduction. 11 (7), 1487-1491 (1996).</li><li>Man, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+superphysiologic+AMH+promotes+premature+luteinization+of+antral+follicles+in+human+ovarian+xenografts.">Chronic superphysiologic AMH promotes premature luteinization of antral follicles in human ovarian xenografts.</a> Science Advances. 8 (9), 7315 (2022).</li><li>Gougeon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+follicular+growth+in+the+human:+A+model+from+preliminary+results.">Dynamics of follicular growth in the human: A model from preliminary results.</a> Human Reproduction. 1 (2), 81-87 (1986).</li><li>Richards, J. S., Ren, Y. A., Candelaria, N., Adams, J. E., Rajkovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+follicular+theca+cell+recruitment,+differentiation,+and+impact+on+fertility:+2017+update.">Ovarian follicular theca cell recruitment, differentiation, and impact on fertility: 2017 update.</a> Endocr Rev. 39 (1), 1-20 (2018).</li><li>Asiabi, P., Dolmans, M. M., Ambroise, J., Camboni, A., Amorim, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+theca+cells+from+ovarian+cells+isolated+from+postmenopausal+women.">In vitro differentiation of theca cells from ovarian cells isolated from postmenopausal women.</a> Human Reproduction. 35 (12), 2793-2807 (2020).</li><li>Dalman, A., Totonchi, M., Valojerdi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+human+theca+stem+cells+and+their+differentiation+into+theca+progenitor+cells.">Establishment and characterization of human theca stem cells and their differentiation into theca progenitor cells.</a> Journal of Cellular Biochemistry. 119 (12), 9853-9865 (2018).</li></ol>

Better resolution of the cellular diversity within the ovarian follicles is clinically important for several reasons. In applying the above protocol to the isolation of the unique phenotypic subtypes that reside within antral stage follicles, several factors should be considered. First, the health and viability of the ovarian tissue from which the antral follicle is derived is critical in determining the quality of the cells and the success of downstream applications. This can be optimized by minimizing the ischemic inte...

The primary functional elements of the human ovary are the follicles, which govern the growth and development of oocytes. Protocols for the isolation of follicular cells have been well established in the context of in vitro fertilization, but these are appropriate only for the collection of cells from luteinized follicles at the point of oocyte retrieval1. We have developed a protocol that enables the isolation of discrete cell populations from antral follicles at different developmental stages that arise from native ovaries or xenotransplanted ovarian tissue2. Although there is consensus that the contributions of follicle resident cells to the cultivation of the oocyte are highly important, few studies have prospectively identified and extracted the unique phenotypic subtypes present in antral-stage follicles. A deeper understanding of the differentiation hierarchy and signal transduction between specialized cells during the different developmental stages could broaden our understanding of ovarian physiology under homeostatic and pathological conditions. Moreover, the discrimination of discrete cellular subtypes and their molecular contributions to follicle growth/maturation may provide a means of generating ex vivo surrogates that reconstruct ovarian function to foster oocyte maturation and/or treat endocrine dysfunction. 
Each unique cell type within the ovary contributes to the complex function of the follicle, which effectively functions as a discrete mini-organ to foster the growth and maturation of the oocyte it contains. The oocyte, the centerpiece of the follicle, is directly enveloped by a continuous layer of granulosa cells (GCs), with the theca cells (TCs) forming a secondary layer of cells that combine with the oocyte and GCs to compose the follicular unit. Although classified into two groups, GCs and TCs contain numerous subtypes. GCs are classified according to their position within the follicle; GCs that surround the oocyte versus those that are adjacent to the basement membrane are designated as oophorous and mural GCs, respectively, and these subtypes display unique transcriptomic signatures. TCs have numerous subtypes that function to provide steroidogenic, metabolic, and structural support. Endothelial, perivascular, and immune cells play a central role in maintaining normal ovarian physiology. The ovarian stroma serves not only as a substrate for follicle growth but also likely provides a source of progenitors that give rise to TCs. This multilayered complex of cellular subtypes within the ovary is what enables its function as both an endocrine and a reproductive organ.
This paper presents a protocol for the identification and purification of granulosa, theca, stromal, endothelial, and hematopoietic cells from antral follicles. We have utilized this protocol to isolate these ovarian cells and analyze them using single-cell sequencing, followed by specific staining in follicles of different developmental stages. The protocol provides a straightforward methodology that is replicable and will enable the high-resolution analysis of both the physiology and pathology of the ovary.

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			<td>Antibiotic-Antimycotic 100x</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td>Anti-Anti</td>
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			<td>Antifade Mountant solution</td>
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			<td>Collagenase from&#160;Clostridium histolyticum</td>
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			<td>C 2674</td>
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			<td>DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
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			<td>Dispase II, powder</td>
			<td>Thermo Fisher Scientific</td>
			<td>17105041</td>
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			<td>DMSO</td>
			<td>Millipore Sigma</td>
			<td>D 2650</td>
			<td>Dimethyl sulfoxide</td>
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			<td>DPBS, no calcium, no magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
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			<td>Enzyme Cell Detachment Medium&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>00-4555-56</td>
			<td>Accutase</td>
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			<td>Fetal Bovine Serum, heat-inactivated</td>
			<td>Thermo Fisher Scientific</td>
			<td>10438026</td>
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			<td>Hanks&#8242; Balanced Salt solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>14175079</td>
			<td>no calcium, no magnesium, no phenol red</td>
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			<td>Leibovitz&#8217;s L-15 medium&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11415064</td>
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			<td>Normal Saline</td>
			<td>Quality Biological</td>
			<td>114-055-101</td>
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			<td>Sucrose</td>
			<td>Millipore Sigma</td>
			<td>S 1888</td>
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			<td>Freezing Medium (100 mL, filtered through a 0.2 micron filter)</td>
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			<td>- 69.64 mL of Leibovitz&#39;s L-15</td>
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			<td>- 17.66 mL of fetal bovine serum</td>
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			<td>- 1 mL of antibiotic-antimycotic</td>
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			<td>Lab Plasticware and Supplies</td>
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			<td>6-well Clear Flat Bottom Not Treated&#160;</td>
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			<td>Cell Strainer 100 &#181;m</td>
			<td>Fisher scientific</td>
			<td>352360</td>
			<td>Corning, Falcon</td>
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			<td>Cryovials</td>
			<td>Thermo Fisher Scientific</td>
			<td>377267</td>
			<td>CryoTube 1.8 mL</td>
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			<td>Petri dish, D x H 150 mm x 25 mm&#160;</td>
			<td>Millipore Sigma</td>
			<td>CLS430599</td>
			<td>60EA</td>
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			<td>Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, 5 mL</td>
			<td>Fisher scientific</td>
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			<td>Corning, Falcon</td>
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			<td>Vacuum Filter/Storage Bottle System, 0.22 &#181;m</td>
			<td>Corning</td>
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			<td>CD34</td>
			<td>R&#38;D Systems</td>
			<td>FAB7227A</td>
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			<td>CD45</td>
			<td>BioLegend</td>
			<td>304019</td>
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			<td>CD55</td>
			<td>BioLegend</td>
			<td>311306</td>
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			<td>CD 99</td>
			<td>BioLegend</td>
			<td>371308</td>
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			<td>PVRL</td>
			<td>BioLegend</td>
			<td>340404</td>
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			<td>Surgical tools</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>long forceps (~150 mm length)</td>
			<td>Fisherbrand</td>
			<td>12-000-128</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr>
			<td>medium forceps (~110 mm length)</td>
			<td>Fisherbrand</td>
			<td>12-000-157</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr>
			<td>number 21 scalpel</td>
			<td>Andwin Scientific&#160;</td>
			<td>EF7281H</td>
			<td>Fisher Scientific</td>
		</tr>
		<tr>
			<td>number 11 scalpel</td>
			<td>Andwin Scientific</td>
			<td>&#160;FH/CX7281A</td>
			<td>Fisher Scientific</td>
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			<td>sharp fine curved scissors</td>
			<td>Roboz Surgical</td>
			<td>RS-5881</td>
			<td></td>
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			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>FACSJazz Flourescence activated cell sorter</td>
			<td>BD</td>
			<td></td>
			<td></td>
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		<tr>
			<td>LSM 710 META Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
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extraction, labeling, and purification of lineage-specific cells from human antral follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

We isolated follicles from the surface of the ovary and enzymatically treated them to isolate the GCs as well as theca and stroma cells surrounding the antral cavity. The cells were collected, and the cell fractions were sorted from the antral follicles (diameters ranging between 0.5 mm and 4 mm) by FACS to &#62;95% purity (Figure 1).
To label and purify unique cellular fractions within the human antral follicles, we combined enzymatic digestion with flow cytometr...

Watch this Scientific Journal Video about Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles at JoVE.com

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...

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