Name: extraction, labeling, and purification of lineage-specific cells from human antral follicles
Uploaded: 2022-11-30T13:40:00.000+00:00
Duration: 6 min 36 s
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 collection of tissue, and approval from the family of the organ donor was obtained following informed consent regarding the use of tissue.
1. Ovarian tissue collection and handling
<ol>
	<li>Collect whole ovaries and the associated tissue, and rinse in sterile saline solution.</li>
	<li>Using sterile tweezers, place the ovaries in a sterile container. Pour sterile, buffered, cold solution (e.g., Leibovitz&#39;s L-15 medium) into the container. Ensure that the tissue is completely covered with the liquid.</li>
	<li>Close the container and double-bag it using sterile plastic bags. Transport the container on ice within an isolated box (e.g., Styrofoam). Transfer the specimens to the lab that will process the ovaries as soon as possible, preferably in a time not exceeding 5 h3,4.</li>
</ol>
2. Processing the ovarian tissue
NOTE: Make sure all the reagents and tools are prepared before the tissue arrives in the lab to reduce the ischemic interval as much as possible. The buffers can be prepared up to 1 week prior to freezing and refrigerated until use.
<ol>
	<li>Preparations for the ovarian processing and freezing
	<ol>
		<li>Set sterilized surgical tools in the biosafety cabinet in preparation for tissue processing: one number 21 scalpel; one number 11 scalpel; sharp fine curved scissors; one long forceps (~150 mm length); and two medium forceps (~110 mm length).</li>
		<li>Prepare 100 mL of tissue processing medium (see the Table of Materials): Leibovitz&#39;s L-15 medium containing antibiotic-antimycotic solution and filtered using a 0.2 &#956;m filter. Keep the medium chilled.</li>
		<li>Label the cryovials, add 1.5 mL of freezing solution (see the Table of Materials) to each vial, and store it at 4 &#176;C.</li>
		<li>Have available a 6-well plate on hand for use.</li>
	</ol>
	</li>
	<li>Upon arrival of the tissue
	<ol>
		<li>Spray the container with 70% ethanol solution, wipe thoroughly, and place it within the biosafety cabinet.</li>
		<li>Wearing sterile gloves, open the container. Place the ovary in a sterile Petri dish, and pour chilled medium (from step 2.1.2) to hydrate the tissue. Ensure that the ovary is half-submerged in the medium.</li>
		<li>Remove any sutures or other material and dissect the residual surrounding tissue away from the ovary.</li>
	</ol>
	</li>
</ol>
3. Isolation of antral follicles
<ol>
	<li>Identify individual follicles for isolation. In choosing healthy follicles, exclude any blood-filled, dark, or non-symmetric follicles, as those are likely to be atretic.</li>
	<li>Isolate the chosen antral follicles from the whole ovary using scalpels, keeping the antrum intact by excising the cortical tissue encompassing the follicle.</li>
	<li>Place each excised intact antral follicle in one well of the 6-well plate. If needed for descriptive purposes, use a ruler placed underneath the dish to determine the follicle diameter.</li>
	<li>Using a scalpel, bisect the intact antral follicle to gain access to the antral cavity, and add 4 mL of enzymatic cell detachment medium. Incubate the plate in a humidified incubator at 5% CO2 and 37 &#176;C for 10 min.</li>
	<li>Repeat the extraction of additional antral follicles as needed.</li>
</ol>
4. Isolation of follicle resident cells
<ol>
	<li>Following incubation, add 4 mL of available medium containing 20% FBS, and flush by repeated, vigorous pipetting of the medium over the bisected follicles with a P1,000 micropipette.</li>
	<li>Collect the medium, and pass it through a 100 &#956;m&#160;filter.</li>
	<li>Centrifuge the filtered supernatant at 300 &#215; g and 4 &#176;C for 3 min. Aspirate the supernatant, and reserve the cell pellet (enriched with GCs) on ice for labeling and fluorescence-activated cell sorting (FACS).</li>
	<li>Place the remaining tissue in a mixture of collagenase (100 U/mL) and dispase (1 U/mL) diluted in a balanced saline solution (e.g., Hanks), and incubate for 30 min at 5% CO2 and 37 &#176;C, mechanically disrupting the tissue by trituration and vigorous pipetting (i.e., 10-15 passes through the pipet tip) twice after 10 min and 20 min.</li>
	<li>Recover media containing the tissue, flush via pipetting through a P1,000 micropipette tip, and strain through a 100 &#956;m&#160;filter.</li>
	<li>Centrifuge at 300 &#215; g and 4 &#176;C for 3 min. Aspirate the supernatant, and put aside the cell pellet (enriched with TCs and stroma cells) on ice for labeling and FACS.</li>
</ol>
5. Fluorescence-activated cell sorting
<ol>
	<li>Resuspend the cell pellets in blocking solution (PBS + 0.1% FBS), and incubate for 10 min at 4 &#176;C.</li>
	<li>Centrifuge at 300 &#215; g and 4 &#176;C for 3 min, and aspirate the supernatant.</li>
	<li>Resuspend the cell pellets in blocking solution containing directly conjugated antibodies (see Table 1 for appropriate antibody combinations to identify GCs and TCs), and incubate on ice for 10 min.</li>
	<li>Wash and centrifuge the cells at 300 &#215; g and 4 &#176;C for 3 min. Resuspend the cells in FACS buffer containing 4&#8242;,6-diamidino-2-phenylindole (DAPI), and run the cell suspension through a FACS instrument to capture a small population of the cells (500-1,000) using the fluorescence intensity of fluorophore-conjugated antibodies for gating.</li>
	<li>For the identification of unique subpopulations, utilize the following gating strategies:
	<ol>
		<li>For the purification of GCs, draw a gate around the CD45+ cells (Figure 1B), and exclude this population from the CD99+ population (Figure 1A and Figure 1C); then, further subdivide the remaining CD99+ fraction into PVRL+/- fractions (Figure 1A and Figure 1C).</li>
		<li>For the purification of TCs and stroma, gate the total ENG+ (Figure 1A) or CD55+ (Figure 1D) population to exclude the CD34+ endothelial fraction (Figure 1E) and distinguish the steroidogenic (ANPEP+) and non-steroidogenic (ANPEP-) cells. Be careful to compensate for bleed-through between fluorophores that are close in the emission spectra.</li>
	</ol>
	</li>
	<li>To validate the purity of the sorted cell fraction, run 5% of the total captured volume through the FACS instrument, and ensure that cells populate the expected gates and are present at &#62;95% purity.</li>
</ol>
6. Processing of the ovarian cortical tissue
<ol>
	<li>Bisect the remainder of the ovary, and excise the medulla using curved fine scissors and scalpels, being careful to avoid damage to the ovarian cortex.</li>
	<li>Continue processing and thinning the ovarian cortex by gentle scraping until minimal medulla remains. Ensure to use the minimum force needed. 
	NOTE: The cortical tissue thickness should be between 1 mm and 1.5 mm.</li>
	<li>Divide the cortical tissue into 2-3 mm wide strips along the length of the ovary.</li>
</ol>
7. Ovarian tissue slow freezing
<ol>
	<li>Preparation for freezing
	<ol>
		<li>Place one cortical strip into each chilled cryogenic vial containing freezing solution.</li>
		<li>Shake the cryogenic vials containing the cortical strips on a rotating plate for 20 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Slow freezing of the ovarian tissue5
	<ol>
		<li>Place the cryovials containing ovarian tissue into a programmable freezer set to start at 0 &#176;C.</li>
		<li>Cool at a rate of 2 &#176;C/min until &#8722;7 &#176;C.</li>
		<li>Maintain the cryovials at this temperature for 10 min.</li>
		<li>Nucleate ice crystal formation by touching each cryovial with a cotton tip that has been briefly immersed in liquid nitrogen (LN2).</li>
		<li>Cool at a rate of 0.3 &#176;C/min until the sample temperature reaches &#8722;40&#176;C.</li>
		<li>Increase the rate of cooling to 10 &#176;C/min until the sample temperature reaches &#8722;140 &#176;C.</li>
		<li>Transfer the cryovials for storage in LN2.</li>
	</ol>
	</li>
</ol>

<ol>
	<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>

All authors declare they have no competing interests.

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 interval, working quickly (preferably in pairs), and preparing the laboratory spaces that will be used for the tissue processing and FACS. Second, these protocols are well-suited to antral-stage follicles precisely because they can be easily identified and extracted from the ovary to efficiently access the antral cavity; a similar approach for the study of early-stage and pre-antral follicles would yield a cell population with much greater heterogeneity. Finally, the number and quality of cells obtained from the follicles will vary widely depending on how developmentally advanced a follicle is and whether or not the follicle has entered a state of atresia. While these limitations make the study of the reconstruction of folliculogenesis a challenge, the efficient and robust isolation of follicle-derived cells will enable further interrogation of the mechanisms underlying human ovarian biology and infertility.
The purification of GCs from the antral follicles has been utilized previously by our group to demonstrate the deleterious influence of super physiologic levels of anti-Mullerian hormone (AMH) on follicle development6. In that study, cryopreserved and thawed ovarian tissue from both fertility preservation patients and organ donors was transplanted into immunocompromised mice in different experimental arms: control or AMH-expressing endothelial cells. Chronic exposure to AMH prompted a phenotype of accelerated luteinization that was reflected in increased follicle size, but this was only clearly elucidated in the molecular signature of follicle-resident GCs.
As the oocyte exits dormancy, GCs acquire a cuboidal morphology7, begin to proliferate, and together with the oocyte, secrete signals that recruit the surrounding stroma cells and promote their differentiation toward a TC fate8. Although the proper differentiation of stroma cells into specialized TCs is known to be essential for fertility, the mechanisms that drive TC specification and govern their interaction with follicle-resident cells during oocyte maturation are not well-defined. In humans, the cellular heterogeneity that exists in the TCs surrounding the follicle remains particularly elusive because, unlike GCs, which are readily accessible in an IVF setting, the isolation and culture of TCs require a direct biopsy of preovulatory follicles. In addition, most studies have relied on mechanical dissociation of the theca layer from isolated antral follicles9,10, yielding a heterogeneous mixture of stromal, theca, vascular, and immune populations.
Single-cell multi-omics approaches have revealed the cellular heterogeneity of numerous tissues and the diverse cell types that foster oocyte development within the ovary exist within follicles across a wide range of sizes and maturation stages. The methods described above complement existing protocols for the isolation and freezing of ovarian tissue for the purpose of fertility preservation and can be implemented in parallel for the procurement of follicle-resident cells for clinical or experimental purposes. The application of these methods will be important for deciphering the molecular mechanisms that govern human folliculogenesis and are a critical prerequisite to approaches aiming to foster oocyte development/maturation from primordial follicles in vitro.

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.

<table><tbody><tr><td>&lt;strong&gt;Chemicals, reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Antibiotic-Antimycotic 100x</td><td>Thermo Fisher Scientific</td><td>15240062</td><td>Anti-Anti</td></tr><tr><td>Antifade Mountant solution</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;P36930</td><td>ProLong Gold</td></tr><tr><td>Collagenase from&amp;nbsp;&lt;em&gt;Clostridium histolyticum&lt;/em&gt;</td><td>Millipore Sigma</td><td>C 2674</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>Dispase II, powder</td><td>Thermo Fisher Scientific</td><td>17105041</td><td></td></tr><tr><td>DMSO</td><td>Millipore Sigma</td><td>D 2650</td><td>Dimethyl sulfoxide</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo Fisher Scientific</td><td>14190144</td><td></td></tr><tr><td>Enzyme Cell Detachment Medium&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>00-4555-56</td><td>Accutase</td></tr><tr><td>Fetal Bovine Serum, heat-inactivated</td><td>Thermo Fisher Scientific</td><td>10438026</td><td></td></tr><tr><td>Hanks&amp;prime; Balanced Salt solution</td><td>Thermo Fisher Scientific</td><td>14175079</td><td>no calcium, no magnesium, no phenol red</td></tr><tr><td>Leibovitz&amp;rsquo;s L-15 medium&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11415064</td><td></td></tr><tr><td>Normal Saline</td><td>Quality Biological</td><td>114-055-101</td><td></td></tr><tr><td>Sucrose</td><td>Millipore Sigma</td><td>S 1888</td><td></td></tr><tr><td>&lt;strong&gt;Freezing Medium&lt;/strong&gt; (100 mL, filtered through a 0.2 micron filter)</td><td></td><td></td><td></td></tr><tr><td>- 69.64 mL of Leibovitz&#039;s L-15</td><td></td><td></td><td></td></tr><tr><td>- 17.66 mL of fetal bovine serum</td><td></td><td></td><td></td></tr><tr><td>- 3.42 g of sucrose</td><td></td><td></td><td></td></tr><tr><td>- 10.65 mL of DMSO</td><td></td><td></td><td></td></tr><tr><td>- 1 mL of antibiotic-antimycotic</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Lab Plasticware and Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>6-well Clear Flat Bottom Not Treated&amp;nbsp;</td><td>Corning</td><td>351146</td><td>Falcon</td></tr><tr><td>Cell Strainer 100 &amp;micro;m</td><td>Fisher scientific</td><td>352360</td><td>Corning, Falcon</td></tr><tr><td>Cryovials</td><td>Thermo Fisher Scientific</td><td>377267</td><td>CryoTube 1.8 mL</td></tr><tr><td>Petri dish, D x H 150 mm x 25 mm&amp;nbsp;</td><td>Millipore Sigma</td><td>CLS430599</td><td>60EA</td></tr><tr><td>Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, 5 mL</td><td>Fisher scientific</td><td>352235</td><td>Corning, Falcon</td></tr><tr><td>Vacuum Filter/Storage Bottle System, 0.22 &amp;micro;m</td><td>Corning</td><td>431154</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ANPEP</td><td>BioLegend</td><td>301703</td><td></td></tr><tr><td>CD34</td><td>R&amp;amp;D Systems</td><td>FAB7227A</td><td></td></tr><tr><td>CD45</td><td>BioLegend</td><td>304019</td><td></td></tr><tr><td>CD55</td><td>BioLegend</td><td>311306</td><td></td></tr><tr><td>CD 99</td><td>BioLegend</td><td>371308</td><td></td></tr><tr><td>PVRL</td><td>BioLegend</td><td>340404</td><td></td></tr><tr><td>&lt;strong&gt;Surgical tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><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&amp;nbsp;</td><td>EF7281H</td><td>Fisher Scientific</td></tr><tr><td>number 11 scalpel</td><td>Andwin Scientific</td><td>&amp;nbsp;FH/CX7281A</td><td>Fisher Scientific</td></tr><tr><td>sharp fine curved scissors</td><td>Roboz Surgical</td><td>RS-5881</td><td></td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FACSJazz Flourescence activated cell sorter</td><td>BD</td><td></td><td></td></tr><tr><td>LSM 710 META Confocal microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

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 cytometric sorting. We previously identified surface proteins that specifically mark follicle-resident fractions2. We have shown that CD99 (red in Figure 1A) specifically labels GCs, and PVRL1 (yellow in Figure 1A) is increasingly localized to the oophorous GC compartment as the antral follicles increase in size2. We have also shown that antibodies against endoglin (ENG, green in Figure 1A) or CD55 (Figure 1D) specifically demarcate all stroma and TCs and that alanine aminopeptidase (ANPEP, Figure 1E) is specifically expressed by androgenic TCs2,6.
The cells were labeled with an antibody directed against CD99 to identify GCs, and antibodies against CD45 were used to exclude hematopoietic cells (Figure 1B,C). Antibodies against PVRL1 enabled the separation of the GC population into oophorous and mural compartments (Figure 1C). Following enzymatic digestion with collagenase/dispase, the cell preparations labeled with antibodies against CD55 (or alternatively ENG), CD34, and ANPEP enabled the capture of all the stroma/TCs (CD55+) and/or androgenic TCs (ANPEP+), with the exclusion of endothelial cells (CD34+) from the TC populations (Figure 1D,E). Using this panel and a stepwise enzymatic dissociation protocol, we labeled and purified hematopoietic, endothelial, granulosa (oophorous and mural), and theca (stromal and androgenic) cells from the antral follicles of freshly resected ovaries.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64402/64402fig01.jpg" /> 
Figure 1: Labeling and purification of lineage-specific cells from human antral follicles. (A) Representative micrograph showing GC (CD99+ PVRL+/-) and TC (ENG+) compartments within human follicles at the antral stage; white stroke boxes are magnified to the right. (B-E) Representative sort plots identifying GCs as CD45-&#160;CD99+ PVRL1+/- cells (a and b) as well as generic (CD34-&#160;CD55+) and androgenic (CD34- CD55+ ANPEP+) TCs (C,D). Gated populations are shared between (B,C) and (D,E); unstained controls are shown to the left in (B-E). Scale bar = (A) 100 &#181;m. Abbreviations: GC = granulosa cell; TC = theca cell; PVRL = nectin; ANPEP = alanine aminopeptidase; Nuc = nuclei; SSC = side scatter; PE = phycoerythrin; APC = allophycocyanin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64402/64402fig01large.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>Cell Type</td>
			<td>Inclusion Antibodies</td>
			<td>Exclusion Antibodies</td>
		</tr>
		<tr>
			<td>Granulosa cells</td>
			<td>CD99 (PVRL+/-)</td>
			<td>CD45</td>
		</tr>
		<tr>
			<td>All Theca Cells</td>
			<td>CD55 or ENG</td>
			<td>CD34 &#38; CD45</td>
		</tr>
		<tr>
			<td>Steroidogenic Theca Cells</td>
			<td>ANPEP</td>
			<td>CD34 &#38; CD45</td>
		</tr>
	</tbody>
</table>
Table 1: List of antibodies that can be used to identify GCs, TCs, and ovarian stroma. Abbreviations: GC = granulosa cell; TC = theca cell.

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 ...

extraction-labeling-purification-lineage-specific-cells-from-human

Nanyang Technological University

Research

JoVE Journal

Biology

1.1K Views.  Weill Cornell Medical College. Het hier gedemonstreerde protocol maakt het labelen en isoleren mogelijk van specifieke cellijnen die aanwezig zijn in ovariële follikels vanaf vroege antrale stadia, waardoor de analyse en cultuur met hoge resolutie mogelijk wordt. Deze zeer nauwkeurige benadering maakt gebruik van combinaties van fluorofoor geconjugeerde antilichamen om celoppervlakmarkers te targeten die robuust en specifiek tot expressie komen tussen discrete granulosa-, stroma- en theca-afstammingslijnen. Begin met het ...

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

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

Developmental Biology

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

An Efficient Method for Extracting Human Fallopian Tube Epithelia for Single-cell Analyses

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if fertilization is successful, the embryo. The epithelial cells that line the inner surface of fallopian tubes are integral to both normal and abnormal fallopian tube processes, including disease initiation. After menopause, fallopian tubes cease to have a significant role in the body and the intraepithelial cell makeup changes. We describe a method in which these epithelial cells can be isolated from fresh fallopian tubes with minimal stromal cell contamination into a single-cell suspension. These cells can be grown in culture or used for further analysis such as flow cytometry and single-cell RNA sequencing. This isolation protocol can be achieved in 4-6 h and yields viable cells that can be used for immediate downstream analysis. This efficient protocol facilitates the isolation of fallopian tube epithelial cells with an enriched epithelial population.

This protocol describes a one-day method for the isolation of human fallopian tube epithelial cells. Isolated epithelial cells can be plated in 2-dimensional (2D) culture or dissociated into single-cell suspensions and utilized in downstream experiments, including flow cytometry and single-cell RNA sequencing.

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if ...

Cancer Research

Gene Expression Analyses in Human Follicles

The ovary is a heterogeneous organ composed of different cell types. To study the molecular mechanisms occurring during folliculogenesis, the localization of proteins and gene expression can be performed on fixed tissue. However, to properly assess gene expression levels in a human follicle, this complex and delicate structure must be isolated. Hence, an adapted protocol previously described by Woodruff&#39;s laboratory has been developed to separate follicles (the oocyte and the granulosa cells) from their surrounding environment. The ovarian cortical tissue is first manually processed to obtain small fragments using two tools: a tissue slicer and a tissue chopper. The tissue is then enzymatically digested with 0.2% collagenase and 0.02% DNase for at least 40 min. This digestion step is performed at 37 &#176;C and 5% CO2 and is accompanied by mechanical pipetting of the medium every 10 min. After incubation, the isolated follicles are collected manually using a calibrated microcapillary pipette under microscope magnification. If follicles are still present in the pieces of tissue, the procedure is completed with manual microdissection. The follicles are collected on ice in a culture medium and are rinsed twice in droplets of phosphate-buffered saline solution. This digestion procedure must be carefully controlled to avoid follicle deterioration. As soon as the structure of the follicles appears to be compromised or after a maximum of 90 min, the reaction is stopped with a 4 &#176;C blocking solution containing 10% fetal bovine serum. A minimum of 20 isolated follicles (sized under 75 &#181;m) should be collected to obtain an adequate amount of total RNA after RNA extraction for real-time quantitative polymerase chain reaction (RT-qPCR). After extraction, the quantification of total RNA from 20 follicles reaches a mean value of 5 ng/&#181;L. The total RNA is then retrotranscribed into cDNA, and the genes of interest are further analyzed using RT-qPCR.

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

The ovary is a heterogeneous organ composed of different cell types. To study the molecular mechanisms occurring during folliculogenesis, the localization ...

Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and endangered species. Research has been mostly limited to antral and large preantral follicles due to difficulty in the isolation of smaller preantral follicles, especially in large mammals such as bovine species. This work presents an efficient approach to retrieve large numbers of small preantral follicles from a single bovine ovary. The cortex of individual bovine ovaries was sliced into 500 &#181;m cubes using a tissue chopper and homogenized for 6 min at 9,000-11,000 rpm using a 10 mm probe. Large debris was separated from the homogenate using a cheese cloth, followed by serial filtration through 300 &#181;m and 40 &#181;m cell strainers. The contents retained in the 40 &#181;m strainer were rinsed into a search dish, where follicles were identified and collected into a drop of medium. The viability of the collected follicles was tested via trypan blue staining. This method enables the isolation of a large number of viable small preantral follicles from a single bovine ovary in approximately 90 min. Importantly, this method is entirely mechanical and avoids the use of enzymes to dissociate the tissue, which may damage the follicles. The follicles obtained using this protocol can be used for downstream applications such as isolation of RNA for RT-qPCR, immunolocalization of specific proteins, and in vitro culture.

Advancing the study of preantral folliculogenesis requires efficient methods of follicle isolation from single ovaries. Presented here is a streamlined, mechanical protocol for follicle isolation from bovine ovaries using a tissue chopper and homogenizer. This method allows collection of a large number of viable preantral follicles from a single ovary.

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and ...

Separation of Hen Ovarian Follicle Layers for Reproductive Research Applications

Separation of Avian Preovulatory Follicle Granulosa and Theca Cell Layers for Downstream Applications

Layer hens (egg-laying chickens) and broiler breeders (breeding stock for meat-producing chickens) are crucial to the world's food supply as a reliable source of protein. They are also an emerging animal model for the study of human reproductive disease. As the field of poultry research develops, the health and function of the layer hen and broiler breeder ovary will be an important point of study for both agricultural and biomedical researchers. One of the challenges presented by this emerging interest is the need for replicable techniques that all researchers can employ in ovarian specimen collection. In particular, a detailed visual process must be established to define the proper separation of the specialized granulosa and theca cell layers from hen follicles to achieve agreement and consistency among researchers. 
This study describes the extraction of preovulatory follicles and ovary tissue in white leghorn hens of prime reproductive age. The separation of these follicles is performed under cold, liquid conditions to congeal the yolk for easier manipulation and to prevent the follicle's own weight from tearing apart cell layers during the separation process. Once the separation is complete, the desired cell layers can be further digested for tissue culture approaches or can be cryopreserved for genomic and proteomic analyses.

Here, we describe a protocol for separating yolk, granulosa cells, and theca cells in avian preovulatory follicles. This precision handling enables critical investigations into the role of these layers in reproductive function, aiding the understanding of follicular development, hormonal regulation, and disease research for enhanced agricultural yield and biomedical insights.

Layer hens (egg-laying chickens) and broiler breeders (breeding stock for meat-producing chickens) are crucial to the world's food supply as a reliable ...

Mouse Antral Oocyte Isolation: A Method to Isolate Antral Oocytes from Freshly Harvested Mouse Ovaries

Source: Monti, M. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/53616" target="_blank">Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization</a>. J. Vis. Exp. (2016)
This video describes the isolation of antral oocytes from freshly harvested mouse ovaries. These antral oocytes can be used for further characterization and developmental studies.

Source: Monti, M. et al. Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization. J. Vis. Exp. (2016)
This video ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video