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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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.......

Representative Results

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 >95% purity (Figure 1).

To label and purify unique cellular fractions within the human antral follicles, we combined enzymatic digestion with flow cytometr.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Chemicals, reagents
Antibiotic-Antimycotic 100xThermo Fisher Scientific15240062Anti-Anti
Antifade Mountant solutionThermo Fisher Scientific P36930ProLong Gold
Collagenase from Clostridium histolyticumMillipore SigmaC 2674
DAPIThermo Fisher ScientificD1306
Dispase II, powderThermo Fisher Scientific17105041
DMSOMillipore SigmaD 2650Dimethyl sulfoxide
DPBS, no calcium, no magnesiumThermo Fisher Scientific14190144
Enzyme Cell Detachment Medium Thermo Fisher Scientific00-4555-56Accutase
Fetal Bovine Serum, heat-inactivatedThermo Fisher Scientific10438026
Hanks′ Balanced Salt solutionThermo Fisher Scientific14175079no calcium, no magnesium, no phenol red
Leibovitz’s L-15 medium Thermo Fisher Scientific11415064
Normal SalineQuality Biological114-055-101
SucroseMillipore SigmaS 1888
Freezing Medium (100 mL, filtered through a 0.2 micron filter)
- 69.64 mL of Leibovitz's L-15
- 17.66 mL of fetal bovine serum
- 3.42 g of sucrose
- 10.65 mL of DMSO
- 1 mL of antibiotic-antimycotic
Lab Plasticware and Supplies
6-well Clear Flat Bottom Not Treated Corning351146Falcon
Cell Strainer 100 µmFisher scientific352360Corning, Falcon
CryovialsThermo Fisher Scientific377267CryoTube 1.8 mL
Petri dish, D x H 150 mm x 25 mm Millipore SigmaCLS43059960EA
Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, 5 mLFisher scientific352235Corning, Falcon
Vacuum Filter/Storage Bottle System, 0.22 µmCorning431154
Antibodies
ANPEPBioLegend301703
CD34R&D SystemsFAB7227A
CD45BioLegend304019
CD55BioLegend311306
CD 99BioLegend371308
PVRLBioLegend340404
Surgical tools
long forceps (~150 mm length)Fisherbrand12-000-128Fisher Scientific
medium forceps (~110 mm length)Fisherbrand12-000-157Fisher Scientific
number 21 scalpelAndwin Scientific EF7281HFisher Scientific
number 11 scalpelAndwin Scientific FH/CX7281AFisher Scientific
sharp fine curved scissorsRoboz SurgicalRS-5881
Instruments
FACSJazz Flourescence activated cell sorterBD
LSM 710 META Confocal microscopeZeiss

References

  1. Aghadavod, E., et al. Isolation of granulosa cells from follicular fluid; Applications in biomedical and molecular biology experiments. Advanced Biomedical Research. 4, 250 (2015).
  2. Man, L., et al.

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