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Biology
ヒト胞状卵胞からの系統特異的細胞の抽出、標識、精製
ヒト胞状卵胞からの系統特異的細胞の抽出、標識、精製
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JoVE Journal Biology
Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

ヒト胞状卵胞からの系統特異的細胞の抽出、標識、精製

Full Text
1,629 Views
06:36 min
November 30, 2022

DOI: 10.3791/64402-v

Limor Man1, Nicole Lustgarten Guahmich1, Eleni Kallinos1, Laury Arazi1, Zev Rosenwaks1,2, Daylon James1,2

1Center for Reproductive Medicine and Infertility,Weill Cornell Medical College, 2Tri-Institutional Stem Cell Derivation Laboratory,Weill Cornell Medical College

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Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study presents protocols for the identification and purification of ovarian cells from antral follicles, which are essential for understanding ovarian biology. It details methods for processing whole ovaries for cryopreservation while enabling the extraction of intact, enzymatically treated antral follicles to liberate various follicle-resident cell types.

Key Study Components

Research Area

  • Ovarian cell biology
  • Cell purification techniques
  • Follicle development and analysis

Background

  • Importance of characterizing ovarian cells
  • Relevance of antral follicles in reproductive health
  • Need for precise cell isolation methods

Methods Used

  • Labeling and isolation of specific cell lineages using fluorophore conjugated antibodies
  • Mouse ovaries as the biological system
  • Techniques including FACS for cell sorting, enzymatic digestion, and cryopreservation

Main Results

  • Successfully isolated and labeled granulosa, stroma, and theca cells from antral follicles
  • Demonstrated high purity (>95%) of isolated cell types
  • Identified specific markers for different cell populations, enhancing understanding of follicle composition

Conclusions

  • This study demonstrates effective methods for the isolation and characterization of ovarian cells.
  • The findings hold relevance for research on endocrine disorders and subsequent therapeutic approaches.

Frequently Asked Questions

What are the main cell types isolated in this study?
The main cell types isolated include granulosa cells, theca cells, stromal cells, endothelial cells, and hematopoietic cells.
Why is the isolation of ovarian cells important?
Isolating ovarian cells is crucial for understanding reproductive biology and the mechanisms underlying ovarian function and disorders.
What is the role of FACS in this protocol?
FACS (Fluorescence-activated cell sorting) is used to achieve high purity in cell populations by sorting based on the expression of cell surface markers.
How are the antral follicles processed?
Antral follicles are excised, enzymatically treated, and then subjected to FACS for cell isolation.
What implications does this research have?
The research can contribute to the understanding of ovarian cell dynamics and can be applied to toxicology studies and models of endocrine disorders.
Are there methods for cryopreservation discussed?
Yes, the study includes protocols for the cryopreservation of ovarian cortical strips for future analysis.
What types of assays can the isolated cells be used for?
The isolated ovarian cells can be utilized for molecular analysis, in vitro culture studies, and to model the ovarian microenvironment.

ここでは、胞状卵胞からの卵巣細胞の同定と精製のためのプロトコルを提示します。皮質ストリップの凍結保存のために卵巣全体を処理すると同時に、顆粒膜、テカ、内皮、造血、間質細胞など、複数の卵胞常在細胞タイプを遊離させるために酵素的に処理される無傷の胞状卵胞を採取する方法について詳しく説明しています。

ここで示すプロトコルは、胞状の初期段階から卵巣卵胞に存在する特定の細胞系譜の標識と単離を可能にし、それによって高分解能の分析と培養を可能にします。この非常に正確なアプローチでは、蛍光色素標識抗体の組み合わせを利用して、個別の顆粒膜、間質、およびλ骨系統の間で堅牢かつ特異的に発現する細胞表面マーカーを標的としています。卵巣を滅菌ペトリ皿に入れることから始め、冷やした培地を注ぎ、組織を水和させ、卵巣が培地に半分沈んでいることを確認します。

縫合糸やその他の材料を取り除き、残っている周囲の組織を卵巣から解剖します。胞状卵胞を分離するには、個々の卵胞を識別し、健康な卵胞を選択します。血液で満たされた、暗い、または非対称の卵胞を除外します。

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