Obstetrics and Gynaecology Department, GROW - School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands.
Reproductive Sciences Laboratory, Division of Genetics and Cell Biology, IRCCS Ospedale San Raffaele, Milan, 20132, Italy.
Hum Reprod. 2020 Feb 29;35(2):364-376. doi: 10.1093/humrep/dez266.
Are the primary cell cultures and cell lines used in endometriosis research of sufficient quality?
Primary cells used in endometriosis research lack purity and phenotypic characterisation, and cell lines are not genotypically authenticated.
The poor reproducibility of in vitro research and the lack of authenticity of the cell lines used represent reasons of concern in the field of reproductive biology and endometriosis research.
STUDY DESIGN, SIZE, DURATION: In the present study, past in vitro research in the field of endometriosis was systematically reviewed to determine whether the appropriate quality controls were considered. In addition, we explored the performance of Paired Box 2 (Pax2) as an endometrium specific marker in endometrial and endometriotic primary cell cultures; we also characterised the most diffused endometriosis cell lines with respect to important markers including the short tandem repeat (STR) profile.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Literature review part: almost 300 published protocols describing the isolation and creation of primary cell cultures from endometriosis were reviewed. Wet-lab part: primary cells isolated from 13 endometriosis patients were analysed by immunohistochemistry, immunofluorescence and FACS for the expression of Pax2. Cell lines Z11 and Z12, the most diffused endometriosis cell lines, were characterised with respect to the expression of Pax2, steroid hormone receptors and STR profile.
From the literature review work, we underscored the lack of sufficient cell purity and phenotypic characterisation of primary cell cultures, which present high risk of contaminations from surrounding non-endometriotic tissues. Past work based on the use of cell lines was reviewed as well, and it emerged that cell line authentication was never performed.In an effort to address these weaknesses for future research, we present data on the performance of Pax2, a suitable marker to exclude ovarian (and other non-endometrial) cell contaminations from primary cell cultures; STR profiles of cell lines Z11 and Z12 were analysed and indicated that the cells were authentic. These profiles are now available for authentication purposes to researchers wishing to perform experiments with these cells.A quality control pipeline to assure sufficient quality of in vitro research in the field of reproductive biology and endometriosis is proposed. We encourage scientists, research institutes, journal reviewers, editors and funding bodies to raise awareness of the problem and adopt appropriate policies to solve it in the future.
LARGE-SCALE DATA: STR profiles of cell lines Z11 and Z12 are deposited at the Cellosaurus database-web.expasy.org.
LIMITATIONS, REASONS FOR CAUTION: There may be additional markers suitable to assess cell quality.
Future in vitro research in endometriosis and the reliability of outcomes can be improved by using the recommendations presented in this study.
STUDY FUNDING/COMPETING INTEREST(S): The study was partly financed by the 'Stichting Fertility Foundation' (The Netherlands). The authors declare no existing conflict of interest.
Non-applicable.
用于子宫内膜异位症研究的原代细胞培养物和细胞系的质量是否足够？
用于子宫内膜异位症研究的原代细胞缺乏纯度和表型特征，细胞系未进行基因分型验证。
体外研究的可重复性差以及所用细胞系的真实性不足是生殖生物学和子宫内膜异位症研究领域令人关注的原因。
研究设计、大小、持续时间：在本研究中，系统地回顾了过去子宫内膜异位症领域的体外研究，以确定是否考虑了适当的质量控制。此外，我们还探讨了配对盒 2 (Pax2) 作为子宫内膜特异性标志物在子宫内膜和子宫内膜异位症原代细胞培养物中的表现；我们还对最流行的子宫内膜异位症细胞系进行了特征描述，包括短串联重复 (STR) 图谱。
参与者/材料、设置、方法：文献综述部分：回顾了近 300 篇描述从子宫内膜异位症中分离和创建原代细胞培养物的已发表方案。湿实验室部分：通过免疫组织化学、免疫荧光和流式细胞术分析了从 13 名子宫内膜异位症患者中分离出的原代细胞中 Pax2 的表达情况。Z11 和 Z12 细胞系是最流行的子宫内膜异位症细胞系，它们的 Pax2 表达、类固醇激素受体和 STR 图谱特征也得到了描述。
从文献综述工作中，我们强调了原代细胞培养物缺乏足够的细胞纯度和表型特征，这存在着高风险的来自周围非子宫内膜组织的污染。我们还回顾了过去基于细胞系使用的工作，结果表明从未进行过细胞系验证。为了解决未来研究中的这些弱点，我们提出了 Pax2 的性能数据，Pax2 是一种合适的标志物，可以排除原代细胞培养物中的卵巢（和其他非子宫内膜）细胞污染；分析了细胞系 Z11 和 Z12 的 STR 图谱，并表明这些细胞是真实的。现在这些图谱可用于希望使用这些细胞进行实验的研究人员进行认证。提出了一个质量控制管道，以确保生殖生物学和子宫内膜异位症领域的体外研究具有足够的质量。我们鼓励科学家、研究机构、期刊审稿人、编辑和资助机构提高认识，并在未来采取适当的政策来解决这个问题。
细胞系 Z11 和 Z12 的 STR 图谱已存入细胞库数据库-web.expasy.org。
局限性、谨慎的原因：可能有其他合适的标记物来评估细胞质量。
通过使用本研究中提出的建议，未来的子宫内膜异位症体外研究和结果的可靠性可以得到提高。
研究资金/利益冲突：该研究部分由“生育基金会”（荷兰）资助。作者没有现有的利益冲突。
不适用。
