Gat Itai, Maghen Leila, Filice Melissa, Wyse Brandon, Zohni Khaled, Jarvi Keith, Lo Kirk C, Gauthier Fisher Andrée, Librach Clifford
Create Fertility Centre, Toronto, Ontario, Canada; Pinchas Borenstein Talpiot Medical Leadership Program, Sheba Medical Center, Ramat Gan, Israel; Sackler school of medicine, Tel Aviv university, Tel Aviv, Israel.
Create Fertility Centre, Toronto, Ontario, Canada.
Fertil Steril. 2017 Mar;107(3):595-605.e7. doi: 10.1016/j.fertnstert.2016.12.028.
To optimize culture conditions for human testicular somatic cells (TSCs) and spermatogonial stem cells.
Basic science study.
Urology clinic and stem cell research laboratory.
PATIENT(S): Eight human testicular samples.
INTERVENTIONS(S): Testicular tissues were processed by mechanical and enzymatic digestion. Cell suspensions were subjected to differential plating (DP) after which floating cells (representing germ cells) were removed and attached cells (representing TSCs) were cultured for 2 passages (P0-P1) in StemPro-34- or DMEM-F12-based medium. Germ cell cultures were established in both media for 12 days.
MAIN OUTCOME MEASURE(S): TSC cultures: proliferation doubling time (PDT), fluorescence-activated cell sorting for CD90, next-generation sequencing for 89 RNA transcripts, immunocytochemistry for TSC and germ cell markers, and conditioned media analysis; germ cell cultures: number of aggregates.
RESULT(S): TSCs had significantly prolonged PDT in DMEM-F12 versus StemPro-34 (319.6 ± 275.8 h and 110.5 ± 68.3 h, respectively). The proportion of CD90-positive cells increased after P1 in StemPro-34 and DMEM-F12 (90.1 ± 10.8% and 76.5 ± 17.4%, respectively) versus after DP (66.3 ± 7%). Samples from both media after P1 clustered closely in the principle components analysis map whereas those after DP did not. After P1 in either medium, CD90-positive cells expressed TSC markers only, and fibroblast growth factor 2 and bone morphogenetic protein 4 were detected in conditioned medium. A higher number of germ cell aggregates formed in DMEM-F12 (59 ± 39 vs. 28 ± 17, respectively).
CONCLUSION(S): Use of DMEM-F12 reduces TSC proliferation while preserving their unique characteristics, leading to improved germ cell aggregates formation compared with StemPro-34, the standard basal medium used in the majority of previous reports.
优化人睾丸体细胞（TSCs）和精原干细胞的培养条件。
基础科学研究。
泌尿外科诊所和干细胞研究实验室。
8份人睾丸样本。
睾丸组织经机械和酶消化处理。细胞悬液进行差异贴壁（DP），之后去除悬浮细胞（代表生殖细胞），将贴壁细胞（代表TSCs）在基于StemPro-34或DMEM-F12的培养基中培养2代（P0 - P1）。两种培养基中均建立生殖细胞培养12天。
TSC培养：增殖倍增时间（PDT）、CD90的荧光激活细胞分选、89种RNA转录本的二代测序、TSC和生殖细胞标志物的免疫细胞化学以及条件培养基分析；生殖细胞培养：聚集体数量。
与StemPro-34相比，TSCs在DMEM-F12中的PDT显著延长（分别为319.6±275.8小时和110.5±68.3小时）。P1后，StemPro-34和DMEM-F12中CD90阳性细胞比例增加（分别为90.1±10.8%和76.5±17.4%），而DP后为（66.3±7%）。P1后两种培养基的样本在主成分分析图中紧密聚集，而DP后的样本则不然。在任何一种培养基中P1后，CD90阳性细胞仅表达TSC标志物，且条件培养基中检测到成纤维细胞生长因子2和骨形态发生蛋白-4。DMEM-F12中形成的生殖细胞聚集体数量更多（分别为59±39和28±17）。
与大多数先前报道中使用的标准基础培养基StemPro-34相比，使用DMEM-F12可降低TSC增殖，同时保留其独特特性，从而导致生殖细胞聚集体形成增加。
