title: Optimal culture conditions are critical for efficient expansion of human testicular somatic and germ cells in vitro.
abstract: OBJECTIVE
  To optimize culture conditions for human testicular somatic cells (TSCs) and spermatogonial stem cells.
  
  
  METHODS
  Basic science study.
  
  
  METHODS
  Urology clinic and stem cell research laboratory.
  
  
  METHODS
  Eight human testicular samples.
  
  
  METHODS
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Itai
authors.initials: I
authors.lastname: Gat
authors.affiliation: Create Fertility Centre, Toronto, Ontario, Canada.
authors.forename: Leila
authors.initials: L
authors.lastname: Maghen
authors.affiliation: Create Fertility Centre, Toronto, Ontario, Canada.
authors.forename: Melissa
authors.initials: M
authors.lastname: Filice
authors.affiliation: Create Fertility Centre, Toronto, Ontario, Canada.
authors.forename: Brandon
authors.initials: B
authors.lastname: Wyse
authors.affiliation: Create Fertility Centre, Toronto, Ontario, Canada; Department of Reproductive Health and Family Planning, National Research Center, Cairo, Egypt.
authors.forename: Khaled
authors.initials: K
authors.lastname: Zohni
authors.affiliation: Division of Urology, Department of Surgery, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Keith
authors.initials: K
authors.lastname: Jarvi
authors.affiliation: Division of Urology, Department of Surgery, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Kirk C
authors.initials: KC
authors.lastname: Lo
authors.affiliation: Create Fertility Centre, Toronto, Ontario, Canada.
authors.forename: Andrée
authors.initials: A
authors.lastname: Gauthier Fisher
authors.affiliation: Create Fertility Centre, Toronto, Ontario, Canada; Department of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada; Department of Obstetrics and Gynecology, Women's College Hospital, Toronto, Ontario, Canada. Electronic address: drlibrach@createivf.com.
authors.forename: Clifford
authors.initials: C
authors.lastname: Librach
mesh_terms: D000072956:Adult Germline Stem Cells / Q000378:metabolism / Q000502:physiology*; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D010641:Phenotype; D013091:Spermatogenesis; D013093:Spermatogonia / Q000378:metabolism / Q000502:physiology*; D013737:Testis / Q000166:cytology*; D013997:Time Factors; D059467:Transcriptome
chemical_list: D015415:Biomarkers; D017077:Culture Media, Conditioned
keywords: Human testicular somatic cells; germ cells; male fertility preservation; spermatogonial stem cells