title: Comparison of enzymatic digestion and mechanical dissociation of human testicular tissues.
abstract: OBJECTIVE
  To compare mechanical dissociation, employing the Medimachine system, and enzymatic digestion of human testicular tissues with respect to the proportion of spermatogonia and somatic cells, with the long-term objective of establishing human spermatogonial cultures.
  
  
  METHODS
  Experimental basic science study.
  
  
  METHODS
  Reproductive biology laboratory.
  
  
  METHODS
  Testicular tissues were obtained from patients with gender dysphoria on the day of sex reassignment surgery. On the basis of the histological evaluation, tissue samples with complete spermatogenesis (fresh, n = 6; cryopreserved, n = 7) and with meiotic arrest (cryopreserved, n = 4) were selected.
  
  
  METHODS
  None.
  
  
  METHODS
  The composition of testicular cell suspensions was assessed performing quantitative real-time polymerase chain reaction (qPCR) analyses for germ cell-specific (FGFR3, SALL4, UTF1, MAGE-A4) and somatic marker genes (ACTA2 and VIM). Additionally, flow-cytometric analyses were used to evaluate the percentage of SALL4-and vimentin-positive cells.
  
  
  RESULTS
  While Medimachine dissociation yielded higher cell numbers in all patient groups, viability of cells was highly variable and correlated with the histological status of the tissue. Interestingly, qPCR analysis revealed a significantly decreased expression of the somatic marker genes ACTA2 and VIM and an increased expression of the spermatogonial marker genes FGFR3 and SALL4 after Medimachine dissociation. These findings were corroborated by flow-cytometric analyses that demonstrated that the proportion of SALL4-positive cells was up to 4 times higher after mechanical dissociation.
  
  
  CONCLUSIONS
  Medimachine dissociation of human testicular tissues is comparably fast and leads to an enrichment of SALL4-positive spermatogonia. The use of this method may therefore constitute an advantage for the establishment of human spermatogonial cell cultures.
authors.affiliation: Institute for Reproductive and Regenerative Biology, Centre of Reproductive Medicine and Andrology, University Hospital Münster, Albert-Schweitzer-Campus, Münster, Germany; Department of Clinical Andrology, Centre of Reproductive Medicine and Andrology, University Hospital Münster, Albert-Schweitzer-Campus, Münster, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Schneider
authors.affiliation: Institute for Reproductive and Regenerative Biology, Centre of Reproductive Medicine and Andrology, University Hospital Münster, Albert-Schweitzer-Campus, Münster, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Redmann
authors.affiliation: Institute for Reproductive and Regenerative Biology, Centre of Reproductive Medicine and Andrology, University Hospital Münster, Albert-Schweitzer-Campus, Münster, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Wistuba
authors.affiliation: Institute for Reproductive and Regenerative Biology, Centre of Reproductive Medicine and Andrology, University Hospital Münster, Albert-Schweitzer-Campus, Münster, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schlatt
authors.affiliation: Department of Clinical Andrology, Centre of Reproductive Medicine and Andrology, University Hospital Münster, Albert-Schweitzer-Campus, Münster, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Kliesch
authors.affiliation: Institute for Reproductive and Regenerative Biology, Centre of Reproductive Medicine and Andrology, University Hospital Münster, Albert-Schweitzer-Campus, Münster, Germany. Electronic address: Nina.Neuhaus@ukmuenster.de.
authors.forename: Nina
authors.initials: N
authors.lastname: Neuhaus
mesh_terms: D002470:Cell Survival / Q000502:physiology; D057075:Enzyme Assays / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008297:Male; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D013093:Spermatogonia / Q000201:enzymology; D013737:Testis / Q000166:cytology / Q000201:enzymology*
keywords: Human testis; Medimachine dissociation; SALL4; spermatogonia