title: Expansion of mouse sertoli cells on microcarriers.
abstract: BACKGROUND
  Sertoli cells (SCs) have been described as the 'nurse cells' of the testis whose primary function is to provide essential growth factors and create an appropriate environment for development of other cells [for example, germinal and nerve stem cells (NSCs), used here]. However, the greatest challenge at present is that it is difficult to obtain sufficient SCs of normal physiological function for cell transplantation and biological medicine, largely due to traditional static culture parameter difficult to be monitored and scaled up.
  
  
  OBJECTIVE
  Operational stirred culture conditions for in vitro expansion and differentiation of SCs need to be optimized for large-scale culture.
  
  
  METHODS
  In this study, the culturing process for primary SC expansion and maintaining lack of differentiation was optimized for the first time, by using microcarrier bead technology in spinner flask culture. Effects of various feeding/refreshing regimes, stirring speeds, seed inoculum levels of SCs, and concentrations of microcarrier used for expansion of mouse SCs were also explored. In addition, pH, osmotic pressure and metabolic variables including consumption rates of glucose, glutamine, amino acids, and formation rates of lactic acid and ammonia, were investigated in culture.
  
  
  RESULTS
  After 6 days, maximal cell densities achieved were 4.6 x 10(6) cells/ml for Cytodex-1 in DMEM/FBS compared to 4.8 x 10(5) cells/ml in static culture. Improved expansion was achieved using an inoculum of 1 x 10(5) cells/ml and microcarrier concentration of 3 mg/ml at stirring speed of 30 rpm. RESULTS indicated that medium replacement (50% changed everyday) resulted in supply of nutrients and removal of waste products inhibiting cell growth, that lead to maintenance of cultures in steady state for several days. These conditions favoured preservation of SCs in the undifferentiated state and significantly increased their physiological activity and trophic function, which were assessed by co-culturing with NSCs and immunostaining.
  
  
  CONCLUSIONS
  Data obtained in this study demonstrate the vast potential of this stirred culture system for efficient, reproducible and cost-effective expansion of SCs in vitro. The system has advantages over static culture, which has major obstacles such as lower cell density, is time-consuming and susceptible to contamination.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: B
authors.initials: B
authors.lastname: Shi
authors.forename: S
authors.initials: S
authors.lastname: Zhang
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: Y
authors.initials: Y
authors.lastname: Zhuang
authors.forename: J
authors.initials: J
authors.lastname: Chu
authors.forename: S
authors.initials: S
authors.lastname: Zhang
authors.forename: X
authors.initials: X
authors.lastname: Shi
authors.forename: J
authors.initials: J
authors.lastname: Bi
authors.forename: M
authors.initials: M
authors.lastname: Guo
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology*; D005947:Glucose / Q000378:metabolism; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008863:Microspheres; D012708:Sertoli Cells / Q000187:drug effects* / Q000378:metabolism* / Q000637:transplantation
chemical_list: D000596:Amino Acids; D015415:Biomarkers; D003470:Culture Media; D005947:Glucose