title: [Culture of pancreatic progenitor cells in hanging drop and on floating filter].
abstract: OBJECTIVE
  To construct a method to culture pancreatic progenitor cells in hanging drop and on floating filter,and to examine if pancreatic progenitor cells can differentiate into mature endocrine cells with this method.
  
  
  METHODS
  Murine embryos at day 12.5 were isolated and digested into single cells,which were then cultured in hanging drop for 24h and formed spheres.Spheres were cultured on the filter for 6 days,which floated in the dish containing medium.During culture,the expressions of pancreas duodenum homeobox-1(PDX-1)and neurogenin3(Ngn3)were determined.The expressions of endocrine and exocrine markers,insulin,glucagon,and carboxypeptidase(CPA)were determined on day 7 by immunohistochemistry.Insulin secretion of spheres stimulated by glucose was detected by ELISA.The changes of pancreatic marker expressions during culture were monitored by real-time polymerase chain reaction(PCR).
  
  
  RESULTS
  One day after the culture,there were still a large amount of PDX-1 positive cells in pancreatic spheres,and these cells proliferated.On day 3,high expression of Ngn3 was detected,and the Ngn3-positive cells did not proliferate.On day 7,The expressions of endocrine and exocrine markers in the differentiated pancreatic progenitor cells were detected,which were consistent with that in vivo.Insulin was secreted by spheres upon the stimulation of glucose.
  
  
  CONCLUSIONS
  In hanging drop and on floating filter,pancreatic progenitor cells can differentiate into mature endocrine cells.
authors.affiliation: Institute of Hematology and Hospital of Blood Diseases,CAMS and PUMC,Tianjin,China.
authors.forename: Feng-xia
authors.initials: FX
authors.lastname: Ma
authors.forename: Fang
authors.initials: F
authors.lastname: Chen
authors.forename: Ying
authors.initials: Y
authors.lastname: Chi
authors.forename: Shao-guang
authors.initials: SG
authors.lastname: Yang
authors.forename: Shi-hong
authors.initials: SH
authors.lastname: Lu
authors.forename: Zhong-chao
authors.initials: ZC
authors.lastname: Han
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D055098:Endocrine Cells / Q000166:cytology*; D018398:Homeodomain Proteins / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D051379:Mice; D010179:Pancreas / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D015534:Trans-Activators / Q000378:metabolism
chemical_list: D018398:Homeodomain Proteins; D007328:Insulin; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein