title: Advances in pancreatic islet monolayer culture on glass surfaces enable super-resolution microscopy and insights into beta cell ciliogenesis and proliferation.
abstract: A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells.
authors.affiliation: Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Phelps
authors.affiliation: Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.|Graduate Program in Biotechnology and Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Chiara
authors.initials: C
authors.lastname: Cianciaruso
authors.affiliation: Nestlé Institute of Health Sciences S.A., EPFL Innovation Park, CH-1015 Lausanne, Switzerland.
authors.forename: Jaime
authors.initials: J
authors.lastname: Santo-Domingo
authors.affiliation: Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Miriella
authors.initials: M
authors.lastname: Pasquier
authors.affiliation: Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.|Graduate Program in Biotechnology and Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.|Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Galliverti
authors.affiliation: Pancreatic Islet Processing Facility, Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Piemonti
authors.affiliation: Cell Isolation and Transplantation Center, Faculty of Medicine, Department of Surgery, Geneva University Hospitals and University of Geneva, CH-1211 Geneva, Switzerland.
authors.forename: Ekaterine
authors.initials: E
authors.lastname: Berishvili
authors.affiliation: BioImaging and Optics Core Facility, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Olivier
authors.initials: O
authors.lastname: Burri
authors.affiliation: Nestlé Institute of Health Sciences S.A., EPFL Innovation Park, CH-1015 Lausanne, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Wiederkehr
authors.affiliation: Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.|Graduate Program in Biotechnology and Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.|Institute for Molecular Engineering, University of Chicago, Chicago, IL 60615, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Hubbell
authors.affiliation: Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.|Graduate Program in Biotechnology and Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Steinunn
authors.initials: S
authors.lastname: Baekkeskov
mesh_terms: D000199:Actins / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D002923:Cilia / Q000378:metabolism*; D018920:Coculture Techniques; D005260:Female; D005898:Glass / Q000737:chemistry*; D005947:Glucose / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism*; D008297:Male; D008853:Microscopy / Q000379:methods*; D008870:Microtubules / Q000378:metabolism; D008875:Middle Aged; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D038081:Organogenesis; D010641:Phenotype; D017207:Rats, Sprague-Dawley; D055815:Young Adult
chemical_list: D000199:Actins; D007328:Insulin; D005947:Glucose; D002118:Calcium