title: Three-dimensional podocyte-endothelial cell co-cultures: Assembly, validation, and application to drug testing and intercellular signaling studies.
abstract: Proteinuria is a common symptom of glomerular diseases and is due to leakage of proteins from the glomerular filtration barrier, a three-layer structure composed by two post-mitotic highly specialized and interdependent cell populations, i.e. glomerular endothelial cells and podocytes, and the basement membrane in between. Despite enormous progresses made in the last years, pathogenesis of proteinuria remains to be completely uncovered. Studies in the field could largely benefit from an in vitro model of the glomerular filter, but such a system has proved difficult to realize. Here we describe a method to obtain and utilize a three-dimensional podocyte-endothelial co-culture which can be largely adopted by the scientific community because it does not rely on special instruments nor on the synthesis of devoted biomaterials. The device is composed by a porous membrane coated on both sides with type IV collagen. Adhesion of podocytes on the upper side of the membrane has to be preceded by VEGF-induced maturation of endothelial cells on the lower side. The co-culture can be assembled with podocyte cell lines as well as with primary podocytes, extending the use to cells derived from transgenic mice. An albumin permeability assay has been extensively validated and applied as functional readout, enabling rapid drug testing. Additionally, the bottom of the well can be populated with a third cell type, which multiplies the possibilities of analyzing more complex glomerular intercellular signaling events. In conclusion, the ease of assembly and versatility of use are the major advantages of this three-dimensional model of the glomerular filtration barrier over existing methods. The possibility to run a functional test that reliably measures albumin permeability makes the device a valid companion in several research applications ranging from drug screening to intercellular signaling studies.
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy; Fondazione D'Amico per la Ricerca sulle Malattie Renali, via Pace 9, 20122 Milan, Italy. Electronic address: li_min@libero.it.
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy; Bio-imaging Unit, Department of Cardiovascular Research, IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, via La Masa 19, 20156 Milan, Italy; Fondazione D'Amico per la Ricerca sulle Malattie Renali, via Pace 9, 20122 Milan, Italy. Electronic address: alecorbelli@hotmail.com.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Corbelli
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy; Fondazione D'Amico per la Ricerca sulle Malattie Renali, via Pace 9, 20122 Milan, Italy. Electronic address: swat4028@yahoo.co.jp.
authors.forename: Shojiro
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy; Fondazione D'Amico per la Ricerca sulle Malattie Renali, via Pace 9, 20122 Milan, Italy. Electronic address: armellonis@libero.it.
authors.forename: Silvia
authors.initials: S
authors.lastname: Armelloni
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy; Fondazione D'Amico per la Ricerca sulle Malattie Renali, via Pace 9, 20122 Milan, Italy. Electronic address: aquamarine0321@libero.it.
authors.forename: Masami
authors.initials: M
authors.lastname: Ikehata
authors.affiliation: Cell Factory, Unit of Cell Therapy and Cryobiology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy. Electronic address: valentina.parazzi@gmail.com.
authors.forename: Valentina
authors.initials: V
authors.lastname: Parazzi
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy; Fondazione D'Amico per la Ricerca sulle Malattie Renali, via Pace 9, 20122 Milan, Italy. Electronic address: chiarapignatari@gmail.com.
authors.forename: Chiara
authors.initials: C
authors.lastname: Pignatari
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy; Fondazione D'Amico per la Ricerca sulle Malattie Renali, via Pace 9, 20122 Milan, Italy. Electronic address: giardinolaura@gmail.com.
authors.forename: Laura
authors.initials: L
authors.lastname: Giardino
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy; Fondazione D'Amico per la Ricerca sulle Malattie Renali, via Pace 9, 20122 Milan, Italy. Electronic address: Deborah.mattinzoli@gmail.com.
authors.forename: Deborah
authors.initials: D
authors.lastname: Mattinzoli
authors.affiliation: Cell Factory, Unit of Cell Therapy and Cryobiology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy. Electronic address: lorenza.lazzari@policlinico.mi.it.
authors.forename: Lorenza
authors.initials: L
authors.lastname: Lazzari
authors.affiliation: Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DiNOGMI), University of Genoa, via G. Gaslini 5, 16148 Genoa, Italy; Medical Genetics Unit, Istituto Giannina Gaslini, via G. Gaslini 5, 16148 Genoa, Italy. Electronic address: aldamaria.puliti@unige.it.
authors.forename: Aldamaria
authors.initials: A
authors.lastname: Puliti
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy; Department of Chemistry, Materials, and Chemical Engineering "G.Natta", Politecnico di Milano, via Mancinelli 7, 20131 Milan, Italy; Fondazione CEN - European Centre for Nanomedicine, Piazza Leonardo da Vinci 32, 20133 Milan, Italy. Electronic address: francesco.cellesi@polimi.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Cellesi
authors.affiliation: Laboratory of Renal Physiopathology, Department of Medical, Surgical, and Health Sciences, Trieste University, via Strada di Fiume 447, 34149 Trieste, Italy. Electronic address: czennaro@units.it.
authors.forename: Cristina
authors.initials: C
authors.lastname: Zennaro
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy; Fondazione D'Amico per la Ricerca sulle Malattie Renali, via Pace 9, 20122 Milan, Italy. Electronic address: pmessa@policlinico.mi.it.
authors.forename: Piergiorgio
authors.initials: P
authors.lastname: Messa
authors.affiliation: Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Pace 9, 20122 Milan, Italy; Fondazione D'Amico per la Ricerca sulle Malattie Renali, via Pace 9, 20122 Milan, Italy. Electronic address: mariapia.rastaldi@policlinico.mi.it.
authors.forename: Maria Pia
authors.initials: MP
authors.lastname: Rastaldi
mesh_terms: D000418:Albumins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D018920:Coculture Techniques; D024042:Collagen Type I / Q000494:pharmacology; D024141:Collagen Type IV / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D018698:Glutamic Acid / Q000494:pharmacology; D006801:Humans; D007678:Kidney Glomerulus / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000187:drug effects; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D050199:Podocytes / Q000187:drug effects / Q000378:metabolism; D017470:Receptors, Glutamate / Q000378:metabolism; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
chemical_list: D000418:Albumins; D024042:Collagen Type I; D024141:Collagen Type IV; D017470:Receptors, Glutamate; D042461:Vascular Endothelial Growth Factor A; D018698:Glutamic Acid; D003907:Dexamethasone; D004317:Doxorubicin
keywords: Albumin permeability; Drug testing; Endothelial cells; Glomerular filtration barrier; Intercellular signaling; Mesenchymal stem cells; Podocytes; Three-dimensional co-culture