title: Triple-layered mixed co-culture model of RPE cells with neuroretina for evaluating the neuroprotective effects of adipose-MSCs.
abstract: Mesenchymal stem cell (MSC) therapy is promising for neuroprotection but there is no report of an appropriate in vitro model mimicking the situation of the in vivo retina that is able to test the effect of MSCs in suspension or encapsulated with/without a drug combination. This study aims to establish a viable mixed co-culture model having three layers: neuroretina explants (NRs), retinal pigment epithelium (RPE) cells and adipose tissue-derived MSCs (AT-MSCs) for evaluating adipose-MSC effects. AT-MSCs were grown on the lower surface of a transwell membrane and RPE cells were grown on the bottom of a culture plate as monocultures. A transwell membrane was inserted into a culture plate well. NR was placed as an organotypic culture on the upper surface of the transwell membrane. Thus, a triple-layered co-culture setup was constructed. In double-layered setups, NR were co-cultured with AT-MSCs or RPE cells. Optimum medium, experiment execution period and transwell membrane permeability (TMP) were determined. MSC effects on RPE cell proliferation and NR reactive gliosis were evaluated. Limitations were discussed. Our study shows that neurobasal A with DMEM (1:1) mixed medium was suitable for viability of all three layers. AT-MSC growth decreased TMP significantly, 30-60 % in 3- to 6-day periods. Spontaneous NR reactive gliosis limits the experiment execution period to 6 days. AT-MSCs maintained their undifferentiated nature and showed no or limited neuroprotective effects. In this study, we successfully assembled viable double- and triple-layered co-culture setups for AT-MSCs, RPE and NR, optimised conditions for their survival and explored setup Limitations.
authors.affiliation: Instituto Universitario de Oftalmobiología Aplicada (IOBA), Universidad de Valladolid, Campus Miguel Delibes, Paseo de Belén, 17, 47011, Valladolid, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Rodriguez-Crespo
authors.forename: Salvatore
authors.initials: S
authors.lastname: Di Lauro
authors.forename: Amar K
authors.initials: AK
authors.lastname: Singh
authors.forename: Maria T
authors.initials: MT
authors.lastname: Garcia-Gutierrez
authors.forename: Manuel
authors.initials: M
authors.lastname: Garrosa
authors.forename: J Carlos
authors.initials: JC
authors.lastname: Pastor
authors.forename: Ivan
authors.initials: I
authors.lastname: Fernandez-Bueno
authors.forename: Girish K
authors.initials: GK
authors.lastname: Srivastava
mesh_terms: D000273:Adipose Tissue / Q000166:cytology*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D049109:Cell Proliferation; D018920:Coculture Techniques / Q000379:methods*; D005455:Fluorescent Antibody Technique; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008954:Models, Biological; D018696:Neuroprotective Agents / Q000378:metabolism*; D010641:Phenotype; D055213:Retinal Pigment Epithelium / Q000166:cytology*; D034421:Sus scrofa
chemical_list: C020932:11-cis-retinal-binding protein; D002352:Carrier Proteins; D005904:Glial Fibrillary Acidic Protein; D018696:Neuroprotective Agents