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Triple-layered mixed co-culture model of RPE cells with neuroretina for evaluating the neuroprotective effects of adipose-MSCs


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.

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The authors would like to thank the following institutions for funding our work: (1) National Plan of R + D + I 2008–2011 and ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (PS09/00938) (MICINN), co-financed by FEDER, Spain; (2) Centro en Red de Medicina Regenerativa y Terapia Celular de Junta de Castilla y León, 47011 Valladolid, Spain; (3) JCYL BIO/39/VA26/10 and VA386A12-2, Junta de Castilla y León, Spain; and (4) AECI, Ministry of Foreign Affairs and Cooperation, Spain. This study was presented in parts to the Association for Research in Vision and Ophthalmology (ARVO) Annual meeting, Orlando, FL, USA, May 2014 and the International Symposium on Ocular Pharmacology and Therapeutics (ISOPT), Rejkyavik, Iceland, June 2014.

Conflict of interest

The authors have no conflicts of interest to declare.

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Correspondence to Girish K. Srivastava.

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Rodriguez-Crespo, D., Di Lauro, S., Singh, A.K. et al. Triple-layered mixed co-culture model of RPE cells with neuroretina for evaluating the neuroprotective effects of adipose-MSCs. Cell Tissue Res 358, 705–716 (2014).

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  • Human adipose-derived mesenchymal stem cells
  • Neuroretina
  • RPE cells
  • Organotypic culture