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Factors influencing mesenchymal stromal cells in in vitro cellular models to study retinal pigment epithelial cell rescue

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Abstract

Mesenchymal stromal cells (MSC) stop or slow retinal pigment epithelium (RPE) and neuroretina (NR) degeneration by paracrine activity in oxidative stress-induced retinal degenerative diseases. However, it is mandatory to develop adequate in vitro models that allow testing new treatment strategies against oxidative stress before performing in vivo studies. The viable double- and triple-layered setups are composed of separate layers of NR, MSC, and RPE (NR–MSC–RPE, NR–RPE, MSC-RPE) partially mimic in vivo retinal conditions. In this study, the paracrine neuroprotective effect of each setup’s microenvironment on hydrogen peroxide (H2O2)-stressed was compared with unstressed RPE cells. RPE cell proliferation viability was assessed on day 1, 3, and 6 using Alamar Blue® (10%), MTT (10%) and a cell viability/cytotoxicity assay kit followed by data analysis. The results showed that RPE cells, highly viable (> 90%) in mixed medium of DMEM and neurobasal A (1:1), lost 50% viability on exposure to 400 µM of H2O2 (P < 0.05). The unexposed groups differed significantly from exposed groups for RPE cell growth (RPE and \({_{\text{H}_2\text{O}_2}}\)RPE (P < 0.0001), NR–MSC–RPE, and NR–MSC–\({_{\text{H}_2\text{O}_2}}\)RPE (P < 0.05), NR–RPE and NR–\({_{\text{H}_2\text{O}_2}}\)RPE (P < 0.01), and MSC–RPE and MSC–\({_{\text{H}_2\text{O}_2}}\)RPE (P < 0.01). NR–\({_{\text{H}_2\text{O}_2}}\)RPE and NR–RPE supported RPE cell proliferation viability better than other setups (P < 0.01) and RPE cells proliferated 0.49-fold more in NR-MSC-\({_{\text{H}_2\text{O}_2}}\)RPE than NR–MSC–RPE. Thus, NR and MSC presence improved significantly each setup’s microenvironment for cell rescue, nevertheless, each setup also showed limitations for its use as an in vitro study tool. Health of microenvironment of such setups depends on many factors including cell-secreted trophic factors.

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Acknowledgements

This research work was performed under the approved research projects (Grant numbers PS09/00938, JCYL BIO/39/VA26/10 and VA386A12-2), and financed by the Centro en Red de Medicina Regenerativa y Terapia Celular de Junta de Castilla y León, 47011 Valladolid, Spain. David Rodriguez-Crespo has defended his Master's thesis on obtaining appropriate institutional approvals, which is available in the University library. The authors would like to say thanks to Miss M. Teresa García-Gutierrez for providing support in the laboratory for executing the experiments.

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Author notes

  1. Girish K. Srivastava and David Rodriguez-Crespo contributed equally to study.

Authors and Affiliations

  1. Retina Group, Instituto Universitario de Oftalmobiología Aplicada (IOBA), Universidad de Valladolid, Campus Miguel Delibes, Paseo de Belén, 17, 47011, Valladolid, Spain

    Girish K. Srivastava, David Rodriguez-Crespo, Ivan Fernandez-Bueno & José Carlos Pastor

  2. Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla Y León, Valladolid, Spain

    Girish K. Srivastava & José Carlos Pastor

Authors
  1. Girish K. Srivastava
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  2. David Rodriguez-Crespo
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  3. Ivan Fernandez-Bueno
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  4. José Carlos Pastor
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Contributions

GS: coordination, designing and performing experiments, data analyzing, manuscript writing, reviewing and submission. DC: Performing experiments and data generation. IFB: Data analysis and manuscript reviewing. JP: Obtaining research funds, reviewing and suggesting manuscript improvement.

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Correspondence to Girish K. Srivastava.

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The authors have no conflicts of interest to declare.

Research involving human participants and/or animals

The authors declare there is no participation of human and animal in this study. A local slaughterhouse (J. Gutierrez S.L. Valladolid, Spain) donated the porcine eyes under an institutional agreement for research purposes and the Ocular Surface Group (OSG) of our institution provided human AT-MSC; these cells were isolated, characterized, and used in the research studies of our research group [15, 16], including publications by the OSG [21, 22].

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Srivastava, G.K., Rodriguez-Crespo, D., Fernandez-Bueno, I. et al. Factors influencing mesenchymal stromal cells in in vitro cellular models to study retinal pigment epithelial cell rescue. Human Cell 35, 1005–1015 (2022). https://doi.org/10.1007/s13577-022-00705-5

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