Research Information System
NICHE 2022, 15 th National and 1 st International Congress of Histology and Embryology, Ankara, Turkey, 26 - 28 May 2022, pp.179-183
Introduction
Mesenchymal stem cells (MSCs) and MSC exosomes (MSC-Exos) are promising therapeutic possibilities for ischemic acute kidney injury (AKI)1, but their safety and efficacy are still debatable on proximal tubules (PT) at the cellular level. Recently microfluidic kidney-on-a-chip systems present smart platforms mimicking tubular microphysiopathological environment to assess theragnostic tools before translation to clinics. A gravity-driven membraneless microfluidic-based 3D culture platform may reproduce acute hypoxic PT injury and real-time assess the therapeutic potency of human bone marrow-derived MSC exosomes (hBMMSC-Exos) as cellular therapeutics.
Purpose
We aimed to isolate, characterize and quantitatively analyze the efficacy of hBMMSC-Exos with a real-time proliferation assay (RTCA) and assess the potency in terms of tubular epithelial permeability, epithelial polarity, expression of injury-specific genes and proliferation on the novel microfluidic acute hypoxic PT injury platform.
Material-Method
hBMMSC-Exos were isolated, characterized. RTCA determined the effective dose and treatment window on acute hypoxic PT injury. 2-lane 3D gravity-driven microfluidic platform was set to mimic PT in vitro. ZO-1, acetylated α-tubulin immunolabelling, permeability index assessed structural; cell proliferation by WST-1, BNIP3, HO-1, HIF1A1 expression by qRT-PCR measured functional integrity of PT.
Results
hBMMSC-Exos induced PT proliferation with ED50 of 172,582 µg/ml at 26th hour. Hypoxia significantly decreased ZO-1, increased permeability index, decreased cell proliferation rate, increased BNIP3, HO-1 and decreased HIF1A1 on 24-48 hours in the microfluidic platform. hBMMSC-Exos reinforced polarity by 1.72-fold increase in ZO-1 (p=0.0121), restored permeability by 20/45-fold against 20/155 kDa dextran (p=0.0004 and p<0.0001, respectively), increased epithelial proliferation 3-fold (p<0.0001) and decreased BNIP3 expression by 0.62-fold compared to control.
Conclusion
The real-time potency assay and 3D gravity-driven microfluidic acute hypoxic PT injury platform precisely demonstrated therapeutic performance window of allogeneic hBMMSC-Exos on ischemic AKI providing molecular, structural and functional cellular data. The novel standardized, non-invasive 2-step system validates cell-based personalized theragnostic tools in a real-time physiological microenvironment prior to safe and efficient clinical usage in nephrology.
Keywords
AKI, Proximal Tubule, Exosome, Microfluidic, Kidney-on-a-chip
References
1. B. Altun et al.; American Journal of Nephrology 35(6) (2012) 531-539.