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Oliver W Gramlich, Wei Zhu, Anthony Burand, James A Ankrum, Markus H Kuehn; Transplantation of cryopreserved MSC rescues retinal ganglion cells following I/R injury. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4402. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mesenchymal stromal cells (MSC) synthesize a number of growth factors and have the potential to be neuroprotective. Cryopreservation of MSC reportedly degrades their neuroprotective abilities but is essential for clinical applications. Here we employ refined MSC cryopreservation techniques and evaluate their capacity to rescue retinal ganglion cells (RGC) in a mouse model of Ischemia/Reperfusion (I/R) injury.
Human MSC were cryopreserved using CryoStor CS5 media and a controlled rate freezing container. Viability after thaw was determined by TUNEL-staining and FACS. The secretome of 2x104 fresh or cryo-MSC was analyzed 48 hours after IFN-γ (100 ng/ml)/TNF-α (50ng/ml) stimulation using the Human Growth factor Array Q1.I/R was induced by elevating the intraocular pressure to 80 mmHg for 1 hour in left eyes of C57BL6/J mice. Two hours after I/R, 3x104 fresh (I/R + MSC; N=10) or cryopreserved MSC (I/R + cryo-MSC; N=8) were transplanted into the retinal cavity. A vehicle control group received PBS injections only (I/R + PBS; N=10). All contralateral eyes served as controls (non-ischemic, N=28). RGC density (RGC/mm2) was determined seven days after I/R in retinal wholemounts via gamma-synuclein immunostaining. Differences were evaluated using Tukey’s post-hoc test.
Post-thawed MSC show marginally reduced viability when compared to fresh MSC (fresh MSC: 99.9 %, cryo-MSC: 99.8%). The profile of growth factor release was similar in both groups, except for BMP-7. BMP-7 secretion was only induced in cryo-MSC but not in fresh MSC.As expected, retinas of vehicle controls display pronounced loss of RGC after I/R injury (RGC/mm2 in I/R + PBS: 309±308, non- ischemic: 2413±413; P=0.0001). RGC survival was significantly higher in I/R eyes after transplantation of both fresh (829±405; P=0.019) and cryo-MSC (845±320; P=0.023) when compared to vehicle-only group.
Our in vitro data demonstrate that our cryopreservation method preserves MSC viability and secretion of potentially neuroprotective factors such as BDNF, GNDF, osteoprotegerin and TGF-β. Furthermore, the ability of cryo-MSC to rescue RGC following I/R is as high as that of fresh MSC. These data suggest that it may be possible to use preserved MSC for the immediate treatment of acute neuropathies.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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