Ultrastructural changes in the spinal cord of rats with experimental allergic encephalomyelitis under the influence of human umbilical cord-derived multipotent mesenchymal stromal cells cryopreserved according to different protocols

Abstract

The transplantation of multipotent mesenchymal stromal cells (MMSCs) is considered to be a possible therapy of multiple sclerosis. For the clinical application of human umbilical cord-derived MMSCs (UC-MMSCs) it is necessary to develop a method of their cryopreservation taking into account the type of cryoprotective media and to investigate the possibility of using these cells for therapeutic purposes in vivo. The purpose of the study was to investigate the effect of UC-MMSCs, cryopreserved in solutions of different composition, on the processes of demyelination and remyelination of the spinal cord of rats with experimental allergic encephalomyelitis (EAE) as a model of multiple sclerosis. Materials and methods. The EAE was modeled by subcutaneous administration of homogenized spinal cord of adult rats with complete Freund's adjuvant. On the 18th day rats with moderate relapsing-remitting form of EAE were suboccipitally injected 1•106 UC-MMSCs, cryopreserved in cryoprotective media containing dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), ethylene glycol, trehalose and sucrose at different composition. On the 35th and 60th days, the studies of ultrastructural changes of the lumbar spinal cord (L3-L5) were performed, assessing the degree of demyelination of nerve fibers by the ratio of myelin sheath (MS) thickness to the diameter of the axis cylinder (AC) of axons. Results. In rats with moderate EAE from the 35th to the 60th day after the modelling of the disorder, destructive changes and signs of demyelination in the spinal cord increased; the MS/AC index corresponded to the average degree of axon demyelination. Suboccipitally administered cryopreserved UC-MMSCs to EAE rats, depending on the used cryopreservation solution, slowed or stopped the demyelination, decreased the MS/AC index to a low degree of axonal demyelination. Reducing the concentration of DMSO in the cryopreservation medium from 10 % to 4 % and adding 6 % trehalose provided a better effectiveness of UC-MMSCs in decreasing the degree of demyelination in EAE. At the same time, the standard solution (10 % DMSO, 90 % FBS) provided these effect, but to a lesser extent. The use of a multicomponent cryopreservation medium containing 15 % ethylene glycol, 3 % DMSO, 10 % sucrose, 12 % trehalose and 60 % FBS did not achieve the goal of maintaining the effects of UC-MMSCs to reduce the degree of demyelination in EAE. Conclusions. To maintain the therapeutic properties of UC-MMSCs, it is advisable to add a reduced concentration of DMSO (4 %) and 6 % trehalose to the cryopreservation medium, supplemented with 90 % fetal bovine serum.