Mesenchymal stem cells (MSCs) are actively used in regenerative medicine to treat neuropathies, diabetes mellitus, burns, ulcers, infertility, premature ovarian failure and climacteric syndrome. MSCs application on different carriers allows to modify their properties for the different pathology treatment. For the biomaterials medical application, it is important to develop shortterm storage methods for their transportation from the biotechnological laboratory to the clinic. The aim of the study was to determine the possibility of subnormothermic and hypothermic storage of placental mesenchymal stem cells in alginate carriers.
Placental MSCs were isolated with enzymatic method from placenta of women after cesarean section with informed consent. Encapsulation in alginates was done by dropping of 1% sodium alginate solution with cells in a 2% calcium chloride solution. The cells in the alginate beans were stored in a medium in subnormothermic (~ + 20ºС) and hypothermic (~ + 4ºС) conditions. Every 24 hours morphological study with trypan blue, neutral red, FDA/PI staining, resazurin recovery test, and glucose test was performed.
When polymerizing a solution of sodium alginate with placental cells at a concentration of 106/ml in a solution of calcium chloride, alginate microspheres with a diameter of 1.5–2.0 mm with placental cells where obtained. Cells were evenly distributed over the microspheres in separate cavities. Cells are stained with the trypan blue, neutral red, FDA/PI according to viability, as are cells that are not encapsulated in microspheres. The non-viable cells where stained with trypan blue, viable cells where stained with neutral red and MTT. A standard MTT test with formazan extraction was not indicative due to incomplete formazan extraction from alginate spheres with dimethyl sulfoxide or ethanol. Resazurin recovery test and glucose test were indicative.
After 72 hours of subnormothermic storage of alginate microspheres with placental cells, it was found that a significant metabolic activity decreasing. In hypothermic storage, metabolic activity was decreased after 48 hours of storage. It can be explained by the adaptability of human enzyme systems to functioning at 37 °C and the temperature decreasing to 4 °C is critical for them, while subnormothermic conditions are able to maintain metabolism at a level sufficient for survival. The morphological study of cells in alginate microspheres was not informative, because of the difficulty cells visualizing in a three-dimensional structure and the density of alginate. Rapid screening tests may include trypan blue staining and MTT test.
Conclusions. Placental cells in alginate microsphers can be stored in subnormothermic (+20°C) conditions no more than 48 hours. Storage more than 48 hours in subnormothermic conditions, or 24 hours in hypothermic conditions leads to a sharp metabolic activity decreasing.
Full text: PDF (Ukr) 499K