Characterization of Placental Mesenchymal Stem Cells Spheroids after Generation, Hypothermic and Subnormothermic Storage

Abstract

Background. Placental mesenchymal stem cells application is a promising area of cell therapy for number diseases. Their using in the form of spheroids results in enhancement of the therapeutic potential, improve the cells survival after transplantation. Medical purposes require the characterization and development of spheroids storage technology. Objective. The aim of the paper is to characterize placental mesenchymal stem cells spheroids after generation and low-temperature storage. Methods. Spheroids were produced by a hanging drop method from placental mesenchymal stem cells, chara­cterized after production and after subnormothermic (20 °C) and hypothermic (4 °C) storage for up to 96 h. The structure, metabolic activity of spheroids according to the data of the MTT test and the resazurin restoration test were studied. Results. The hanging drop method makes it possible to produce from 1×106 placental mesenchymal stem cells about 825.3 ± 56.2 spheroids in the size of 50–150 microns and 62.3 ± 5.1 spheroids of 150–300 microns in size, having a spherical shape, not stained with trypan blue, stained with neutral red and fluorescein diacetate. The spheroids adhere to the plastic and destroying through short-time centrifugation. During subnormothermic storage spheroids, structural characteristics and metabolic activity change after 48 h, during hypothermic – after 24 h of storage. Conclusions. The hanging drop method makes it possible to produce about 900 spheroids from 1 million placental MSCs with stable metabolic activity. For medical purposes, subornorthomeric (20 °C) storage not more than 24 h is recommended for placental MSC spheroid. Longer storage leads to a sharp decreasing of their metabolic activity and destroying to the separated cells.

Keywords

• Spheroids
• Mesenchymal stem cells
• Placenta
• Hypothermia
• Subnormothermia