Characterization of Placental Mesenchymal Stem Cells Spheroids after Generation, Hypothermic and Subnormothermic Storage
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.
Silini AR, Cargnoni A, Magatti M, Pianta S, Parolini O. The long path of human placenta, and its derivatives, in regenerative medicine. Front Bioeng Biotechnol. 2015 Oct 19;3:162. DOI: 10.3389/fbioe.2015.00162
Pogozhykh O, Prokopyuk V, Figueiredo C, Pogozhykh D. Placenta and placental derivatives in regenerative therapies - experimental studies, history, and prospects. Stem Cells Int. 2018;2018:14. DOI: 10.1155/2018/4837930
Kim TH, Choi JH, Jun Y, Lim SM, Park S, Paek JY, et al. 3D-cultured human placenta-derived mesenchymal stem cell spheroids enhance ovary function by inducing folliculogenesis. Sci Rep. 2018 Oct 17;8(1):15313. DOI: 10.1038/s41598-018-33575-9
Petrenko Y, Syková E, Kubinová Š. The therapeutic potential of three dimensional multipotent mesenchymal stromal cellspheroids. Stem Cell Res Ther. 2017;8(1):94. DOI: 10.1186/s13287-017-0558-6
Giwa S, Lewis JK, Alvarez L, Langer R, Roth AE, Church GM, et al. The promise of organ and tissue preservation to transform medicine. Nat Biotechnol. 2017 Jun 7;35(6):530-42. DOI: 10.1038/nbt.3889
Pogozhykh O, Prokopyuk V, Prokopyuk O, Kuleshova L, Goltsev A, Figueiredo C, et al. Towards biobanking technologies for natural and bioengineered multicellular placental constructs. Biomaterials. 2018;185:39-50. DOI: 10.1016/j.biomaterials.2018.08.060
van de Kerkhove MP, Hoekstra R, van Nooijen FC, Spoelstra FO, Doorschodt BM, van Wijk AC, et al. Subnormothermic preservation maintains viability and function in a porcine hepatocyte culture model simulating bioreactor transport. Cell Transplant. 2006;15(2):161-8. DOI: 10.3727/000000006783982089
Pogozhykh D, Prokopyuk V, Pogozhykh O, Mueller T, Prokopyuk O. Influence of factors of cryopreservation and hypothermic storage on survival and functional parameters of multipotent stromal cells of placental origin. PLoS One. 2015;10(10):1. DOI: 10.1371/journal.pone.0139834
Foty R. A simple hanging drop cell culture protocol for generation of 3D spheroids. J Vis Exp. 2011 May 6;(51):1. DOI: 10.3791/2720
Pravdyuk AI, Petrenko YA, Fuller BJ, Petrenko AY. Cryopreservation of alginate encapsulated mesenchymal stromal cells. Cryobiology. 2013;66(3):215-22. DOI: 10.1016/j.cryobiol.2013.02.002
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