Saturday September 25, 2021 - 19:00 to 20:00
Viability of human STEM cells used to produce microchimeric piglets after 10 years of cryopreservation
Guillermo Ramis1,2, Laura Martínez-ALarcón2,3, Lucía Hernández-Cuesta1, Ana M. García-Hernández2,4, Antonio Muñoz1,2, Pablo Ramirez2,3.
1Producción Animal, Universidad de Murcia, Murcia, Spain; 2Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain; 3Servicio de Cirugía, Hospital Clínico Universitario Virgen de la Arrixaca, Murcia, Spain; 4Servicio de Hematología. Sala Blanca, Hospital Clínico Universitario Virgen de la Arrixaca, Murcia, Spain
Introduction: STEM cells play an important role in current regenerative medicine, and have been used previously in xenotransplantation to produce microchimeric piglets (Abellaneda et al., 2013). Different sources of cells have been proposed for this purpose: Bone Marrow Mesenchymal cultured cells, Umbilical Cord Mononuclear cells or Bone Marrow STEM cells.
The aim of this work was to describe the viability of different cells to produce microchimeric piglets, cryopreserved over more than 10 years.
Material and methods: Three samples of Bone Marrow derived Mesenchymal cells (BM-MC) and five samples of Umbilical Cord Total Mononuclear cells (UC-TMC) were recovered from the cryopreservation service of the University of Murcia. These cells were preserved in 2009-2010, following previously described protocols. The BM-MC samples were defreezed in laminar flow chamber, washed and resuspended as previously described and viability was calculated by flow cytometry and visual count in Neubauer’s chamber. UC-TMC were thawed in bain marie, washed and resuspended. BM-MC were cultured, and viability was calculated on passages 7 to 9.
Results: Results are shown in tables 1 (BM-MC) and 2 (UC-TMC). There was a reduction in the number of cells recovered after thaw, but in general, all samples showed enough cells to be used in microchimeric production. The BM-MC showed a good performance in culture, with a good expansion and viability. One of the samples showed a much higher account than the registered in the freeze procedure, which, probably is the results of a record mistake. The BM-MC could be cultured and expanded, both for their phenotypic characterization and to increase the availability of cells. They showed good viability, although it began to decline after the eighth passage.
Discussion and Conclussions: One of the drawbacks of using STEM cells in experimental protocols such as the production of microchimeric piglets is obtaining these cells. Both the obtaining of umbilical cord and bone marrow cells are subject to donation and represent an added difficulty to xenotransplantation experimentation. Being able to preserve part of the cells obtained for use in later experiments would be a very important advantage. This experience shows that the freezing protocols used allow viable cells up to 10 years after freezing, which would allow a continuous source of experimental cells. However, cells should be used, and re-preserved, in the first passages since after the eighth they showed loss of viability.