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Surface markers of colony forming marrow cells cryopreserved at -196°C since 1974, and the differentiation to dendritic cells and others, and the functional activity of the marrow derived mesenchymal stromal cells.

Number: pap. ID: 866

Author(s) : SUMIDA S., KITAMUEA T., MOTOMURA N., et al.

Summary

Establishment of the long-term cryopreservation technology for cells, tissues, and organs will be an ultimate object of cryobiology. This study was conducted to solve some problems in the bone marrow transplantation using the bone marrow cells, which were harvested for the autologous transplantation from 266 advanced cancer patients before conditioning therapy and had been preserved in liquid phase of liquid nitrogen since 1973. The survival and proliferation ability of thawed marrow cells were confirmed by the colony forming units in culture and the flow-cytometric analysis of cell surface markers at day 0, 3, 6, 9, 14 and 21 using the mouse antibodies conjugated with FITC: CD1, 2, 3, 4, 11b, 14, 16, 20, 30, 38, 57, HLA-DR, and glycophorin A, and with PE: CD5, 7, 8, 10, 11c, 13, 19, 22, 33, and 34. The subcultures of colony cells were tried. The results: 1) Granulocyte-Macrophage CFU-C -and Erythroblast-CFU-C were observed as self maintaining and differentiation capacity under favourable conditions in all cases, which had been preserved in liquid phase of liquid nitrogen for 29 years or more. 2) Overall recovery % of thawed marrow stem cells gave the regression equation: Y = -0.0002X + 0.0362, which suggests that freezing in liquid nitrogen hardly influences the CFU-C recovery. 3) The increased expression of surface markers: CD 11b, 13, and 33 clarified that the growth factors in the media effectively stimulated the proliferation and differentiation of those cells expressing those surface makers. But, the increased titers of those surface markers decreased again on Day 14 in some cases. 4) The cells, which had no stimulation by any colony stimulation factors, expressed few markers and appeared in vitro as the cell debris under the reversed microscopy and on the cytogram of flow cytometry. 5) Some colony cells were successfully sub-cultured to the second generation. 6) The mature dendritic cells were surrounded by many small nucleated cells that was reminiscent to antigen presentation and the another dendrites differentiated to spindle-shaped fibroblasts and made the colonies. 7). Mesenchymal stromal cells (BMSCs) or fibroblast were recruited to construct interesting frameworks on the bottom surface of Petri dish on Day 7 or later in culture. 8) Although 3 of 30 conservation bags were ruptured during thawing process in a +40°C water bath, no obstacle including bacterial contamination was not caused in the culture preparation afterward. 8) The cryopreservation of stem cells and cord blood cells too at -60 to -80°C in the mechanical deep freezer and in the vapour phase of liquid nitrogen did not constantly succeed the formation of colony, probably due to long-termed duration of unstable temperatures as already reported. Finally, the authors like to emphasize that the bone marrow stem cells rescue not only the iatrogenically ablated hematopoietic systems of the patients but also the accidental radiation injury of special workers, who work in the vicinity of the nuclear reactor by any chance, the cryopreserved autologous marrow or peripheral progenitor cells will play an important and an indispensable role of therapeutic procedure.

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  • Original title: Surface markers of colony forming marrow cells cryopreserved at -196°C since 1974, and the differentiation to dendritic cells and others, and the functional activity of the marrow derived mesenchymal stromal cells.
  • Record ID : 30001689
  • Languages: English
  • Source: Proceedings of the 23rd IIR International Congress of Refrigeration: Prague, Czech Republic, August 21-26, 2011. Overarching theme: Refrigeration for Sustainable Development.
  • Publication date: 2011/08/21

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