Long-term preservation of canine bone marrow: in vitro studies

In vitro studies were performed on canine bone marrow frozen with DMSO and stored in liquid nitrogen for 2 to 6 months. The results are compared with previously reported parallel in vivo experiments that demonstrated no loss of stem cells. When studies were performed immediately after thawing, there was no substantial drop in the count of nucleated cells and, except for megakaryocytes, there was no alteration of the bone marrow morphology. After two washes, and removal of DMSO, the nucleated cell count dropped to 50% of its previous value. Optic and electron microscopy showed severe damage in mature myeloid elements. In some instances, the cells had a condensed nucleus similar to the red-purple inclusion body of LE cells (as observed in systemic lupus erythematosus), and electron microscopy showed heavy chromatin clumping. On the other hand, both optic and electron microscopy showed a good preservation of lymphocytes, plasmocytes, and erythroid precursors. Two-hour DNA synthesis slightly dropped after storage, and this drop appeared more consistent when related to a constant volume of bone marrow (50 microliters) rather than to a constant number of nucleated cells (10(6)). In five instances frozen and thawed bone marrow was grown in short-term cultures, and analysis of 98 metaphases showed no major aberrations of the chromosomes and only 2% of minor aberrations, such as breakages and fragments. These data, compared with the results of previous in vivo experiments that showed no loss of stem cells after 5 months storage, suggest that stem cells are less sensitive to freezing and thawing injury than myeloid elements and/or that it might be safer for the thawed bone marrow not to be manipulated before infusion.