C-1011: Viability and differentiation of human blood and marrow cells cryopreserved since 1972, and homing effects of cryopreserved cells

Abstract

Our frozen allogeneic bone marrow transplantation program was started in 1972, but the initial clinical results were so poor that we switched to frozen autologous bone marrow transplantation (FABMT), which resulted in treatment of 293 advanced cancer patients. The bone marrow cells of those patients who had died (due to deterioration from cancer) remained in the frozen state until today. Those 200 units of bone marrow cells in cryopreservation bags with their pilot tubes have been stored in a liquid nitrogen stocker for up to 42 years. All cancer patients in this study signed informed consent of this treatment, and expired before 1999. Recently, in 2011–2013, we thawed and cultured those frozen marrow cells to confirm the long term (41 year) viability as elucidated by colony formation of erythroid, monocyte, and mesenchymal stroma cells (MSCs), and as MSCs to form their confluent networks. Finally, we established a novel technique to preserve bone marrow. As previously reported, the bone marrow cells of patients who had solid cancer tumors formed colonies of erythroids, mononuclear cells (small, large, and adipocyte), and mesenchymal stroma cells after thawing and culture in a 5% CO2 incubator. The frozen bone marrow cells formed splendid colonies, not inferior, when compared with fresh marrow cells which were currently collected for controls, and also differentiated the blood cells including red, mononuclear, and mesenchymal cells. As for the subculture of the colony cells, even passage 5 (P5) cells was possible. P2 cells cryoprotected in a 10 ∼ 15% Me 2 SO in IMDM were successfully cryopreserved at −80 °C or −196°C again for months, so the cryopreservation of an additional long term will be possible for marrow cells more than 40 years using the repetitive freeze-thaw-in vivo (in the cancer patients) or in vitro subculture method (RFTIVIVSCM) based on our clinical experiences of FABMT. As adjuvants/supplements, 1) adult human sera (AHS), 2) Limulus tridentatus (member of Brady-telic lines, a living fossil) fresh blood cell lysate (LL), and 3) feeder layer of human saphenous vein fragment (HVT), HeLa cells, and K 562 cells (myeloid leukemia cell line) were tested. LL slightly suppressed colony formation but promoted mesenchymal cell appearanced proliferation