019 Viability and differentiation of human bone marrow cells cryopreserved since 1972, Part-3

Abstract

Aim: A 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) to rescue 293 advanced cancer patients who were lethally myelosuppressed due to high dose chemotherapy. However, many unused bone marrow cells from patients who had died due to deterioration of cancer remained in a frozen state. More than 200 units in cryopreservation bags with their pilot tubes were stored in a liquid nitrogen refrigerator for up to 41 years. All cancer patients in this study had agreed to the informed consent of this treatment and had expired before 1999. We have published our study results of blood and bone marrow cell freezing in 1972–1983 (1). Our current technique of freezing bone marrow for autologous transplantation (2), and high dose chemotherapy with autologous marrow transplantation in patients with poor-prognosis tumors (3) have been published (1. S. Sumida. Transfusion of Blood by Freezing. Lippincott-George Thieme Publ. 1973–1974. 2. S. Sumida, et al. Low Temp. Med. 1982: 8–12. 3. S. Sumida, et al. Jap. J. Cancer Oncol. 1984; 14 Suppl. 1:47–51 ). Recently, in 2011–2013, we thawed and cultured those stored marrow cells to confirm the long term (41 years) viability of such thawed marrow cells as elucidated by colony formation of erythroid, monocyte, and mesenchymal cells (MSCs), and as MSCs to form confluent networks of MSCs. Methods and results: As previously reported, the bone marrow cells of patients who had solid cancer tumors formed colonies of erythoids, mononuclear cells (small, large, and adipocyte), and mesenchymal cells after thawing and culture in a 5% CO 2 incubator. The frozen bone marrow cells formed splendid colonies, not inferior, when compared with fresh marrow cells, 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 a further long term will be possible. 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), and of HeLa cells were tested. AHS and HVT stimulated colony growth, LL slightly suppressed colony formation but promoted mesenchymal cell appearance. We also report the experience of rescuing over 100 myelo-ablated patients due to high dose chemotherapy by use of FABMT since the 1980s. Conclusions: This research demonstrates that even marrow cells frozen for 41 years or more could play a role in regeneration of hematopoiesis as evidenced by our long term culture studies. Clinical studies with cells that were thawed and infused into original patients were successful and suggest the patients’ circulation would be the best environment for the proliferation and differentiation for marrow stem cells. Source of funding: Partly supported by the Research Expense Grant 2012 by Ministry of Education and Science in Japan. Conflict of interest: None declared. cryomedicine@nifty.com