Cryo-Forum

From: Proceedings 10th World Congress of Cryosurgery
Cryopreserving hematopoietic stem cells for autologous or allogeneic transplantation

November 1998
M Korbling, University of Texas, M.D. Anderson Cancer Center, Houston, Texas

Hematopoietic stem cells are defmed as being pluripotent and indefinitely self-renewing. Such stem cells are rare encompassing approximately 0.00 1% of total nucleated cells (or 1 stem cell out of 100,000 nucleated cells). The need for cryopreserving stem cells is both to schedule myeloablative treatment independently from stem cell harvest, and to prophylactically keep stem cells alive for potential later use in case of relapsing disease. Freezing stem cells coincides with the formation of ice crystals inside the cells leading to destruction of cell organelles and to cell death. This can be prevented by:

1. The use of cryoprotectants such as dimethyl sulfoxide (DMSO) with or without hydroxyethyl starch (HES) which helps to stabilize the movement of water across the cell membrane during freezing,

2. Cooling stem cells at a controlled rate, and compensating for the heat that is released as the medium in which the cells are

suspended changes fi7om liquid to solid phase.

The concentration of DMSO contained in the cell suspension to be frozen is usually between 5% and 10%. More recently, the addition of 4-6% HES has been proposed. Stem cells are frozen at a controlled freezing rate of 1C to 2C/min. To compensate for the heat of fusion during the phase transition, the freezing chamber is shock cooled as pre-programmed. Stem cells are either stored immersed in liquid nitrogen at -196C, or in the vapor phase of liquid nitrogen which usually maintains a temperature of -150C to -180C. Satisfactory storage temperatures of -130C to -150C can also be achieved using specially designed electrical freezers. Stem cells cryopreserved in this way retain their viability for at least 5 years, and many transplants have been successfully performed with stem cells stored for 10 to 18 years. In stem cell apheresis products the mean recovery of CD34+ cells after freezing, thawing and washing is 86.6% (range, 24.7 to 257.8) (rr--31), 115.5% for CD3+ cells, 121.4% for CD4+ cells, 105.6% for CD8+ cells, 118.1% for CD19+ cells and 102.4% for CD3+ 56+ cells. There is no selective loss of any lymphoid subset during freezing and thawing. There is no difference in cell recovery between large volume (200 ml bags) and small volume (5 ral vial) freezing. Stem cell freezing is a well-established technology with a high safety standard. New freezing technologies are needed to protect more mature and differentiated myeloid cells (i.e. granulocytes) and even solid organs.


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