Long‐term storage of peripheral blood stem cells frozen and stored with a conventional liquid nitrogen technique compared with cells frozen and stored in a mechanical freezer

Abstract

Cryopreservation of hematopoietic progenitor cells using liquid nitrogen and controlled-rate freezing requires complex equipment and highly trained staff and is expensive. We compared the liquid nitrogen method with methods using a combination of dimethyl sulfoxide (DMSO) and hydroxyethyl starch (HES) for cryopreservation followed by storage in mechanical freezers. Peripheral blood stem cells (PBSCs) were collected from normal donors by apheresis and allocated to one of four preservation and storage conditions: 1) 10% DMSO with freezing in liquid nitrogen and storage in liquid nitrogen, 2) 5% DMSO and 6% HES with freezing and storage in a -80 degrees C mechanical freezer, 3) 5% DMSO and 6% HES with freezing in a -80 degrees C mechanical freezer and storage in a -135 degrees C mechanical freezer, or 4) 5% DMSO and 6% HES with freezing and storage both in a 135 degrees C mechanical freezer. Cells were stored for 5 years during which total nucleated cells (TNCs), cell viability, CD34+ cell content, and colony-forming unit-granulocyte-macrophage content were determined. There were some significant differences in the variables measured during freezing and the 5 years of storage compared to the values before freezing and storage; however, these differences were not consistent and do not favor one protocol over the others. Samples stored for 24 hours before cryopreservation showed a significant decrease in TNCs, but no other significant changes during the 5 years. In vitro measurements indicate that PBSCs can be successfully frozen and stored using a combination of DMSO and HES providing smaller amounts of DMSO and allowing simplified freezing and storage conditions.