13. Peripheral blood derived hematopoietic progenitor cells (HPC): An overview of a successful application of cryobiology

Abstract

Hematopoietic progenitor cells (HPC) are primitive pluripotent cells capable of self renewal as well as of differentiation and maturation into all hematopoietic lineages. They can be found in bone marrow, in fetal liver, in umbilical cord blood, and in the mononuclear cell fraction of circulating blood. They are characterized by their colony-forming capacities in different in vitro cell culture assays and by the surface antigen marker CD34. Today, HPC are successfully cryopreserved using methods based on a dimethyl sulphoxide (Me2SO) method proposed by Ashwood-Smith (1961) for bone marrow. Cryopreserved peripheral HPC (in combination with patient high-dose chemotherapy or irradiation) have become a “standard” blood component for the treatment of several malignant diseases in more than 10,000 patients in Europe per year, e.g. lymphoma, myeloma, leukemia, and germ cell tumors. Nowadays HPC are usually obtained by apheresis from peripheral blood after stimulation with a cytokine named G-CSF (granulocyte colony stimulating factor). During cooling, heat is removed either by computer-controlled and liquid nitrogen (LN2) operated machines or in mechanical (−80 °C) refrigerators. Stiff et al. (1983) have demonstrated that the addition of 6% hydroxyethyl starch (HES) reduced the “original” concentration of Me2SO (10%) by half. A serious problem regarding frozen peripheral HPC is the danger of clotting after thawing. Although it is highly advisable to use as little anticoagulant – mostly ACD-A (acid–citrate–dextrose, formula A) or heparin – as possible during the apheresis procedure from the patient’s point of view, this may lead to problems during the following processing and after thawing. So if low anticoagulant concentrations have been used during the apheresis procedure, we recommend adding additional ACD-A to the final apheresis product. There are reports in the literature (e.g., Douay et al., 1982; Goldman et al., 1978) on Me2SO toxicity at room temperature. As a consequence of these and other reports, in clinical protocols diluted and precooled Me2SO solutions are usually added at 0–4 °C. In an investigation with highly pure Me2SO (pharmaceutical grade), however, no impairment (colony assay) within the first hour was found when 5% or 10% of Me2SO were added at either 4 or 37 °C (Rowley and Anderson, 1993). Cool-down kinetics depend on several parameters: geometry (“undefined,” cylindrical, or plate shaped), sample thickness, composition (type and concentration of cryoprotectants), and thermal properties (heat capacity, heat conductivity) of the freezing bag and (if used) the surrounding metal envelope. Therefore, temperature time-histories measured in small reference samples (e.g., tubes) may differ significantly from those measured in the corresponding product bags. With regard to cryoprotectant concentration and cooling rates, we did not find significant differences in cell recovery and clonogenicity (Sputtek et al., 1997), provided at least 5% Me2SO was present and cooling rates did not exceed 5 °C/min. Our most recent data supports the conclusion that a reliable −150 °C freezer is as suitable for long-term storage of HPC as is storage in the vapor phase over LN2. There was no systematic decline in viability measured in terms of six different parameters after more than 16 months storage in a −150 °C freezer (Sputtek et al. 2011). In our hands cell counts dropped significantly within 5 h after thawing compared to a high recovery when post thaw storage was performed at 20 °C and 0 °C, respectively. Therefore, we recommend to continue the common practice of thawing the frozen HPC units at the patient’s bedside.