Abstract
Progenitor cell therapies show great promise, but their potential for clinical applications requires improved storage and transportation. Desiccated cells stored at ambient temperature would provide economic and practical advantages over approaches employing cell freezing and subzero temperature storage. The objectives of this study were to assess a method for loading the stabilizing sugar, trehalose, into hematopoietic stem and progenitor cells (HPC) and to evaluate the effects of subsequent freeze-drying and storage at ambient temperature on differentiation and clonogenic potential. HPC were isolated from human umbilical cord blood and loaded with trehalose using an endogenous cell surface receptor, termed P2Z. Solution containing trehalose-loaded HPC was placed into vials, which were transferred to a tray freeze-dryer and removed during each step of the freeze-drying process to assess differentiation and clonogenic potential. Control groups for these experiments were freshly isolated HPC. Control cells formed 1450±230 CFU-GM, 430±140 BFU-E, and 50±40 CFU-GEMM per 50 µL. Compared to the values for the control cells, there was no statistical difference observed for cells removed at the end of the freezing step or at the end of primary drying. There was a gradual decrease in the number of CFU-GM and BFU-E for cells removed at different temperatures during secondary drying; however, there were no significant differences in the number of CFU-GEMM. To determine storage stability of lyophilized HPC, cells were stored for 4 weeks at 25°C in the dark. Cells reconstituted immediately after lyophilization produced 580±90 CFU-GM (∼40%, relative to unprocessed controls p<0.0001), 170±70 BFU-E (∼40%, p<0.0001), and 41±22 CFU-GEMM (∼82%, p = 0.4171), and cells reconstituted after 28 days at room temperature produced 513±170 CFU-GM (∼35%, relative to unprocessed controls, p<0.0001), 112±68 BFU-E (∼26%, p<0.0001), and 36±17 CFU-GEMM (∼82%, p = 0.2164) These studies are the first to document high level retention of CFU-GEMM following lyophilization and storage for 4 weeks at 25°C. This type of flexible storage stability would potentially permit the ability to ship and store HPC without the need for refrigeration.
Highlights
Hematopoietic stem and progenitor cells (HPC) contain populations of cells with long-term and short-term regeneration capacities as well as committed progenitors
As a first step toward preserving HPC by freeze drying, we previously investigated the permeabilization of a model cell line (TF-1) to trehalose[48]
Expression of P2ZR and permeabilization of HPC We investigated the expression of P2Z receptor on HPC using indirect staining and flow cytometric analysis (Figure 1)
Summary
Hematopoietic stem and progenitor cells (HPC) contain populations of cells with long-term and short-term regeneration capacities as well as committed progenitors. HPC give rise to all the blood cell types including lymphoid (T-cells, B-cells, NK-cells, dendritic) and myeloid (monocyctes/macrophages, neutrophils, megakaryocytes, granulocytes, eosinophils, erythrocytes)[1]. There is a growing need for producing, storing and shipping large numbers of HPC to ensure a steady supply for use in clinical applications. Simple preservation techniques, such as refrigeration or tissue culture, have drawbacks including limited shelf life, high cost and risk of contamination. Cryopreservation has been successfully utilized for long-term storage of HPC This approach is based on the principle that chemical, biological and physical processes are sufficiently ‘‘suspended’’ at cryogenic temperatures (2196uC) because liquid water does not exist below 2130uC[9]. There is insufficient thermal energy for chemical and metabolic processes to proceed at practical relevant rates[9,13]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
