Figures
Figure 1. Overview of the experimental design and HSPC-fractions investigated. (a) Overview of experimental design. (b) Cryovariables investigated in this work. (c) Summary of analyses done on samples post-thaw. HSPC-fractions tracked herein by flow cytometry and their relative content in HSC, progenitors and mature cells, are presented. (d) Representative flow cytometry analyses of ex vivo expanded CD34+ cells post-expansion before freezing. Gating strategy used to track viable cells and frequency of CD34+-subfractions are presented. HSPC: hematopoietic stem and progenitor cell; UCB: umbilical cord blood; CRF: controlled-rate freezer; CPP: CryoProtectPure-STEM; CSL: CryoScarLess; CFU: colony-forming unit; TNC: total nucleated cell; HSC: hematopoietic stem cell; DMSO: dimethyl sulfoxide.
Figure 2. Impact of different freezing procedures on the viability and recovery of ex vivo expanded cryopreserved HSPCs. (a–d) Total number of nucleated cells, CD34+ cells, CD34+CD45RA– cells, and CD34+CD45RA–CD90+ cells post-thaw in indicated conditions. (e) Total number of CFU post-thaw. Data are presented as mean ± standard error of the mean (SEM, n = 3–4). No significant differences are detected. HSPC: hematopoietic stem and progenitor cell; CRF: controlled-rate freezer; CFU: colony-forming unit; TNC: total nucleated cell.
Figure 3. Impact of different thawing methods on the viability and recovery of ex vivo expanded cryopreserved HSPCs. (a–d) Viability of TNC, CD34+ cells, CD34+CD45RA– cells, and CD34+CD45RA–CD90+ cells post-thaw. (e–h) Total number of indicated viable cells post-thaw. (i) Total number of CFU post-thaw. Data are presented as mean ± standard error of the mean (SEM, n = 4). No significant differences are detected. HSPC: hematopoietic stem and progenitor cell; CFU: colony-forming unit; TNC: total nucleated cell.
Figure 4. Impact of different freezing solutions on the viability and recovery of ex vivo expanded cryopreserved HSPCs. (a–d) Viability of TNC, CD34+ cells, CD34+CD45RA– cells, and CD34+CD45RA–CD90+ cells post-thaw. (e–h) Total number of indicated viable cells post-thaw. (i) Total number of CFU post-thaw. Data are presented as mean ± standard error of the mean (SEM, n = 3–4). Significant differences were determined by one-way ANOVA; *P < 0.05, **P < 0.01 and *** P < 0.001. HSPC: hematopoietic stem and progenitor cell; CPP: CryoProtectPure-STEM; CSL: CryoScarLess; CFU: colony-forming unit; TNC: total nucleated cell; DMSO: dimethyl sulfoxide; ANOVA: analysis of variance.
Figure 5. Investigation of delayed-onset cell death. (a, b) Viability measured by flow cytometry by Sytox exclusion 1.5 h and 20 h post-thaw. Comparisons between the different freezing procedures (a) and different thawing procedures (b) are presented. (c) Impact of different cryosolutions on the incidence of necrosis and apoptosis 20 h post-thaw. Representative cytometry analyses for each cryosolutions tested and summarized proportions of live, apoptotic and necrotic cells are presented. Data are presented as mean ± standard error of the mean (SEM, n = 4). Significant differences were determined by one-way ANOVA; *P < 0.05, **P < 0.01 and ***P < 0.001. CRF: controlled-rate freezer; CPP: CryoProtectPure-STEM; CSL: CryoScarLess; DMSO: dimethyl sulfoxide; ANOVA: analysis of variance.