MOLECULAR AND FUNCTIONAL INTEGRITY OF CRYOPRESERVED CHRONIC MYELOID LEUKAEMIA CD34-POSITIVE HAEMATOPOIETIC STEM CELLS
(Abstract release date: 05/19/16)
EHA Library. Hodkinson C. 06/09/16; 134709; PB1809
Dr. Clare Hodkinson
Contributions
Contributions
Abstract
Abstract: PB1809
Type: Publication Only
Background
Biopreservation is a billion dollar industry, despite this, access to large numbers of high quality biospecimens remains a challenge for many researchers. Chronic myeloid leukaemia (CML) is a rare condition of approximately 1 new case per 100,000 in the UK per year. CML is a clonal disorder originating from a CD34-positive haematopoietic stem cell. Studies of molecular targets and functional processes in tumour-initiating stem cells have greatly enhanced the understanding of oncogenesis and disease progression, but the impact of freezing, thawing and long-term storage on tumour-initiating cells is under reported. Knowledge of these bioprocesses is critical to the development of high-quality biobank collections.
Aims
To investigate the effect of cryopreservation on CML CD34-positive cells and the utility of such material for downstream molecular and functional analyses.
Methods
Mononuclear cells (MNCs) and CD34-positive cells were isolated from excess cryopreserved autologous peripheral blood stem cell (PBSC) harvests, collected between 1990 and 2006, fresh whole blood from untreated CML patients, and healthy NHS blood donors. Cells were cryopreserved by controlled-rate freezing, stored in gas-phase liquid nitrogen and underwent three cycles of freeze-thaw (FT). Molecular and functional assays were performed at each cycle. RNA integrity number (RIN), as a measure of total RNA quality, were assessed on an Agilent Bioanalyzer, and semi-quantitative PCR, using GUSB as a reference gene, was performed to evaluate BCR-ABL gene expression. CD34-positive cell frequency was determined by two-colour flow cytometry using CD34 PE-Cy5.5 and the vital dye Calcein. Analysis of CD34-positive cell clonogenic potential was conducted using a colony-forming unit (CFU) assay, whereby cells were incubated in complete CFU media, supplemented with 3U/ml erythropoietin, for 16 days.
Results
Mean RIN values for normal and CML cell subsets were ≥7. RIN values for normal MNCs and CD34-positive cells were significantly higher (p<.01) compared to CML cellular subsets; there was no significant effect of repeat FT on total RNA derived from either normal or diseased cell subsets. GUSB and BCR-ABL expression were significantly higher (p<.05) in freshly isolated CML cell subsets compared to FT cycles 2 and 3, but not cycle 1. A downward trend in the ratio of BCR-ABL:GUSB expression was observed with repeat FT cycles but this did not reach statistical significance. The frequency of live CD34-positive cells remained stable with successive FT cycles. No difference in CFU potential was observed for CD34-positive cells in healthy donors between fresh and FT cycle 1, but significantly fewer (p<.05) colonies were observed for successive FT cycles. Cryopreservation of CML CD34-positive cells resulted in a significant reduction (p<0.5) in CFU potential compared to fresh material.
Conclusion
Long-term storage and cryopreservation has minimal impact on the quality of genetic material for use in downstream molecular analysis. However, gene expression data indicates a potential global downregulation of gene regulation post freeze-thaw and functional studies suggest that while CD34-positive cells are sufficiently robust to withstand cryopreservation they experience a loss in proliferative potential, to which CML derived cells are more sensitive. This implies that there are different handling and storage requirements for cell subsets derived from healthy and diseased individuals, highlighting the need to standardise biospecimen preparation and storage for meaningful analyses where access to fresh material is limited.
Session topic: E-poster
Keyword(s): Autologous stem cell collection, CD34+ cells, Chronic myeloid leukemia, Cryopreservation
Type: Publication Only
Background
Biopreservation is a billion dollar industry, despite this, access to large numbers of high quality biospecimens remains a challenge for many researchers. Chronic myeloid leukaemia (CML) is a rare condition of approximately 1 new case per 100,000 in the UK per year. CML is a clonal disorder originating from a CD34-positive haematopoietic stem cell. Studies of molecular targets and functional processes in tumour-initiating stem cells have greatly enhanced the understanding of oncogenesis and disease progression, but the impact of freezing, thawing and long-term storage on tumour-initiating cells is under reported. Knowledge of these bioprocesses is critical to the development of high-quality biobank collections.
Aims
To investigate the effect of cryopreservation on CML CD34-positive cells and the utility of such material for downstream molecular and functional analyses.
Methods
Mononuclear cells (MNCs) and CD34-positive cells were isolated from excess cryopreserved autologous peripheral blood stem cell (PBSC) harvests, collected between 1990 and 2006, fresh whole blood from untreated CML patients, and healthy NHS blood donors. Cells were cryopreserved by controlled-rate freezing, stored in gas-phase liquid nitrogen and underwent three cycles of freeze-thaw (FT). Molecular and functional assays were performed at each cycle. RNA integrity number (RIN), as a measure of total RNA quality, were assessed on an Agilent Bioanalyzer, and semi-quantitative PCR, using GUSB as a reference gene, was performed to evaluate BCR-ABL gene expression. CD34-positive cell frequency was determined by two-colour flow cytometry using CD34 PE-Cy5.5 and the vital dye Calcein. Analysis of CD34-positive cell clonogenic potential was conducted using a colony-forming unit (CFU) assay, whereby cells were incubated in complete CFU media, supplemented with 3U/ml erythropoietin, for 16 days.
Results
Mean RIN values for normal and CML cell subsets were ≥7. RIN values for normal MNCs and CD34-positive cells were significantly higher (p<.01) compared to CML cellular subsets; there was no significant effect of repeat FT on total RNA derived from either normal or diseased cell subsets. GUSB and BCR-ABL expression were significantly higher (p<.05) in freshly isolated CML cell subsets compared to FT cycles 2 and 3, but not cycle 1. A downward trend in the ratio of BCR-ABL:GUSB expression was observed with repeat FT cycles but this did not reach statistical significance. The frequency of live CD34-positive cells remained stable with successive FT cycles. No difference in CFU potential was observed for CD34-positive cells in healthy donors between fresh and FT cycle 1, but significantly fewer (p<.05) colonies were observed for successive FT cycles. Cryopreservation of CML CD34-positive cells resulted in a significant reduction (p<0.5) in CFU potential compared to fresh material.
Conclusion
Long-term storage and cryopreservation has minimal impact on the quality of genetic material for use in downstream molecular analysis. However, gene expression data indicates a potential global downregulation of gene regulation post freeze-thaw and functional studies suggest that while CD34-positive cells are sufficiently robust to withstand cryopreservation they experience a loss in proliferative potential, to which CML derived cells are more sensitive. This implies that there are different handling and storage requirements for cell subsets derived from healthy and diseased individuals, highlighting the need to standardise biospecimen preparation and storage for meaningful analyses where access to fresh material is limited.
Session topic: E-poster
Keyword(s): Autologous stem cell collection, CD34+ cells, Chronic myeloid leukemia, Cryopreservation
Abstract: PB1809
Type: Publication Only
Background
Biopreservation is a billion dollar industry, despite this, access to large numbers of high quality biospecimens remains a challenge for many researchers. Chronic myeloid leukaemia (CML) is a rare condition of approximately 1 new case per 100,000 in the UK per year. CML is a clonal disorder originating from a CD34-positive haematopoietic stem cell. Studies of molecular targets and functional processes in tumour-initiating stem cells have greatly enhanced the understanding of oncogenesis and disease progression, but the impact of freezing, thawing and long-term storage on tumour-initiating cells is under reported. Knowledge of these bioprocesses is critical to the development of high-quality biobank collections.
Aims
To investigate the effect of cryopreservation on CML CD34-positive cells and the utility of such material for downstream molecular and functional analyses.
Methods
Mononuclear cells (MNCs) and CD34-positive cells were isolated from excess cryopreserved autologous peripheral blood stem cell (PBSC) harvests, collected between 1990 and 2006, fresh whole blood from untreated CML patients, and healthy NHS blood donors. Cells were cryopreserved by controlled-rate freezing, stored in gas-phase liquid nitrogen and underwent three cycles of freeze-thaw (FT). Molecular and functional assays were performed at each cycle. RNA integrity number (RIN), as a measure of total RNA quality, were assessed on an Agilent Bioanalyzer, and semi-quantitative PCR, using GUSB as a reference gene, was performed to evaluate BCR-ABL gene expression. CD34-positive cell frequency was determined by two-colour flow cytometry using CD34 PE-Cy5.5 and the vital dye Calcein. Analysis of CD34-positive cell clonogenic potential was conducted using a colony-forming unit (CFU) assay, whereby cells were incubated in complete CFU media, supplemented with 3U/ml erythropoietin, for 16 days.
Results
Mean RIN values for normal and CML cell subsets were ≥7. RIN values for normal MNCs and CD34-positive cells were significantly higher (p<.01) compared to CML cellular subsets; there was no significant effect of repeat FT on total RNA derived from either normal or diseased cell subsets. GUSB and BCR-ABL expression were significantly higher (p<.05) in freshly isolated CML cell subsets compared to FT cycles 2 and 3, but not cycle 1. A downward trend in the ratio of BCR-ABL:GUSB expression was observed with repeat FT cycles but this did not reach statistical significance. The frequency of live CD34-positive cells remained stable with successive FT cycles. No difference in CFU potential was observed for CD34-positive cells in healthy donors between fresh and FT cycle 1, but significantly fewer (p<.05) colonies were observed for successive FT cycles. Cryopreservation of CML CD34-positive cells resulted in a significant reduction (p<0.5) in CFU potential compared to fresh material.
Conclusion
Long-term storage and cryopreservation has minimal impact on the quality of genetic material for use in downstream molecular analysis. However, gene expression data indicates a potential global downregulation of gene regulation post freeze-thaw and functional studies suggest that while CD34-positive cells are sufficiently robust to withstand cryopreservation they experience a loss in proliferative potential, to which CML derived cells are more sensitive. This implies that there are different handling and storage requirements for cell subsets derived from healthy and diseased individuals, highlighting the need to standardise biospecimen preparation and storage for meaningful analyses where access to fresh material is limited.
Session topic: E-poster
Keyword(s): Autologous stem cell collection, CD34+ cells, Chronic myeloid leukemia, Cryopreservation
Type: Publication Only
Background
Biopreservation is a billion dollar industry, despite this, access to large numbers of high quality biospecimens remains a challenge for many researchers. Chronic myeloid leukaemia (CML) is a rare condition of approximately 1 new case per 100,000 in the UK per year. CML is a clonal disorder originating from a CD34-positive haematopoietic stem cell. Studies of molecular targets and functional processes in tumour-initiating stem cells have greatly enhanced the understanding of oncogenesis and disease progression, but the impact of freezing, thawing and long-term storage on tumour-initiating cells is under reported. Knowledge of these bioprocesses is critical to the development of high-quality biobank collections.
Aims
To investigate the effect of cryopreservation on CML CD34-positive cells and the utility of such material for downstream molecular and functional analyses.
Methods
Mononuclear cells (MNCs) and CD34-positive cells were isolated from excess cryopreserved autologous peripheral blood stem cell (PBSC) harvests, collected between 1990 and 2006, fresh whole blood from untreated CML patients, and healthy NHS blood donors. Cells were cryopreserved by controlled-rate freezing, stored in gas-phase liquid nitrogen and underwent three cycles of freeze-thaw (FT). Molecular and functional assays were performed at each cycle. RNA integrity number (RIN), as a measure of total RNA quality, were assessed on an Agilent Bioanalyzer, and semi-quantitative PCR, using GUSB as a reference gene, was performed to evaluate BCR-ABL gene expression. CD34-positive cell frequency was determined by two-colour flow cytometry using CD34 PE-Cy5.5 and the vital dye Calcein. Analysis of CD34-positive cell clonogenic potential was conducted using a colony-forming unit (CFU) assay, whereby cells were incubated in complete CFU media, supplemented with 3U/ml erythropoietin, for 16 days.
Results
Mean RIN values for normal and CML cell subsets were ≥7. RIN values for normal MNCs and CD34-positive cells were significantly higher (p<.01) compared to CML cellular subsets; there was no significant effect of repeat FT on total RNA derived from either normal or diseased cell subsets. GUSB and BCR-ABL expression were significantly higher (p<.05) in freshly isolated CML cell subsets compared to FT cycles 2 and 3, but not cycle 1. A downward trend in the ratio of BCR-ABL:GUSB expression was observed with repeat FT cycles but this did not reach statistical significance. The frequency of live CD34-positive cells remained stable with successive FT cycles. No difference in CFU potential was observed for CD34-positive cells in healthy donors between fresh and FT cycle 1, but significantly fewer (p<.05) colonies were observed for successive FT cycles. Cryopreservation of CML CD34-positive cells resulted in a significant reduction (p<0.5) in CFU potential compared to fresh material.
Conclusion
Long-term storage and cryopreservation has minimal impact on the quality of genetic material for use in downstream molecular analysis. However, gene expression data indicates a potential global downregulation of gene regulation post freeze-thaw and functional studies suggest that while CD34-positive cells are sufficiently robust to withstand cryopreservation they experience a loss in proliferative potential, to which CML derived cells are more sensitive. This implies that there are different handling and storage requirements for cell subsets derived from healthy and diseased individuals, highlighting the need to standardise biospecimen preparation and storage for meaningful analyses where access to fresh material is limited.
Session topic: E-poster
Keyword(s): Autologous stem cell collection, CD34+ cells, Chronic myeloid leukemia, Cryopreservation
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