title: Suitability of small diagnostic peripheral-blood samples for cell-therapy studies.
abstract: Primary hematopoietic stem and progenitor cells (HSPCs) are key components of cell-based therapies for blood disorders and are thus the authentic substrate for related research. We propose that ubiquitous small-volume diagnostic samples represent a readily available and as yet untapped resource of primary patient-derived cells for cell- and gene-therapy studies.
  
  
  
  In the present study we compare isolation and storage methods for HSPCs from normal and thalassemic small-volume blood samples, considering genotype, density-gradient versus lysis-based cell isolation and cryostorage media with different serum contents. Downstream analyses include viability, recovery, differentiation in semi-solid media and performance in liquid cultures and viral transductions.
  
  
  
  We demonstrate that HSPCs isolated either by ammonium-chloride potassium (ACK)-based lysis or by gradient isolation are suitable for functional analyses in clonogenic assays, high-level HSPC expansion and efficient lentiviral transduction. For cryostorage of cells, gradient isolation is superior to ACK lysis, and cryostorage in freezing media containing 50% fetal bovine serum demonstrated good results across all tested criteria. For assays on freshly isolated cells, ACK lysis performed similar to, and for thalassemic samples better than, gradient isolation, at a fraction of the cost and hands-on time. All isolation and storage methods show considerable variation within sample groups, but this is particularly acute for density gradient isolation of thalassemic samples.
  
  
  
  This study demonstrates the suitability of small-volume blood samples for storage and preclinical studies, opening up the research field of HSPC and gene therapy to any blood diagnostic laboratory with corresponding bioethics approval for experimental use of surplus material.
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; Gene Expression and Therapy Group, King's College London, United Kingdom.
authors.forename: Coralea
authors.initials: C
authors.lastname: Stephanou
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; Cyprus School of Molecular Medicine, Nicosia, Cyprus.
authors.forename: Panayiota
authors.initials: P
authors.lastname: Papasavva
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.
authors.forename: Myria
authors.initials: M
authors.lastname: Zachariou
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; Gene Expression and Therapy Group, King's College London, United Kingdom.
authors.forename: Petros
authors.initials: P
authors.lastname: Patsali
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.
authors.forename: Marilena
authors.initials: M
authors.lastname: Epitropou
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.
authors.forename: Petros
authors.initials: P
authors.lastname: Ladas
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.
authors.forename: Ruba
authors.initials: R
authors.lastname: Al-Abdulla
authors.affiliation: Thalassaemia Centre, Ministry of Health, Archbishop Makarios III Hospital, Nicosia, Cyprus.
authors.forename: Soteroulla
authors.initials: S
authors.lastname: Christou
authors.affiliation: Gene Expression and Therapy Group, King's College London, United Kingdom.
authors.forename: Michael N
authors.initials: MN
authors.lastname: Antoniou
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; Cyprus School of Molecular Medicine, Nicosia, Cyprus. Electronic address: Lederer@cing.ac.cy.
authors.forename: Carsten W
authors.initials: CW
authors.lastname: Lederer
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; Cyprus School of Molecular Medicine, Nicosia, Cyprus.
authors.forename: Marina
authors.initials: M
authors.lastname: Kleanthous
mesh_terms: D001793:Blood Preservation / Q000379:methods / Q000592:standards; D001800:Blood Specimen Collection / Q000379:methods* / Q000592:standards*; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods* / Q000592:standards*; D002470:Cell Survival; D064987:Cell- and Tissue-Based Therapy / Q000379:methods*; D002478:Cells, Cultured; D015925:Cryopreservation; D005240:Feasibility Studies; D005615:Freezing; D006412:Hematopoietic Stem Cells / Q000473:pathology / Q000502:physiology; D006801:Humans; D007958:Leukocyte Count; D007962:Leukocytes / Q000473:pathology* / Q000502:physiology; D012698:Serologic Tests; D013789:Thalassemia / Q000097:blood* / Q000473:pathology
keywords: colony-forming cell assay; cryopreservation; density-gradient separation; erythrocyte lysis by ACK buffer; hematopoietic stem and progenitor cell; in vitro expansion; lentiviral transduction; β-thalassemia