Suitability of small diagnostic peripheral-blood samples for cell-therapy studies

Coralea Stephanou; Panayiota Papasavva; Myria Zachariou; Petros Patsali; Marilena Epitropou; Petros Ladas; Ruba Al-Abdulla; Soteroulla Christou; Michael N. Antoniou; Carsten W. Lederer; Marina Kleanthous

doi:10.1016/j.jcyt.2016.11.007

Suitability of small diagnostic peripheral-blood samples for cell-therapy studies

Coralea Stephanou, Panayiota Papasavva, Myria Zachariou, Petros Patsali, Marilena Epitropou, Petros Ladas, Ruba Al-Abdulla, Soteroulla Christou, Michael N. Antoniou, Carsten W. Lederer, Marina Kleanthous

Medical & Molecular Genetics

Research output: Contribution to journal › Article › peer-review

8 Citations (Scopus)

Abstract

AbstractBackground aims 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. Methods 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. Results 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. Discussion 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.

Original language	English
Pages (from-to)	311-326
Number of pages	16
Journal	CYTOTHERAPY
Volume	19
Issue number	2
DOIs	https://doi.org/10.1016/j.jcyt.2016.11.007
Publication status	Published - Feb 2017

Keywords

β-thalassemia
colony-forming cell assay
cryopreservation
density-gradient separation
erythrocyte lysis by ACK buffer
hematopoietic stem and progenitor cell
in vitro expansion
lentiviral transduction

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10.1016/j.jcyt.2016.11.007

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@article{6b46582e99314eeda235a30f7a075c8e,

title = "Suitability of small diagnostic peripheral-blood samples for cell-therapy studies",

abstract = "AbstractBackground aims 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. Methods 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. Results 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. Discussion 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.",

keywords = "β-thalassemia, colony-forming cell assay, cryopreservation, density-gradient separation, erythrocyte lysis by ACK buffer, hematopoietic stem and progenitor cell, in vitro expansion, lentiviral transduction",

author = "Coralea Stephanou and Panayiota Papasavva and Myria Zachariou and Petros Patsali and Marilena Epitropou and Petros Ladas and Ruba Al-Abdulla and Soteroulla Christou and Antoniou, {Michael N.} and Lederer, {Carsten W.} and Marina Kleanthous",

year = "2017",

month = feb,

doi = "10.1016/j.jcyt.2016.11.007",

language = "English",

volume = "19",

pages = "311--326",

journal = "CYTOTHERAPY",

issn = "1465-3249",

publisher = "Informa Healthcare",

number = "2",

}

TY - JOUR

T1 - Suitability of small diagnostic peripheral-blood samples for cell-therapy studies

AU - Stephanou, Coralea

AU - Papasavva, Panayiota

AU - Zachariou, Myria

AU - Patsali, Petros

AU - Epitropou, Marilena

AU - Ladas, Petros

AU - Al-Abdulla, Ruba

AU - Christou, Soteroulla

AU - Antoniou, Michael N.

AU - Lederer, Carsten W.

AU - Kleanthous, Marina

PY - 2017/2

Y1 - 2017/2

N2 - AbstractBackground aims 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. Methods 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. Results 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. Discussion 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.

AB - AbstractBackground aims 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. Methods 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. Results 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. Discussion 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.

KW - β-thalassemia

KW - colony-forming cell assay

KW - cryopreservation

KW - density-gradient separation

KW - erythrocyte lysis by ACK buffer

KW - hematopoietic stem and progenitor cell

KW - in vitro expansion

KW - lentiviral transduction

U2 - 10.1016/j.jcyt.2016.11.007

DO - 10.1016/j.jcyt.2016.11.007

M3 - Article

SN - 1465-3249

VL - 19

SP - 311

EP - 326

JO - CYTOTHERAPY

JF - CYTOTHERAPY

IS - 2

ER -

Suitability of small diagnostic peripheral-blood samples for cell-therapy studies

Abstract

Keywords

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