Stephanou Coralea, Papasavva Panayiota, Zachariou Myria, Patsali Petros, Epitropou Marilena, Ladas Petros, Al-Abdulla Ruba, Christou Soteroulla, Antoniou Michael N, Lederer Carsten W, Kleanthous Marina
Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; Gene Expression and Therapy Group, King's College London, United Kingdom.
Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; Cyprus School of Molecular Medicine, Nicosia, Cyprus.
Cytotherapy. 2017 Feb;19(2):311-326. doi: 10.1016/j.jcyt.2016.11.007.
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.
原发性造血干细胞和祖细胞（HSPCs）是血液疾病细胞治疗的关键组成部分，因此是相关研究的真正底物。我们提出，普遍存在的小体积诊断样本代表了一种现成的、尚未开发的原发性患者来源细胞资源，可用于细胞和基因治疗研究。
在本研究中，我们比较了从正常和地中海贫血小体积血样中分离和储存HSPCs的方法，考虑了基因型、密度梯度分离与基于裂解的细胞分离以及不同血清含量的冷冻保存培养基。下游分析包括活力、回收率、在半固体培养基中的分化以及在液体培养和病毒转导中的性能。
我们证明，通过氯化铵钾（ACK）裂解或梯度分离法分离的HSPCs适用于克隆形成试验中的功能分析、高水平的HSPC扩增和高效的慢病毒转导。对于细胞的冷冻保存，梯度分离优于ACK裂解，并且在含有50%胎牛血清的冷冻培养基中进行冷冻保存，在所有测试标准下都显示出良好的结果。对于新鲜分离细胞的检测，ACK裂解的效果与梯度分离相似，对于地中海贫血样本，ACK裂解的效果优于梯度分离，且成本和操作时间仅为梯度分离的一小部分。所有分离和储存方法在样本组内都显示出相当大的差异，但对于地中海贫血样本的密度梯度分离，这种差异尤为明显。
本研究证明了小体积血样适用于储存和临床前研究，为任何具有相应生物伦理批准以实验性使用剩余材料的血液诊断实验室开辟了HSPC和基因治疗的研究领域。
