Abstract
BackgroundCellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues. This approach is of great importance in studies on β-thalassemia, since the recruitment of patients and collection of specimens can represent a crucial and often limiting factor in the experimental planning.MethodsErythroid precursor cells were obtained from 72 patients, mostly β-thalassemic, expanded and cryopreserved. Expression of globin genes was analyzed by real time RT-qPCR. Hemoglobin production was studied by HPLC.ResultsIn this paper we describe the production and validation of a Thal-Biobank constituted by expanded erythroid precursor cells from β-thalassemia patients. The biobanked samples were validated for maintenance of their phenotype after (a) cell isolation from same patients during independent phlebotomies, (b) freezing step in different biobanked cryovials, (c) thawing step and analysis at different time points. Reproducibility was confirmed by shipping the frozen biobanked cells to different laboratories, where the cells were thawed, cultured and analyzed using the same standardized procedures. The biobanked cells were stratified on the basis of their baseline level of fetal hemoglobin production and exposed to fetal hemoglobin inducers.ConclusionThe use of biobanked cells allows stratification of the patients with respect to fetal hemoglobin production and can be used for determining the response to the fetal hemoglobin inducer hydroxyurea and to gene therapy protocols with reproducible results.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1016-4) contains supplementary material, which is available to authorized users.
Highlights
Cellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues
This protocol has been widely applied to develop novel fetal hemoglobin (HbF) inducers [7, 8], to validate gene therapy strategies based on novel lentiviral vectors [9, 10] and to perform gene editing using approaches based on zing finger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-CAS9 [11,12,13,14,15]
Production of the cellular Thal‐Biobank The cellular Thal-Biobank is composed of 779 biobanked specimens from 8 healthy donors and 72 patients with sickle cell anemia (SCA) and β-thalassemia
Summary
Cellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues. The number of biobanks is rapidly increasing worldwide, helping to create collaborative networks The creation of these banks can tackle very important biomedical issues that require large numbers of tissue samples of the Cellular biobanking is a key step in a variety of “OMICS” analyses, as well as in the screening of bioactive molecules for the development of novel therapeutic. The biological assay used to test therapeutic molecules on primary erythroid cells from β-thalassemia patients is based on one method called “two-phase culture procedure” developed by the Fibach’s research group [5, 6] In this system, peripheral blood mononuclear cells (PBMCs) isolated from 20 to 25 ml of patients’ blood are seeded in a Phase I culture for about 7 days and differentiated into hemoglobin-producing erythroid cell for a further 7–10 day period (Phase II) by exposure to erythropoietin [5, 6]. This protocol has been widely applied to develop novel fetal hemoglobin (HbF) inducers [7, 8], to validate gene therapy strategies based on novel lentiviral vectors [9, 10] and to perform gene editing using approaches based on ZFN, TALEN, and CRISPR-CAS9 [11,12,13,14,15]
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