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Cai Y, Prochazkova M, Kim YS, Jiang C, Ma J, Moses L, Martin K, Pham V, Zhang N, Highfill SL, Somerville RP, Stroncek DF, Jin P. Assessment and comparison of viability assays for cellular products. Cytotherapy 2024; 26:201-209. [PMID: 38085197 PMCID: PMC10872314 DOI: 10.1016/j.jcyt.2023.11.008] [Citation(s) in RCA: 8] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2023] [Revised: 11/06/2023] [Accepted: 11/14/2023] [Indexed: 02/03/2024]
Abstract
BACKGROUND AIMS Accurate assessment of cell viability is crucial in cellular product manufacturing, yet selecting the appropriate viability assay presents challenges due to various factors. This study compares and evaluates different viability assays on fresh and cryopreserved cellular products, including peripheral blood stem cell (PBSC) and peripheral blood mononuclear cell (PBMC) apheresis products, purified PBMCs and cultured chimeric antigen receptor and T-cell receptor-engineered T-cell products. METHODS Viability assays, including manual Trypan Blue exclusion, flow cytometry-based assays using 7-aminoactinomycin D (7-AAD) or propidium iodide (PI) direct staining or cell surface marker staining in conjunction with 7-AAD, Cellometer (Nexcelom Bioscience LLC, Lawrence, MA, USA) Acridine Orange/PI staining and Vi-CELL BLU Cell Viability Analyzer (Beckman Coulter, Inc, Brea, CA, USA), were evaluated. A viability standard was established using live and dead cell mixtures to assess the accuracy of these assays. Furthermore, precision assessment was conducted to determine the reproducibility of the viability assays. Additionally, the viability of individual cell populations from cryopreserved PBSC and PBMC apheresis products was examined. RESULTS All methods provided accurate viability measurements and generated consistent and reproducible viability data. The assessed viability assays were demonstrated to be reliable alternatives when evaluating the viability of fresh cellular products. However, cryopreserved products exhibited variability among the tested assays. Additionally, analyzing the viability of each subset of the cryopreserved PBSC and PBMC apheresis products revealed that T cells and granulocytes were more susceptible to the freeze-thaw process, showing decreased viability. CONCLUSIONS The study demonstrates the importance of careful assay selection, validation and standardization, particularly for assessing the viability of cryopreserved products. Given the complexity of cellular products, choosing a fit-for-purpose viability assay is essential.
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Affiliation(s)
- Yihua Cai
- Center for Cellular Engineering, Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland, USA
| | - Michaela Prochazkova
- Center for Cellular Engineering, Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland, USA
| | - Yong-Soo Kim
- Center for Cellular Engineering, Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland, USA
| | - Chunjie Jiang
- Center for Cellular Engineering, Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland, USA
| | - Jinxia Ma
- Center for Cellular Engineering, Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland, USA
| | - Larry Moses
- Center for Cellular Engineering, Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland, USA
| | - Kathryn Martin
- Center for Cellular Engineering, Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland, USA
| | - Victoria Pham
- Center for Cellular Engineering, Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland, USA
| | - Nan Zhang
- Center for Cellular Engineering, Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland, USA
| | - Steven L Highfill
- Center for Cellular Engineering, Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland, USA
| | - Robert P Somerville
- Center for Cellular Engineering, Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland, USA
| | - David F Stroncek
- Center for Cellular Engineering, Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland, USA
| | - Ping Jin
- Center for Cellular Engineering, Department of Transfusion Medicine, National Institutes of Health Clinical Center, Bethesda, Maryland, USA.
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Bahsoun S, Brown MJ, Coopman K, Akam EC. Cryopreservation of Human Bone Marrow Derived Mesenchymal Stem Cells at High Concentration Is Feasible. Biopreserv Biobank 2023; 21:450-457. [PMID: 36094454 DOI: 10.1089/bio.2022.0017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Introduction: For stem cell therapies to be adopted in mainstream health care, robust, reliable, and cost-effective storage and transport processes must be developed. Cryopreservation remains the best current platform for this purpose, and freezing cells at high concentration may have many benefits, including savings on cost and storage space, facilitating transport logistics, and reducing cryoprotectant volume. Cells, such as mesenchymal stem cells (MSCs), are typically frozen at 1 million cells per milliliter (mL), but the aim of this study is to examine the post-thaw attributes of human bone marrow derived MSCs (hBM-MSCs) frozen at 1, 5, and 10 million cells per mL. Methods: Thawed cells were assessed for their morphology, phenotypic marker expression, viability, apoptosis level, metabolic activity, proliferation, and osteogenic and adipogenic differentiation. Results: In this study, for the first time, it is shown that all assessed cells expressed the typical MSC markers (CD90, CD105, and CD73) and lacked the expression of CD14, CD20, CD34, CD45, and HLA-DR. In addition, all cells showed elongated fibroblastic morphology. Post-thaw viability was retained with no difference among the three concentrations. Moreover, no significant statistical difference was observed in the post-thaw apoptosis level, metabolic activity, proliferation, and osteogenic potential, indicating that these cells are amenable to cryopreservation at higher concentrations. Conclusion: The results of this study are of paramount importance to the development of manufacturing processes around a useful freezing concentration when cells are targeted to be stored for at least 6 months.
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Affiliation(s)
- Soukaina Bahsoun
- School of Sport, Exercise and Health Sciences, Loughborough University, Loughborough, Leicestershire, United Kingdom
| | - Marie-Juliet Brown
- School of Sport, Exercise and Health Sciences, Loughborough University, Loughborough, Leicestershire, United Kingdom
| | - Karen Coopman
- Centre for Biological Engineering, Loughborough University, Loughborough, Leicestershire, United Kingdom
| | - Elizabeth C Akam
- School of Sport, Exercise and Health Sciences, Loughborough University, Loughborough, Leicestershire, United Kingdom
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Erkurt M, Uysal A, Kuku İ, Kaya E, Berber İ, Sarıcı A, Biçim S, Hidayet E, Kaya A, Merter M. The effect of cryopreserved and noncryopreserved stem cells on the outcome of autologous stem-cell transplantation in multiple myeloma patients: A single-center experience. THE EGYPTIAN JOURNAL OF HAEMATOLOGY 2022. [DOI: 10.4103/ejh.ejh_2_22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
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Erol OD, Pervin B, Seker ME, Aerts-Kaya F. Effects of storage media, supplements and cryopreservation methods on quality of stem cells. World J Stem Cells 2021; 13:1197-1214. [PMID: 34630858 PMCID: PMC8474714 DOI: 10.4252/wjsc.v13.i9.1197] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 04/21/2021] [Accepted: 08/24/2021] [Indexed: 02/06/2023] Open
Abstract
Despite a vast amount of different methods, protocols and cryoprotective agents (CPA), stem cells are often frozen using standard protocols that have been optimized for use with cell lines, rather than with stem cells. Relatively few comparative studies have been performed to assess the effects of cryopreservation methods on these stem cells. Dimethyl sulfoxide (DMSO) has been a key agent for the development of cryobiology and has been used universally for cryopreservation. However, the use of DMSO has been associated with in vitro and in vivo toxicity and has been shown to affect many cellular processes due to changes in DNA methylation and dysregulation of gene expression. Despite studies showing that DMSO may affect cell characteristics, DMSO remains the CPA of choice, both in a research setting and in the clinics. However, numerous alternatives to DMSO have been shown to hold promise for use as a CPA and include albumin, trehalose, sucrose, ethylene glycol, polyethylene glycol and many more. Here, we will discuss the use, advantages and disadvantages of these CPAs for cryopreservation of different types of stem cells, including hematopoietic stem cells, mesenchymal stromal/stem cells and induced pluripotent stem cells.
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Affiliation(s)
- Ozgur Dogus Erol
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Burcu Pervin
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Mehmet Emin Seker
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Fatima Aerts-Kaya
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
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Roddie C, O'Reilly M, Dias Alves Pinto J, Vispute K, Lowdell M. Manufacturing chimeric antigen receptor T cells: issues and challenges. Cytotherapy 2019; 21:327-340. [PMID: 30685216 DOI: 10.1016/j.jcyt.2018.11.009] [Citation(s) in RCA: 107] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2018] [Revised: 11/25/2018] [Accepted: 11/26/2018] [Indexed: 12/12/2022]
Abstract
Clinical trials of adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have delivered unprecedented responses in patients with relapsed refractory B-cell malignancy. These results have prompted Food and Drug Administration (FDA) approval of two CAR T-cell products in this high-risk patient population. The widening range of indications for CAR T-cell therapy and increasing patient numbers present a significant logistical challenge to manufacturers aiming for reproducible delivery systems for high-quality clinical CAR T-cell products. This review discusses current and novel CAR T-cell processing methodologies and the quality control systems needed to meet the increasing clinical demand for these exciting new therapies.
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Affiliation(s)
- Claire Roddie
- Research Department of Haematology, University College London, London, UK; Department of Haematology, University College London Hospitals National Health Service (NHS) Foundation Trust, London.
| | - Maeve O'Reilly
- Research Department of Haematology, University College London, London, UK; Department of Haematology, University College London Hospitals National Health Service (NHS) Foundation Trust, London
| | | | - Ketki Vispute
- Research Department of Haematology, University College London, London, UK
| | - Mark Lowdell
- Research Department of Haematology, University College London, London, UK
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Rohner N, Passweg JR, Tsakiris DA, Halter JP, Heim D, Buser AS, Infanti L, Holbro A. The value of the post-thaw CD34+ count with and without DMSO removal in the setting of autologous stem cell transplantation. Transfusion 2018; 59:1052-1060. [PMID: 30556582 DOI: 10.1111/trf.15107] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2018] [Revised: 11/06/2018] [Accepted: 11/12/2018] [Indexed: 11/28/2022]
Abstract
BACKGROUND CD34+ cell count correlates with engraftment potency after autologous stem cell transplantation. Assessment of CD34+ mainly occurs after apheresis and before cryopreservation with dimethyl sulfoxide (DMSO). The influence of postthaw CD34+ cell numbers over time to engraftment is not well studied, and determination of postthaw CD34+ cell counts is challenging for a variety of reasons. The aim of this retrospective study was to systematically assess the value of postthaw CD34+ cell counts in autologous grafts with and without DMSO removal. STUDY DESIGN AND METHODS Between January 2008 and December 2015, 236 adult patients underwent a total of 292 autologous stem cell transplantations. Median age at transplantation was 56 years, and the main indication was multiple myeloma (60%). DMSO removal was done in 96 grafts (33%), either by centrifugation or by Sepax method. RESULTS Patients receiving grafts containing DMSO showed a significantly faster platelet (p = 0.02) and RBC (p = 0.001) engraftment. DMSO removal was not associated with fewer infusion-related adverse events. We observed a good correlation between CD34+ cell count after apheresis and CD34+ cell count after thawing/washing (r = 0.931). Ninety grafts (31%) showed a significant loss of viable CD34+ cells, which translated into a delayed engraftment. CONCLUSION DMSO removal was associated with delayed platelet and RBC engraftment without preventing adverse events. CD34+ cell enumeration after thawing remains difficult to perform, but grafts showing higher cell loss during cryopreservation and thawing are associated with slower engraftment. Prospective studies on the role of DMSO removal and postthaw CD34+ enumeration using defined protocols are needed.
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Affiliation(s)
- Nicolai Rohner
- Division of Hematology, University Hospital Basel, Switzerland
| | - Jakob R Passweg
- Division of Hematology, University Hospital Basel, Switzerland
| | | | - Jörg P Halter
- Division of Hematology, University Hospital Basel, Switzerland
| | - Dominik Heim
- Division of Hematology, University Hospital Basel, Switzerland
| | - Andreas S Buser
- Division of Hematology, University Hospital Basel, Switzerland.,Blood Transfusion Center, Swiss Red Cross, Basel, Switzerland
| | - Laura Infanti
- Division of Hematology, University Hospital Basel, Switzerland.,Blood Transfusion Center, Swiss Red Cross, Basel, Switzerland
| | - Andreas Holbro
- Division of Hematology, University Hospital Basel, Switzerland.,Blood Transfusion Center, Swiss Red Cross, Basel, Switzerland
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Barik M, Bajpai M, Patnaik S, Mishra P, Behera P, Dwivedi SN. Development of new method and protocol for cryopreservation related to embryo and oocytes freezing in terms of fertilization rate: A comparative study including review of literature. Adv Biomed Res 2016; 5:117. [PMID: 27512686 PMCID: PMC4964661 DOI: 10.4103/2277-9175.185576] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2013] [Accepted: 11/09/2015] [Indexed: 11/04/2022] Open
Abstract
Background: Cryopreservation is basically related to meritorious thin samples or small clumps of cells that are cooled quickly without loss. Our main objective is to establish and formulate an innovative method and protocol development for cryopreservation as a gold standard for clinical uses in laboratory practice and treatment. The knowledge regarding usefulness of cryopreservation in clinical practice is essential to carry forward the clinical practice and research. Materials and Methods: We are trying to compare different methods of cryopreservation (in two dozen of cells) at the same time we compare the embryo and oocyte freezing interms of fertilization rate according to the International standard protocol. Results: The combination of cryoprotectants and regimes of rapid cooling and rinsing during warming often allows successful cryopreservation of biological materials, particularly cell suspensions or thin tissue samples. Examples include semen, blood, tissue samples like tumors, histological cross-sections, human eggs and human embryos. Although presently many studies have reported that the children born from frozen embryos or “frosties,” show consistently positive results with no increase in birth defects or development abnormalities is quite good enough and similar to our study (50–85%). Conclusions: We ensure that cryopreservation technology provided useful cell survivability, tissue and organ preservation in a proper way. Although it varies according to different laboratory conditions, it is certainly beneficial for patient's treatment and research. Further studies are needed for standardization and development of new protocol.
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Affiliation(s)
- Mayadhar Barik
- Department of Pediatric Surgery, AIIMS, New Delhi, India
| | - Minu Bajpai
- Department of Pediatric Surgery, AIIMS, New Delhi, India
| | - Santosh Patnaik
- Department of Ocular Pharmacology and Pharmacy, AIIMS, New Delhi, India
| | - Pravash Mishra
- Department of Anatomy, AIIMS, Bhubaneshwar, Odisha, India
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Morgenstern DA, Ahsan G, Brocklesby M, Ings S, Balsa C, Veys P, Brock P, Anderson J, Amrolia P, Goulden N, Cale CM, Watts MJ. Post-thaw viability of cryopreserved peripheral blood stem cells (PBSC) does not guarantee functional activity: important implications for quality assurance of stem cell transplant programmes. Br J Haematol 2016; 174:942-51. [DOI: 10.1111/bjh.14160] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2016] [Accepted: 04/05/2016] [Indexed: 12/21/2022]
Affiliation(s)
- Daniel A. Morgenstern
- Department of Paediatric Oncology/Haematology; Great Ormond Street Hospital; London UK
| | - Gulrukh Ahsan
- Cell Therapy Laboratory; Great Ormond Street Hospital; London UK
| | | | - Stuart Ings
- Wolfson Cellular Therapy Unit; University College London Hospitals; London UK
| | - Carmen Balsa
- Wolfson Cellular Therapy Unit; University College London Hospitals; London UK
| | - Paul Veys
- Paediatric Bone Marrow Transplantation; Great Ormond Street Hospital; London UK
| | - Penelope Brock
- Formerly; Department of Paediatric Oncology/Haematology; Great Ormond Street Hospital; London UK
| | - John Anderson
- Department of Paediatric Oncology/Haematology; Great Ormond Street Hospital; London UK
| | - Persis Amrolia
- Paediatric Bone Marrow Transplantation; Great Ormond Street Hospital; London UK
| | - Nicholas Goulden
- Formerly; Department of Paediatric Oncology/Haematology; Great Ormond Street Hospital; London UK
| | | | - Michael J. Watts
- Wolfson Cellular Therapy Unit; University College London Hospitals; London UK
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Dijkstra-Tiekstra MJ, Hazelaar S, Gkoumassi E, Weggemans M, de Wildt-Eggen J. Comparison of cryopreservation bags for hematopoietic progenitor cells using a WBC-enriched product. Transfus Apher Sci 2015; 52:187-93. [DOI: 10.1016/j.transci.2014.12.022] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2014] [Revised: 09/25/2014] [Accepted: 12/17/2014] [Indexed: 10/24/2022]
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Storch E, Mark T, Avecilla S, Pagan C, Rhodes J, Shore T, van Besien K, Cushing M. A novel hematopoietic progenitor cell mobilization and collection algorithm based on preemptive CD34 enumeration. Transfusion 2015; 55:2010-6. [PMID: 25808119 DOI: 10.1111/trf.13076] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2014] [Revised: 01/25/2015] [Accepted: 02/02/2015] [Indexed: 12/23/2022]
Abstract
BACKGROUND The collection of autologous peripheral blood (PB) stem cells can be challenging in the subgroup of patients deemed "poor mobilizers" with granulocyte-colony-stimulating factor. Plerixafor, a CXCR-4 antagonist, is an alternative mobilizing agent, but is costly, and the optimal mobilization algorithm has yet to be determined. STUDY DESIGN AND METHODS To address the question we developed a protocol measuring PB CD34 on Day 4 of mobilization. We examined 26 patients before initiating the protocol versus 24 patients after initiation. RESULTS Significant differences (p ≤ 0.05) included fewer days of collection (1.25 days vs. 2.42 days), lower total blood volume processed (25.9 L vs. 57.2 L), lower total product volume (324 mL vs. 691 mL), lower RBC content (9 mL vs. 18 mL), and lower granulocyte percentage per collection (35% vs. 11%). There were no significant differences between the two groups in demographics, baseline platelet count, total CD34, or CD34/kg harvested. CONCLUSION Use of a protocol to assess PB CD34 1 day before collection allows for preemptive administration of plerixafor to augment mobilization. Subsequently, days of collection and processed blood volume are reduced while there is less RBC and granulocyte contamination in the collected stem cell product.
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Affiliation(s)
| | - Tomer Mark
- Department of Medicine, New York-Presbyterian Hospital, New York, New York
| | | | | | - Joanna Rhodes
- Department of Medicine, New York-Presbyterian Hospital, New York, New York
| | - Tsiporah Shore
- Department of Medicine, New York-Presbyterian Hospital, New York, New York
| | - Koen van Besien
- Department of Medicine, New York-Presbyterian Hospital, New York, New York
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Dijkstra-Tiekstra MJ, Setroikromo AC, Kraan M, Gkoumassi E, de Wildt-Eggen J. Optimization of the freezing process for hematopoietic progenitor cells: effect of precooling, initial dimethyl sulfoxide concentration, freezing program, and storage in vapor-phase or liquid nitrogen on in vitro white blood cell quality. Transfusion 2014; 54:3155-63. [DOI: 10.1111/trf.12756] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2014] [Revised: 05/09/2014] [Accepted: 05/09/2014] [Indexed: 01/10/2023]
Affiliation(s)
| | - Airies C. Setroikromo
- Division of Research, Department of Transfusion Monitoring; Sanquin Blood Supply; Groningen the Netherlands
| | - Marcha Kraan
- Division of Research, Department of Transfusion Monitoring; Sanquin Blood Supply; Groningen the Netherlands
| | - Effimia Gkoumassi
- Division of Research, Department of Transfusion Monitoring; Sanquin Blood Supply; Groningen the Netherlands
| | - Janny de Wildt-Eggen
- Division of Research, Department of Transfusion Monitoring; Sanquin Blood Supply; Groningen the Netherlands
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12
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Motta JPR, Paraguassú-Braga FH, Bouzas LF, Porto LC. Evaluation of intracellular and extracellular trehalose as a cryoprotectant of stem cells obtained from umbilical cord blood. Cryobiology 2014; 68:343-8. [DOI: 10.1016/j.cryobiol.2014.04.007] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2013] [Revised: 04/10/2014] [Accepted: 04/13/2014] [Indexed: 01/11/2023]
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Holbro A, Graf L, Topalidou M, Bucher C, Passweg JR, Tsakiris DA. Cryopreserved stem cell products containing dimethyl sulfoxide lead to activation of the coagulation system without any impact on engraftment. Transfusion 2013; 54:1508-14. [PMID: 24304039 DOI: 10.1111/trf.12511] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2013] [Revised: 10/14/2013] [Accepted: 10/22/2013] [Indexed: 11/30/2022]
Abstract
BACKGROUND Dimethyl sulfoxide (DMSO) is extensively used as a cryoprotectant in stem cell preservation. Little is known on direct hemostatic changes in recipients of hematopoietic stem cell transplantation (HSCT), immediately after DMSO administration. The objectives of the current study were to measure hemostatic changes during HSCT. STUDY DESIGN AND METHODS In this prospective analysis, changes in plasma biomarkers, platelets (PLTs), or endothelial cells (D-dimers, thrombin-antithrombin complex [TAT], microparticle activity as thrombin-generation potential [MPA], whole blood aggregation, von Willebrand factor) were measured before and immediately after HSCT. Furthermore, associations with clinical complications were recorded. RESULTS A total of 54 patients were included in the study. Mean MPA and TAT increased significantly immediately after HSCT, returning to baseline the day after the procedure (p<0.01). No significant differences in engraftment for neutrophils and PLTs were found in patients presenting a high increase of TAT or MPA compared with those presenting with a smaller increase. Patients with a high increase in TAT and MPA had received a greater number of total mononucleated cells (p<0.001) and higher transplant volumes (p=0.002). CONCLUSIONS Infusion of stem cells containing DMSO reversibly activated coagulation, measured as thrombin generation. This finding was not associated with acute adverse events and did not influence engraftment. Further studies are needed to compare variable DMSO concentrations as well as DMSO-free products, to better address the influence of DMSO on hemostasis.
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Affiliation(s)
- Andreas Holbro
- Department of Hematology and Diagnostic Hematology, University Hospital Basel, Basel, Switzerland; Blood Transfusion Centre, Swiss Red Cross, Basel, Switzerland
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14
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Abstract
Cell therapies are typically collected in one location, processed in a second location, and then administered in a third location. The ability to preserve the cells is critical to their clinical application. It improves patient access to therapies by increasing the genetic diversity of cells available. In addition, the ability to preserve cells improves the "manufacturability" of a cell therapy product by permitting the cells to be stored until the patient is ready for administration of the therapy, permitting inventory control of products, and improving management of staffing at cell therapy facilities. Finally, the ability to preserve cell therapies improves the safety of cell therapy products by extending the shelf life of a product and permitting completion of safety and quality control testing before release of the product for use. The support of the National Blood Foundation has been critical to our work on improving the quality of frozen and thawed cell therapy products through the development of a microfluidic device to remove dimethlysulfoxide (DMSO). We are also involved in research to replace DMSO with other agents that are less toxic to cells and patients. Finally, the need to advance the preservation of cell therapies was a driving force behind the development of the Biopreservation Core Resource (http://www.biocor.net), a national resource in biopreservation. New interest in translation of cell therapies from the bench to the patient's bedside has the potential to drive the transformation of preservation science, technology, and practice.
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Affiliation(s)
- Allison Hubel
- Biopreservation Core Resource, and the Mechanical Engineering Department, University of Minnesota, Minneapolis, Minnesota 55455, USA.
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15
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Abstract
BACKGROUND Autologous, and in some cases allogeneic, hemopoietic stem cells (HSC) are stored for varying periods of time prior to infusion. For periods of greater than 48 h, storage requires cryopreservation. It is essential to optimize cell storage and ensure the quality of the product for subsequent reinfusion. METHODS A number of important variables may affect the subsequent quality of infused HSC and therapeutic cells (TC). This review discusses these and also reviews the regulatory framework that now aims to ensure the quality of stem cells and TC for transplantation. RESULTS Important variables included cell concentration, temperature, interval from collection to cryopreservation, manipulations performed. They also included rate of freezing and whether controlled-rate freezing was employed. Parameters studied were type of cryoprotectant utilized [dimethyl sulphoxide (DMSO) is most commonly used, sometimes in combination with hydroxyethyl starch (HES)]; and storage conditions. It is also important to assess the quality of stored stem cells. Measurements employed included the total cell count (TNC), mononuclear cell count (MNC), CD34+ cells and colony-forming units - granulocyte macrophage (CFU-GM). Of these, TNC and CD34+ are the most useful. However, the best measure of the quality of stored stem cells is their subsequent engraftment. The quality systems used in stem cell laboratories are described in the guidance of the Joint Accreditation Committee of ISCT (Europe) and the EBMT (JACIE) and the EU Directive on Tissues and Cells plus its supporting commission directives. Inspections of facilities are carried out by the appropriate national agencies and JACIE. CONCLUSION For high-quality storage of HSC and TC, processing facilities should use validated procedures that take into account critical variables. The quality of all products must be assessed before and after storage.
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Affiliation(s)
- Derwood Pamphilon
- Bristol Institute for Transfusion Sciences, University of Bristol, and English National Blood Service, UK
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Pamphilon DH, Selogie E, Szczepiorkowski ZM. Transportation of cellular therapy products: report of a survey by the cellular therapies team of the Biomedical Excellence for Safer Transfusion (BEST) collaborative. Vox Sang 2010; 99:168-73. [PMID: 20230598 DOI: 10.1111/j.1423-0410.2010.01329.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
BACKGROUND AND OBJECTIVES Most cell therapy products (CTP) are infused or processed shortly after collection but in some cases this may be delayed for up to 48 h. A number of variables such as temperature and cell concentration are of critical importance for the integrity of CTP during this time. MATERIALS AND METHODS We conducted a survey of cellular therapy laboratories to ascertain current practices for CTP transportation. RESULTS There were 194 respondents of whom 90% shipped or received CTP--84% allogeneic, 71% autologous and 62% therapeutic cells. Processing facilities shipped or received the following products--hematopoietic progenitor cells (HPC), Marrow 73%; HPC, Apheresis 90%; HPC, Cord Blood 54% and others 14%. Other CTP included donor lymphocytes, mesenchymal stem cells (MSC), natural killer cells, buffy coat neutrophils and virus-specific cytotoxic T lymphocytes (CTL). More than 70% of respondents believed that it was acceptable for CTP to be held for up to 2 h without checking the temperature or cell density and a similar proportion agreed that putting products in containers to control parameters such as temperature within this time period was unnecessary. The majority of centres shipped or received between 1 and 10 CTP annually and 66% received products taking more than 2 h to ship. Of these, 82% specified the conditions for temperature in transit whilst 57% monitored temperature in transit and 74% of these used a data logger. The temperature range most commonly specified was 18-24 degrees C. The majority of processing facilities did not request an adjustment to the cell density even for products taking more than 2 h to reach their facility. More than 90% of respondents tested HPC for CD34(+) cells, viability and sterility; 40-48% performed colony-forming unit-granulocyte macrophage (CFU-GM) analysis. Only viability was thought by > 50% of respondents to be impacted by temperature, cell density and other parameters. CONCLUSION Understanding current practice will help in the design of future studies for CTP storage and transportation.
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Affiliation(s)
- D H Pamphilon
- NHS Blood and Transplant, North Bristol Park, Northway, Filton, Bristol, UK.
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Fleming Glass KK, Longmire EK, Hubel A. OPTIMIZATION OF A MICROFLUIDIC DEVICE FOR DIFFUSION-BASED EXTRACTION OF DMSO FROM A CELL SUSPENSION. INTERNATIONAL JOURNAL OF HEAT AND MASS TRANSFER 2008; 51:5749-5757. [PMID: 19884964 PMCID: PMC2621076 DOI: 10.1016/j.ijheatmasstransfer.2008.04.018] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
This study considers the use of a two-stream microfluidic device for extraction of dimethyl sulphoxide (DMSO) from a cryopreserved cell suspension. The DMSO diffuses from a cell suspension stream into a neighboring wash stream flowing in parallel. The model of Fleming et al.[14] is employed to determine and discuss optimal geometry and operating conditions for a case requiring removal of 95% DMSO from suspension streams with volumetric flow rates up to 2.5 ml/min. The effects of Peclet number, flow rate fraction, and cell volume fraction are analyzed, and expansion of the analysis to other applications is discussed.
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Affiliation(s)
- K K Fleming Glass
- Department of Mechanical Engineering, 1100 Mechanical Engineering, 111 Church Street, University of Minnesota, Minneapolis, MN 55455
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Fleming KK, Longmire EK, Hubel A. Numerical characterization of diffusion-based extraction in cell-laden flow through a microfluidic channel. J Biomech Eng 2007; 129:703-11. [PMID: 17887896 DOI: 10.1115/1.2768373] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Cells are routinely cryopreserved in dimethyl sulfoxide (DMSO), a cryoprotective agent, for medical applications. Infusion of a DMSO-laden cell suspension results in adverse patient reactions, but current DMSO extraction processes result in significant cell losses. A diffusion-based numerical model was employed to characterize DMSO extraction in fully developed channel flow containing a wash stream flowing parallel to a DMSO-laden cell suspension. DMSO was allowed to diffuse across cell membranes as well as across the channel depth. A variety of cases were considered with the ultimate goal of characterizing the optimal geometry and flow conditions to process clinical volumes of cell suspension in a reasonable time (2-3 ml/min). The results were dependent on four dimensionless parameters: depth fraction of the DMSO-laden stream, Peclet number, cell volume fraction in the DMSO-laden stream, and cell membrane permeability parameter. Smaller depth fractions led to faster DMSO extraction but channel widths that were not practical. Higher Peclet numbers led to longer channels but smaller widths. For the Peclet values and channel depths considered (>or=500 microm) and appropriate permeability values, diffusion across cell membranes was significantly faster than diffusion across the channel depth. Cell volume fraction influenced the cross-stream diffusion of DMSO by limiting the fluid volume fraction available in the contaminant stream but did not play a significant role in channel geometry or operating requirements. The model was validated against preliminary experiments in which DMSO was extracted from suspensions of B-lymphoblast cells. The model results suggest that a channel device with practical dimensions can remove a sufficient level of contaminant within a mesoscale volume of cells in the required time.
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Affiliation(s)
- K K Fleming
- Department of Mechanical Engineering, University of Minnesota, 1100 Mechanical Engineering, 111 Church Street, Minneapolis, Minnesota 55455, USA
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Fleming KK, Hubel A. Cryopreservation of Hematopoietic Stem Cells: Emerging Science, Technology and Issues. Transfus Med Hemother 2007. [DOI: 10.1159/000104213] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
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Fleming KK, Hubel A. Cryopreservation of hematopoietic and non-hematopoietic stem cells. Transfus Apher Sci 2006; 34:309-15. [PMID: 16829201 DOI: 10.1016/j.transci.2005.11.012] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2005] [Accepted: 11/23/2005] [Indexed: 11/19/2022]
Abstract
Recent studies illustrate the potential for improving the cryopreservation of stem cells. Reduced DMSO concentrations in the cryopreservation medium, post thaw washing of cells and increased cell concentration have been actively studied. Standardization of cell processing has led to the study of liquid storage prior to cryopreservation, validation of mechanical (uncontrolled rate freezing) freezing, and cryopreservation bag failure. Finally, the need for the systematic study and optimization of preservation processes has not been fulfilled. As the sources and applications of stem cells (hematopoietic and non-hematopoietic) continue to be developed, the need for effective preservation methods will only grow.
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Affiliation(s)
- K K Fleming
- Department of Mechanical Engineering, University of Minnesota, 1100 Mechanical Engineering, 111 Church Street SE, Minneapolis, MN 55455, USA
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Valeri CR, Ragno G. Cryopreservation of human blood products. Transfus Apher Sci 2006; 34:271-87. [PMID: 16872903 DOI: 10.1016/j.transci.2005.11.010] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2005] [Accepted: 11/23/2005] [Indexed: 11/30/2022]
Affiliation(s)
- C Robert Valeri
- Naval Blood Research Laboratory Inc., 195 Bournehurst Drive, Plymouth, MA 02360, USA.
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Beaujean F, Pico J, Norol F, Divine M, Le Forestier C, Duedari N. Characteristics of peripheral blood progenitor cells frozen after 24 hours of liquid storage. JOURNAL OF HEMATOTHERAPY 1996; 5:681-6. [PMID: 9117257 DOI: 10.1089/scd.1.1996.5.681] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Peripheral blood progenitor cells (PBPC) harvested for autologous transplantation are usually cryopreserved within 2-4 h of collection. However, there are conditions in which it would be useful to freeze PBPC after a liquid storage period. This study was performed to evaluate whether prior storage for just 24 h damages frozen PBPC. First, leukapheresis products were obtained from 9 patients and divided into three fractions. The first fraction was frozen within 2 h and used as a control. The second and the third fractions were stored either at 4 degrees C or at 22 degrees C for 24 h before freezing. Cell counts, CFU-GM values, and pH were studied after collection, after storage, and after cryopreservation. Similar results were obtained at 4 degrees C and 22 degrees C. However, pH decreased most markedly at 22 degrees C. Mean postcryopreservation CFU-GM recoveries were not significantly different and were, respectively, 74.03% (control), 96.39% (4 degrees C), and 80.33% (22 degrees C). These observations were confirmed in an additional study of frozen PBPC collected from 12 patients and previously stored for 24 h at 4 degrees C. These data indicate that blood progenitors may be stored for 24 h and subsequently frozen without quantitative and qualitative impairment.
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Affiliation(s)
- F Beaujean
- Laboratoire de Thérapie Cellulaire, ETS Sud-Est Francilien, Hôpital Henri Mondor, Créteil, France
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