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Capelli C, Frigerio S, Lisini D, Nava S, Gaipa G, Belotti D, Cabiati B, Budelli S, Lazzari L, Bagnarino J, Tanzi M, Comoli P, Perico N, Introna M, Golay J. A comprehensive report of long-term stability data for a range ATMPs: A need to develop guidelines for safe and harmonized stability studies. Cytotherapy 2022; 24:544-556. [PMID: 35177338 DOI: 10.1016/j.jcyt.2021.12.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2021] [Revised: 12/01/2021] [Accepted: 12/06/2021] [Indexed: 11/03/2022]
Abstract
BACKGROUND AIMS Advanced therapy medicinal products (ATMPs) are novel drugs based on genes, cells or tissues developed to treat many different diseases. Stability studies of each new ATMP need to be performed to define its shelf life and guarantee efficacy and safety upon infusion, and these are presently based on guidelines originally drafted for standard pharmaceutical drugs, which have properties and are stored in conditions quite different from cell products. The aim of this report is to provide evidence-based information for stability studies on ATMPs that will facilitate the interlaboratory harmonization of practices in this area. METHODS We have collected and analyzed the results of stability studies on 19 different cell-based experimental ATMPs, produced by five authorized cell factories forming the Lombardy "Plagencell network" for use in 36 approved phase I/II clinical trials; most were cryopreserved and stored in liquid nitrogen vapors for 1 to 13 years. RESULTS The cell attributes collected in stability studies included cell viability, immunophenotype and potency assays, in particular immunosuppression, cytotoxicity, cytokine release and proliferation/differentiation capacity. Microbiological attributes including sterility, endotoxin levels and mycoplasma contamination were also analyzed. All drug products (DPs), cryopreserved in various excipients containing 10% DMSO and in different primary containers, were very stable long term at <-150°C and did not show any tendency for diminished viability or efficacy for up to 13.5 years. CONCLUSIONS Our data indicate that new guidelines for stability studies, specific for ATMPs and based on risk analyses, should be drafted to harmonize practices, significantly reduce the costs of stability studies without diminishing safety. Some specific suggestions are presented in the discussion.
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Affiliation(s)
- Chiara Capelli
- Center of Cellular Therapy "G. Lanzani", ASST Papa Giovanni XXIII, Bergamo, Italy; Fondazione per la Ricerca Ospedale di Bergamo, Bergamo, Italy
| | - Simona Frigerio
- Cell Therapy Production Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy
| | - Daniela Lisini
- Cell Therapy Production Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy
| | - Sara Nava
- Cell Therapy Production Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy
| | - Giuseppe Gaipa
- Laboratory of Cell and Gene Therapy Stefano Verri, ASST Monza Ospedale San Gerardo, Monza, Italy
| | - Daniela Belotti
- Laboratory of Cell and Gene Therapy Stefano Verri, ASST Monza Ospedale San Gerardo, Monza, Italy
| | - Benedetta Cabiati
- Laboratory of Cell and Gene Therapy Stefano Verri, ASST Monza Ospedale San Gerardo, Monza, Italy
| | - Silvia Budelli
- Laboratory of Regenerative Medicine - Cell Factory, Department of Transfusion Medicine and Hematology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano, Italy
| | - Lorenza Lazzari
- Laboratory of Regenerative Medicine - Cell Factory, Department of Transfusion Medicine and Hematology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano, Italy
| | - Jessica Bagnarino
- UOSD Cell Factory, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; Pediatric Hematology/Oncology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
| | - Matteo Tanzi
- UOSD Cell Factory, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; Pediatric Hematology/Oncology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
| | - Patrizia Comoli
- UOSD Cell Factory, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; Pediatric Hematology/Oncology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
| | - Norberto Perico
- Aldo & Cele Daccò Clinical Research Center for Rare Diseases, Istituto Ricerche Farmacologiche Mario Negri IRCCS, Bergamo, Italy
| | - Martino Introna
- Center of Cellular Therapy "G. Lanzani", ASST Papa Giovanni XXIII, Bergamo, Italy.
| | - Josée Golay
- Center of Cellular Therapy "G. Lanzani", ASST Papa Giovanni XXIII, Bergamo, Italy; Fondazione per la Ricerca Ospedale di Bergamo, Bergamo, Italy
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Kaushal R, Jahan S, McGregor C, Pineault N. Dimethyl sulfoxide-free cryopreservation solutions for hematopoietic stem cell grafts. Cytotherapy 2021; 24:272-281. [PMID: 34654640 DOI: 10.1016/j.jcyt.2021.09.002] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2021] [Revised: 08/18/2021] [Accepted: 09/05/2021] [Indexed: 02/08/2023]
Abstract
BACKGROUND AIMS The use of effective methods for the cryopreservation of hematopoietic stem cells (HSCs) is vital to retain the maximum engraftment activity of cord blood units (CBUs). Current protocols entail the use of dimethyl sulfoxide (DMSO) as intracellular cryoprotective agent (CPA) and dextran and plasma proteins as extracellular CPAs, but DMSO is known to be cytotoxic, and its infusion in patients is associated with mild to moderate side effects. However, new, commercially available, DMSO-free cryopreservation solutions have been developed, but their capacity to protect HSCs remains poorly investigated. METHODS Herein the authors compared the capacity of four DMSO-free freezing media to cryopreserve cord blood (CB) HSCs: CryoProtectPureSTEM (CPP-STEM), CryoScarless (CSL), CryoNovo P24 (CN) and Pentaisomaltose (PIM). Clinical-grade DMSO/dextran solution was used as control. RESULTS Of the four cryopreservation solutions tested, the best post-thaw cell viability, recovery of viable CD45+ and CD34+ cells and potency were achieved with CPP-STEM, which was equal or superior to that seen with the control DMSO. CSL provided the second best post-thaw results followed by PIM, whereas CN was associated with modest viability and potency. Further work with CPP-STEM revealed that CB CD34-enriched HSCs and progenitors cryopreserved with CPP-STEM maintained high viability and growth expansion activity. In line with this, a pilot transplantation assay confirmed that CPP-STEM-protected CB grafts supported normal short- and long-term engraftment kinetics. CONCLUSIONS The authors' results suggest that new, valuable alternatives to DMSO are now available for the cryopreservation of HSCs and grafts, including CBUs.
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Affiliation(s)
- Richa Kaushal
- Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada; Centre for Innovation, Canadian Blood Services, Ottawa, Canada
| | - Suria Jahan
- Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada; Centre for Innovation, Canadian Blood Services, Ottawa, Canada
| | | | - Nicolas Pineault
- Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada; Centre for Innovation, Canadian Blood Services, Ottawa, Canada.
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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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Mommaerts K, Bellora C, Lambert P, Türkmen S, Schwamborn JC, Betsou F. Method Optimization of Skin Biopsy-Derived Fibroblast Culture for Reprogramming Into Induced Pluripotent Stem Cells. Biopreserv Biobank 2021; 20:12-23. [PMID: 34407379 DOI: 10.1089/bio.2020.0159] [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
Background: Fibroblasts can be isolated from skin biopsies using a chemical dissociation, a physical dissociation, or a combination of both techniques. They can be reprogrammed into induced pluripotent stem cells (iPSCs) through the introduction of defined sets of key transcription factors. This study aimed to identify the optimal protocol for skin biopsy dissociation, fibroblast culture, and fibroblast cryopreservation in the scope of reprogramming into iPSCs and in the context of biobank accreditation. Methods: First, four dissociation techniques typically used in the laboratory (explant based, enzymatic, and/or mechanical) and two cryopreservation media containing 10% dimethyl sulfoxide, either commercial or homemade, were evaluated in terms of post-thaw recovery, viability, growth curves, and karyotyping analyses of the fibroblasts. Next, the clones reprogrammed from the fibroblasts isolated with the two optimal dissociation methods and cryopreservation media were further assessed by reprogramming quality before cryopreservation and post-thaw pluripotency comparison. Results: Fibroblasts isolated from skin biopsies using an explant-based or enzymatic dissociation method showed higher viability, higher proliferative potential, and higher genome stability post-thaw compared to the other dissociation techniques. Fibroblasts obtained by the explant-based dissociation technique showed a slightly higher reprogramming quality. The iPSC reprogrammed from explant-based dissociated fibroblasts showed successful recovery of iPSC clones. No difference between the two cryopreservation media was detected for the tested endpoints, with the exception of a higher visual count of colonies at the end of the reprogramming for the explant-based dissociation method. Conclusions: This article presents a formal method optimization for biospecimen processing in the context of accreditation in laboratories and biobanks. We validated skin biopsy-derived fibroblast isolation, culture, and cryopreservation for downstream mRNA reprogramming into iPSCs. The explant-based dissociation technique and homemade medium are selected as optimal to isolate and cryopreserve fibroblasts from skin biopsies in the scope of reprogramming into iPSCs.
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Affiliation(s)
- Kathleen Mommaerts
- Integrated Biobank of Luxembourg (IBBL), Luxembourg Institute of Health, Dudelange, Luxembourg.,Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg
| | - Camille Bellora
- Integrated Biobank of Luxembourg (IBBL), Luxembourg Institute of Health, Dudelange, Luxembourg
| | - Pauline Lambert
- Integrated Biobank of Luxembourg (IBBL), Luxembourg Institute of Health, Dudelange, Luxembourg
| | - Seval Türkmen
- Hematooncogenetics, National Center of Genetics (NCG), Laboratoire National de Santé (LNS), Dudelange, Luxembourg
| | - Jens C Schwamborn
- Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg
| | - Fay Betsou
- Integrated Biobank of Luxembourg (IBBL), Luxembourg Institute of Health, Dudelange, Luxembourg
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Jahan S, Kaushal R, Pasha R, Pineault N. Current and Future Perspectives for the Cryopreservation of Cord Blood Stem Cells. Transfus Med Rev 2021; 35:95-102. [PMID: 33640254 DOI: 10.1016/j.tmrv.2021.01.003] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2020] [Revised: 01/22/2021] [Accepted: 01/24/2021] [Indexed: 12/29/2022]
Abstract
Hematopoietic stem cell (HSC) transplantation is a well-established procedure for the treatment of many blood related malignancies and disorders. Before transplantation, HSC are collected and cryopreserved until use. The method of cryopreservation should preserve both the number and function of HSC and downstream progenitors responsible for long- and short-term engraftment, respectively. This is especially critical for cord blood grafts, since the cell number associated with this stem cell source is often limiting. Loss of function in cryopreserved cells occurs following cryoinjuries due to osmotic shock, dehydration, solution effects and mechanical damage from ice recrystallization during freezing and thawing. However, cryoinjuries can be reduced by 2 mitigation strategies; the use of cryoprotectants (CPAs) and use of control rate cooling. Currently, slow cooling is the most common method used for the cryopreservation of HSC graft. Moreover, dimethyl-sulfoxide (DMSO) and dextran are popular intracellular and extracellular CPAs used for HSC grafts, respectively. Yet, DMSO is toxic to cells and can cause significant side effects in stem cells' recipients. However, new CPAs and strategies are emerging that may soon replace DMSO. The aim of this review is to summarise key concepts in cryobiology and recent advances in the field of HSC cryobiology. Other important issues that need to be considered are also discussed such as transient warming events and thawing of HSC grafts.
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Affiliation(s)
- Suria Jahan
- Canadian Blood Services, Centre for Innovation, Ottawa, Ontario, Canada; Biochemistry, Microbiology and Immunology department, University of Ottawa, Ottawa, Ontario, Canada
| | - Richa Kaushal
- Canadian Blood Services, Centre for Innovation, Ottawa, Ontario, Canada; Biochemistry, Microbiology and Immunology department, University of Ottawa, Ottawa, Ontario, Canada
| | - Roya Pasha
- Canadian Blood Services, Centre for Innovation, Ottawa, Ontario, Canada
| | - Nicolas Pineault
- Canadian Blood Services, Centre for Innovation, Ottawa, Ontario, Canada; Biochemistry, Microbiology and Immunology department, University of Ottawa, Ottawa, Ontario, Canada.
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Valyi-Nagy K, Betsou F, Susma A, Valyi-Nagy T. Optimization of Viable Glioblastoma Cryopreservation for Establishment of Primary Tumor Cell Cultures. Biopreserv Biobank 2020; 19:60-66. [PMID: 33107762 PMCID: PMC7892309 DOI: 10.1089/bio.2020.0050] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Background: Technologies related to the establishment of primary tumor cell cultures from solid tumors, including glioblastoma, are increasingly important to oncology research and practice. However, processing of fresh tumor specimens for establishment of primary cultures on the day of surgical collection is logistically difficult. The feasibility of viable cryopreservation of glioblastoma specimens, allowing for primary culture establishment weeks to months after surgical tumor collection and freezing, was demonstrated by Mullins et al. in 2013, with a success rate of 59% that was not significantly lower than that achieved with fresh tumor tissue. However, research targeting optimization of viable glioblastoma cryopreservation protocols for establishment of primary tumor cultures has been limited. Objectives: The objective of this study was to optimize glioblastoma cryopreservation methods for viable cryobanking and to determine if two-dimensional (2D) or three-dimensional (3D) culture conditions were more supportive of glioblastoma growth after thawing of frozen tumor specimens. Methods: Portions of eight human glioblastoma specimens were cryopreserved by four different protocols differing in the time of enzymatic digestion (before or after cryopreservation), and in the type of cryopreservation media (CryoStor CS10 or 10% dimethyl sulfoxide and 90% fetal calf serum). After 1 month, frozen tissues were thawed, enzymatically digested, if not digested before, and used for initiation of 2D or 3D primary tumor cultures to determine viability. Results: Among the tested cryopreservation and culturing protocols, the most efficient combinations of cryopreservation and culture were those associated with the use of CryoStor CS10 cryopreservation medium, enzymatic digestion before freezing, and 2D culturing after thawing with a successful culture rate of 8 out of 8 cases (100%). Two-dimensional cultures were in general more efficient for the support of tumor cell growth after thawing than 3D cultures. Conclusions: This study supports development of evidence-based viable glioblastoma cryopreservation methods for use in glioblastoma biobanking and research.
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Affiliation(s)
- Klara Valyi-Nagy
- Department of Pathology, University of Illinois at Chicago, Chicago, Illinois, USA.,ISBER Biospecimen Science Working Group, Vancouver, British Columbia, Canada
| | - Fay Betsou
- ISBER Biospecimen Science Working Group, Vancouver, British Columbia, Canada.,Integrated BioBank of Luxembourg, Dudelange, Luxembourg
| | - Alexandru Susma
- Department of Pathology, University of Illinois at Chicago, Chicago, Illinois, USA
| | - Tibor Valyi-Nagy
- Department of Pathology, University of Illinois at Chicago, Chicago, Illinois, USA
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7
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Vawda R, Badner A, Hong J, Mikhail M, Dragas R, Xhima K, Jose A, Fehlings MG. Harnessing the Secretome of Mesenchymal Stromal Cells for Traumatic Spinal Cord Injury: Multicell Comparison and Assessment of In Vivo Efficacy. Stem Cells Dev 2020; 29:1429-1443. [PMID: 32962528 PMCID: PMC7703247 DOI: 10.1089/scd.2020.0079] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Cell therapy offers significant promise for traumatic spinal cord injury (SCI), which despite many medical advances, has limited treatment strategies. Able to address the multifactorial and dynamic pathophysiology of SCI, cells present various advantages over standard pharmacological approaches. However, the use of live cells is also severely hampered by logistical and practical considerations. These include specialized equipment and expertise, standardization of cell stocks, sustained cell viability post-thawing, and cryopreservation-induced delayed-onset cell death. For this reason, we suggest a novel and clinically translatable alternative to live-cell systemic infusion, which retains the efficacy of the latter while overcoming many of its limitations. This strategy involves the administration of concentrated cell secretome and exploits the trophic mechanism by which stromal cells function. In this study, we compare the efficacy of intravenously delivered concentrated conditioned media (CM) from human umbilical cord matrix cells (HUCMCs), bone marrow mesenchymal stromal cells, as well as newborn and adult fibroblasts in a rat model of moderately severe cervical clip compression/contusion injury (C7--T1, 35 g). This is further paired with a thorough profile of the CM cytokines, chemokines, and angiogenic factors. The HUCMC-derived CM was most effective at limiting acute (48 h post-SCI) vascular pathology, specifically lesion volume, and functional vascularity. Principle component analysis (PCA), hierarchical clustering, and interaction analysis of proteins highly expressed in the HUCMC secretome suggest involvement of the MAPK/ERK, JAK/STAT, and immune cell migratory pathways. This "secretotherapeutic" strategy represents a novel and minimally invasive method to target multiple organ systems and several pathologies shortly after traumatic SCI.
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Affiliation(s)
- Reaz Vawda
- Department of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, Canada
| | - Anna Badner
- Department of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, Canada.,Institute of Medical Science, School of Medicine, University of Toronto, Toronto, Canada
| | - James Hong
- Department of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, Canada.,Institute of Medical Science, School of Medicine, University of Toronto, Toronto, Canada
| | - Mirriam Mikhail
- Department of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, Canada
| | - Rachel Dragas
- Department of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, Canada.,Institute of Medical Science, School of Medicine, University of Toronto, Toronto, Canada
| | - Kristiana Xhima
- Department of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, Canada
| | - Alejandro Jose
- Department of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, Canada
| | - Michael G Fehlings
- Department of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, Canada.,Institute of Medical Science, School of Medicine, University of Toronto, Toronto, Canada.,Spinal Program, Toronto Western Hospital, University Health Network, Toronto, Canada
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He A, Powell S, Kyle M, Rose M, Masmila E, Estrada V, Sicklick JK, Molinolo A, Kaushal S. Cryopreservation of Viable Human Tissues: Renewable Resource for Viable Tissue, Cell Lines, and Organoid Development. Biopreserv Biobank 2020; 18:222-227. [PMID: 32302515 DOI: 10.1089/bio.2019.0062] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The availability of viable human tissues is critical to support translational research focused on personalized care. Most studies have relied on fresh frozen or formalin-fixed paraffin-embedded tissues for histopathology, genomics, and proteomics. Yet, basic, translational, and clinical research downstream assays such as tumor progression/invasion, patient-derived xenograft, organoids, immunoprofiling, and vaccine development still require viable tissue, which are time-sensitive and rare commodities. We describe the generation of two-dimensional (2D) and three-dimensional (3D) cultures to validate a viable freeze cryopreservation technique as a standard method of highest quality specimen preservation. After surgical resection, specimens were minced, placed in CryoStor™ media, and frozen using a slow freezing method (-1°C/min in -80°C) for 24 hours and then stored in liquid nitrogen. After 15-18 months, the tissues were thawed, dissociated into single-cell suspensions, and evaluated for cell viability. To generate primary 2D cultures, cells were plated onto Collagen-/Matrigel-coated plates. To develop 3D cultures (organoids), the cells were plated in reduced serum RPMI media on nonadherent plates or in Matrigel matrix. The epithelial nature of the cells was confirmed by using immunohistochemistry for cytokeratins. DNA and RNA isolation was performed using QIAGEN AllPrep kits. We developed primary lines (2D and 3D) of colon, thyroid, lung, renal, and liver cancers that were positive for cytokeratin staining. 3D lines were developed from the same cohort of tumor types in both suspended media and Matrigel matrix. Multiple freeze-thaw cycles did not significantly alter the viability and growth of 2D and 3D lines. DNA/RNA recovery was similar to its fresh frozen cohort. In this study, we validated 2D and 3D tissue cultures as methods to corroborate the feasibility of viable cryopreservation of tumor tissue. This proof-of-principle study, if more widely implemented, should improve accessibility of human viable tumor tissue/cells in a time-independent manner for many basic, preclinical, and translational assays.
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Affiliation(s)
- Andy He
- Biorepository and Tissue Technology Shared Resources (BTTSR), Moores Cancer Center, UC San Diego, La Jolla, California, USA
| | - Samantha Powell
- Biorepository and Tissue Technology Shared Resources (BTTSR), Moores Cancer Center, UC San Diego, La Jolla, California, USA
| | - Mason Kyle
- Biorepository and Tissue Technology Shared Resources (BTTSR), Moores Cancer Center, UC San Diego, La Jolla, California, USA
| | - Michael Rose
- Biorepository and Tissue Technology Shared Resources (BTTSR), Moores Cancer Center, UC San Diego, La Jolla, California, USA
| | - Edgar Masmila
- Biorepository and Tissue Technology Shared Resources (BTTSR), Moores Cancer Center, UC San Diego, La Jolla, California, USA
| | - Valeria Estrada
- Biorepository and Tissue Technology Shared Resources (BTTSR), Moores Cancer Center, UC San Diego, La Jolla, California, USA
| | - Jason K Sicklick
- Biorepository and Tissue Technology Shared Resources (BTTSR), Moores Cancer Center, UC San Diego, La Jolla, California, USA
| | - Alfredo Molinolo
- Biorepository and Tissue Technology Shared Resources (BTTSR), Moores Cancer Center, UC San Diego, La Jolla, California, USA
| | - Sharmeela Kaushal
- Biorepository and Tissue Technology Shared Resources (BTTSR), Moores Cancer Center, UC San Diego, La Jolla, California, USA
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Alanazi IO, Benabdelkamel H, Alghamdi W, Alfadda AA, Mahbubani KT, Almalik A, Alradwan I, Altammami M, Slater NKH, Masood A. A proteomic approach towards understanding crypoprotective action of Me2SO on the CHO cell proteome. Cryobiology 2020; 94:107-115. [PMID: 32259523 DOI: 10.1016/j.cryobiol.2020.04.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2019] [Revised: 02/25/2020] [Accepted: 04/01/2020] [Indexed: 10/24/2022]
Abstract
Chinese hamster ovary (CHO) cell lines are the most widely used in vitro cells for research and production of recombinant proteins such as rhGH, tPA, and erythropoietin. We aimed to investigate changes in protein profiles after cryopreservation using 2D-DIGE MALDI-TOF MS and network pathway analysis. The proteome changes that occur in CHO cells between freshly prepared cells and cryopreserved cells with and without Me2SO were compared to determine the key proteins and pathways altered during recovery from cryopreservation. A total of 54 proteins were identified and successfully matched to 37 peptide mass fingerprints (PMF). 14 protein spots showed an increase while 23 showed decrease abundance in the Me2SO free group compared to the control. The proteins with increased abundance included vimentin, heat shock protein 60 kDa, mitochondrial, heat shock 70 kDa protein 9, protein disulfide-isomerase A3, voltage-dependent anion-selective channel protein 2. Those with a decrease in abundance were myotubularin, glutathione peroxidase, enolase, phospho glyceromutase, chloride intracellular channel protein 1. The main canonical functional pathway affected involved the unfolded protein response, aldosterone Signaling in Epithelial Cells, 14-3-3-mediated signaling. 2D-DIGE MALDI TOF mass spectrometry and network pathway analysis revealed the differential proteome expression of FreeStyle CHO cells after cryopreservation with and without 5% Me2SOto involve pathways related to post-translational modification, protein folding and cell death and survival (score = 56, 22 focus molecules). This study revealed, for the first time to our knowledge the proteins and their regulated pathways involved in the cryoprotective action of 5% Me2SO. The use of 5% Me2SO as a cryoprotectant maintained the CHO cell proteome in the cryopreserved cells, similar to that of fresh CHO cells.
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Affiliation(s)
- Ibrahim O Alanazi
- The National Center for Genomic Technology (NCGT), Life Science and Environment Research Institute, King Abdulaziz City for Science and Technology (KACST), PO Box 6086, Riyadh, Saudi Arabia
| | - Hicham Benabdelkamel
- Proteomics Resource Unit, Obesity Research Center, College of Medicine, King Saud University, PO Box 2925, Riyadh, 11461, Saudi Arabia
| | - Waleed Alghamdi
- Technology Transfer Office, King Abdulaziz City for Science and Technology (KACST), PO Box 6086, Riyadh, Saudi Arabia.
| | - Assim A Alfadda
- Proteomics Resource Unit, Obesity Research Center, College of Medicine, King Saud University, PO Box 2925, Riyadh, 11461, Saudi Arabia
| | - Krishnaa T Mahbubani
- Department of Chemical Engineering and Biotechnology, University of Cambridge, New Museums Site, Pembroke Street, Cambridge, CB2 3RA, United Kingdom
| | - Abdulaziz Almalik
- Institute of Biotechnology and Environment, King Abdulaziz City for Science and Technology (KACST), PO Box 6086, Riyadh, Saudi Arabia
| | - Ibrahim Alradwan
- Institute of Biotechnology and Environment, King Abdulaziz City for Science and Technology (KACST), PO Box 6086, Riyadh, Saudi Arabia
| | - Musaad Altammami
- Institute of Biotechnology and Environment, King Abdulaziz City for Science and Technology (KACST), PO Box 6086, Riyadh, Saudi Arabia
| | - Nigel K H Slater
- Department of Chemical Engineering and Biotechnology, University of Cambridge, New Museums Site, Pembroke Street, Cambridge, CB2 3RA, United Kingdom
| | - Afshan Masood
- Proteomics Resource Unit, Obesity Research Center, College of Medicine, King Saud University, PO Box 2925, Riyadh, 11461, Saudi Arabia.
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High Fidelity Cryopreservation and Recovery of Primary Rodent Cortical Neurons. eNeuro 2018; 5:eN-MNT-0135-18. [PMID: 30263951 PMCID: PMC6158653 DOI: 10.1523/eneuro.0135-18.2018] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2018] [Revised: 08/30/2018] [Accepted: 09/04/2018] [Indexed: 12/12/2022] Open
Abstract
Cell cryopreservation improves reproducibility and enables flexibility in experimental design. Although conventional freezing methodologies have been used to preserve primary neurons, poor cell viability and reduced survival severely limited their utility. We screened several high-performance freezing media and found that CryoStor10 (CS10) provided superior cryoprotection to primary mouse embryonic cortical neurons compared to other commercially-available or traditional reagents, permitting the recovery of 68.8% of cells relative to a fresh dissection. We characterized developmental, morphometric, and functional indicators of neuron maturation and found that, without exception, neurons recovered from cryostorage in CS10 media faithfully recapitulate in vitro neurodevelopment in-step with neurons obtained by fresh dissection. Our method establishes cryopreserved neurons as a reliable, efficient, and equivalent model to fresh neuron cultures.
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11
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Patient-derived xenograft cryopreservation and reanimation outcomes are dependent on cryoprotectant type. J Transl Med 2018; 98:947-956. [PMID: 29520054 PMCID: PMC6072591 DOI: 10.1038/s41374-018-0042-7] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2016] [Revised: 01/14/2018] [Accepted: 02/16/2018] [Indexed: 01/22/2023] Open
Abstract
Patient-derived xenografts (PDX) are being increasingly utilized in preclinical oncologic research. Maintaining large colonies of early generation tumor-bearing mice is impractical and cost-prohibitive. Optimal methods for efficient long-term cryopreservation and subsequent reanimation of PDX tumors are critical to any viable PDX program. We sought to compare the performance of "Standard" and "Specialized" cryoprotectant media on various cryopreservation and reanimation outcomes in PDX tumors. Standard (10% DMSO media) and Specialized (Cryostor®) media were compared between overall and matched PDX tumors. Primary outcome was reanimation engraftment efficiency (REE). Secondary outcomes included time to tumor formation (TTF), time to harvest (TTH), and potential loss of unique PDX lines. Overall 57 unique PDX tumors underwent 484 reanimation engraftment attempts after previous cryopreservation. There were 10 unique PDX tumors cryopreserved with Standard (71 attempts), 40 with Specialized (272 attempts), and 7 with both (141 attempts). Median frozen time of reanimated tumors was 29 weeks (max. 177). Tumor pathology, original primary PDX growth rates, frozen storage times, and number of implantations per PDX model were similar between cryoprotectant groups. Specialized media resulted in superior REE (overall: 82 vs. 39%, p < 0.0001; matched: 97 vs. 36%, p < 0.0001; >52 weeks cryostorage: 59 vs. 9%, p < 0.0001), shorter TTF (overall 24 vs. 54 days, p = 0.0051; matched 18 vs. 53 days, p = 0.0013) and shorter TTH (overall: 64 vs. 89 days, p = 0.009; matched: 47 vs. 88 days, p = 0.0005) compared to Standard. Specialized media demonstrated improved REE with extended duration cryostorage (p = 0.048) compared to Standard. Potential loss of unique PDX lines was lower with Specialized media (9 vs. 35%, p = 0.017). In conclusion, cryopreservation with a specialized cryoprotectant appears superior to traditional laboratory-based media and can be performed with reliable reanimation even after extended cryostorage.
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12
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Baust JG, Gage AA, Klossner D, Clarke D, Miller R, Cohen J, Katz A, Polascik T, Clarke H, Baust JM. Issues Critical to the Successful Application of Cryosurgical Ablation of the Prostate. Technol Cancer Res Treat 2016; 6:97-109. [PMID: 17375972 DOI: 10.1177/153303460700600206] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
The techniques of present-day cryosurgery performed with multiprobe freezing apparatus and advanced imaging techniques yield predictable and encouraging results in the treatment of prostatic and renal cancers. Nevertheless, and not unique to cryosurgical treatment, the rates of persistent disease demonstrate the need for improvement in technique and emphasize the need for proper management of the therapeutic margin. The causes of persistent disease often relate to a range of factors including selection of patients, understanding of the extent of the tumor, limitations of the imaging techniques, and failure to freeze the tumor periphery in an efficacious manner. Of these diverse factors, the one most readily managed, but subject to therapeutic error, is the technique of freezing the tumor and appropriate margin to a lethal temperature [Baust, J. G., Gage, A. A. The Molecular Basis of Cryosurgery. BJU Int 95, 1187–1191 (2005)]. This article describes the recent experiments that examine the molecular basis of cryosurgery, clarifies the actions of the components of the freeze-thaw cycle, and defines the resultant effect on the cryogenic lesion from a clinical perspective. Further, this review addresses the important issue of management of the margin of the tumor through adjunctive therapy. Accordingly, a goal of this review is to identify the technical and future adjunctive therapeutic practices that should improve the efficacy of cryoablative techniques for the treatment of malignant lesions.
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Affiliation(s)
- J G Baust
- Institute of Biomedical Technology, SUNY Binghamton, Binghamton, NY, USA.
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13
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Fritsch G, Frank N, Dmytrus J, Frech C, Pichler H, Witt V, Geyeregger R, Scharner D, Trbojevic D, Zipperer E, Printz D, Worel N. Relevance of flow cytometric enumeration of post-thaw leucocytes: influence of temperature during cell staining on viable cell recovery. Vox Sang 2016; 111:187-96. [PMID: 27037580 DOI: 10.1111/vox.12398] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2015] [Revised: 02/11/2016] [Accepted: 02/11/2016] [Indexed: 11/29/2022]
Abstract
BACKGROUND AND OBJECTIVES Our post-thaw cell recovery rates differed substantially in interlaboratory comparisons of identical samples, potentially due to different temperatures during cell staining. MATERIALS AND METHODS Viable CD34(+) cells and leucocyte (WBC) subtypes were quantified by multiparameter single-platform flow cytometry in leucapheresis products collected from 30 adult lymphoma and myeloma patients, and from 10 paediatric patients. After thawing, cells were prepared for analysis within 30 min between thawing and acquisition, at either 4°C or at room temperature. RESULTS For cell products cryopreserved in conventional freezing medium (10% final DMSO), viable cell recovery was clearly lower after staining at 4°C than at RT. Of all WBC subtypes analysed, CD4(+) T cells showed the lowest median recovery of 4% (4°C) vs. 25% (RT), followed by CD3, CD34 and CD8 cells. The recovery was highest for CD3γδ cells with 44% (4°C) vs. 71% (RT). In the 10 samples cryopreserved in synthetic freezing medium (5% final DMSO), median recovery rates were 89% for viable CD34 (both at 4°C and RT) and 79% (4°C) vs 68% (RT) for WBC. CONCLUSIONS The post-thaw environment and, potentially, the cryoprotectant impact the outcome of cell enumeration, and results from the analysis tube may not be representative of the cells infused into a patient.
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Affiliation(s)
- G Fritsch
- Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria
| | - N Frank
- Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria
| | - J Dmytrus
- Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria
| | - C Frech
- Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria
| | - H Pichler
- St. Anna Kinderspital, Universitätskinderklinik, Vienna, Austria
| | - V Witt
- St. Anna Kinderspital, Universitätskinderklinik, Vienna, Austria
| | - R Geyeregger
- Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria
| | - D Scharner
- Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria
| | - D Trbojevic
- Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria
| | - E Zipperer
- Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria
| | - D Printz
- Children's Cancer Research Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria
| | - N Worel
- Dept. for Blood Group Serology and Transfusion Medicine, Medical University, Vienna, Austria
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14
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Smagur A, Mitrus I, Ciomber A, Panczyniak K, Fidyk W, Sadus-Wojciechowska M, Holowiecki J, Giebel S. Comparison of the cryoprotective solutions based on human albumin vs. autologous plasma: its effect on cell recovery, clonogenic potential of peripheral blood hematopoietic progenitor cells and engraftment after autologous transplantation. Vox Sang 2015; 108:417-24. [DOI: 10.1111/vox.12238] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2014] [Revised: 10/15/2014] [Accepted: 11/29/2014] [Indexed: 12/30/2022]
Affiliation(s)
- A. Smagur
- Department of Bone Marrow Transplantation and Oncohematology; Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch; Gliwice Poland
| | - I. Mitrus
- Department of Bone Marrow Transplantation and Oncohematology; Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch; Gliwice Poland
| | - A. Ciomber
- Department of Bone Marrow Transplantation and Oncohematology; Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch; Gliwice Poland
| | - K. Panczyniak
- Analytics and Clinical Biochemistry Department; Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch; Gliwice Poland
| | - W. Fidyk
- Department of Bone Marrow Transplantation and Oncohematology; Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch; Gliwice Poland
| | - M. Sadus-Wojciechowska
- Department of Bone Marrow Transplantation and Oncohematology; Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch; Gliwice Poland
| | - J. Holowiecki
- Department of Bone Marrow Transplantation and Oncohematology; Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch; Gliwice Poland
| | - S. Giebel
- Department of Bone Marrow Transplantation and Oncohematology; Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch; Gliwice Poland
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15
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Biobanking: The Future of Cell Preservation Strategies. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2015; 864:37-53. [PMID: 26420612 DOI: 10.1007/978-3-319-20579-3_4] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
With established techniques cryopreservation is often viewed as an "old school" discipline yet modern cryopreservation is undergoing another scientific and technology development growth phase. In this regard, today's cryopreservation processes and cryopreserved products are found at the forefront of research in the areas of discovery science, stem cell research, diagnostic development and personalized medicine. As the utilization of cryopreserved cells continues to increase, the demands placed on the biobanking industry are increasing and evolving at an accelerated rate. No longer are samples providing for high immediate post-thaw viability adequate. Researchers are now requiring samples where not only is there high cell recovery but that the product recovered is physiologically and biochemically identical to its pre-freeze state at the genominic, proteomic, structural, functional and reproductive levels. Given this, biobanks are now facing the challenge of adapting strategies and protocols to address these needs moving forward. Recent studies have shown that the control and direction of the molecular response of cells to cryopreservation significantly impacts final outcome. This chapter provides an overview of the molecular stress responses of cells to cryopreservation, the impact of the apoptotic and necrotic cell death continuum and how studies focused on the targeted modulation of common and/or cell specific responses to freezing temperatures provide a path to improving sample quality and utility. This line of investigation has provided a new direction and molecular-based foundation guiding new research, technology development and procedures. As the use of and the knowledge base surrounding cryopreservation continues to expand, this path will continue to provide for improvements in overall efficacy and outcome.
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Baust JG, Gage AA, Bjerklund Johansen TE, Baust JM. Mechanisms of cryoablation: clinical consequences on malignant tumors. Cryobiology 2013; 68:1-11. [PMID: 24239684 DOI: 10.1016/j.cryobiol.2013.11.001] [Citation(s) in RCA: 109] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2013] [Revised: 11/04/2013] [Accepted: 11/05/2013] [Indexed: 02/09/2023]
Abstract
While the destructive actions of a cryoablative freeze cycle are long recognized, more recent evidence has revealed a complex set of molecular responses that provides a path for optimization. The importance of optimization relates to the observation that the cryosurgical treatment of tumors yields success only equivalent to alternative therapies. This is also true of all existing therapies of cancer, which while applied with curative intent; provide only disease suppression for periods ranging from months to years. Recent research has led to an important new understanding of the nature of cancer, which has implications for primary therapies, including cryosurgical treatment. We now recognize that a cancer is a highly organized tissue dependent on other supporting cells for its establishment, growth and invasion. Further, cancer stem cells are now recognized as an origin of disease and prove resistant to many treatment modalities. Growth is dependent on endothelial cells essential to blood vessel formation, fibroblasts production of growth factors, and protective functions of cells of the immune system. This review discusses the biology of cancer, which has profound implications for the diverse therapies of the disease, including cryosurgery. We also describe the cryosurgical treatment of diverse cancers, citing results, types of adjunctive therapy intended to improve clinical outcomes, and comment briefly on other energy-based ablative therapies. With an expanded view of tumor complexity we identify those elements key to effective cryoablation and strategies designed to optimize cancer cell mortality with a consideration of the now recognized hallmarks of cancer.
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Affiliation(s)
- J G Baust
- Institute of Biomedical Technology, State University of New York at Binghamton, Binghamton, NY 13902, United States; Department of Biological Sciences, Binghamton University, Binghamton, NY 13902, United States.
| | - A A Gage
- Department of Surgery, State University of New York at Buffalo, Medical School, Buffalo, NY 14214, United States
| | | | - J M Baust
- CPSI Biotech, Owego, NY 13827, United States
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17
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Modelling improvements in cell yield of banked umbilical cord blood and the impact on availability of donor units for transplantation into adults. Stem Cells Int 2013; 2013:124834. [PMID: 23509469 PMCID: PMC3590636 DOI: 10.1155/2013/124834] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2012] [Accepted: 01/11/2013] [Indexed: 11/26/2022] Open
Abstract
Umbilical cord blood (UCB) is used increasingly in allogeneic transplantation. The size of units remains limiting, especially for adult recipients. Whether modest improvements in the yield of cells surviving storage and thawing allow more patients to proceed to transplant was examined. The impact of improved cell yield on the number of available UCB units was simulated using 21 consecutive anonymous searches. The number of suitable UCB units was calculated based on hypothetical recipient weight of 50 kg, 70 kg, and 90 kg and was repeated for a 10%, 20%, and 30% increase in the fraction of cells surviving storage. Increasing the percentage of cells that survive storage by 30% lowered the threshold of cells needed to achieve similar engraftment rates and increased numbers of UCB units available for patients weighing 50 (P = 0.011), 70 (P = 0.014), and 90 kg (P = 0.003), controlling for differences in HLA compatibility. Moreover, if recipients were 90 kg, 12 out of 21 patients had access to at least one UCB unit that met standard criteria, which increased to 19 out of 21 patients (P = 0.035) when the fraction of cells surviving storage and thawing increased by 30%. Modest increases in the yield of cells in banked UCB units can significantly increase donor options for adult patients undergoing HSCT.
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18
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Porosity and mechanically optimized PLGA based in situ hardening systems. Eur J Pharm Biopharm 2012; 82:554-62. [DOI: 10.1016/j.ejpb.2012.08.006] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2011] [Revised: 08/07/2012] [Accepted: 08/20/2012] [Indexed: 11/19/2022]
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19
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Malpique R, Tostões R, Beier AFJ, Serra M, Brito C, Schulz JC, Björquist P, Zimmermann H, Alves PM. Surface-based cryopreservation strategies for human embryonic stem cells: a comparative study. Biotechnol Prog 2012; 28:1079-87. [PMID: 22718690 DOI: 10.1002/btpr.1572] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2012] [Revised: 06/12/2012] [Indexed: 12/30/2022]
Abstract
Human embryonic stem cells (hESC) hold tremendous potential in the emerging fields of gene and cell therapy as well as in basic scientific research. One of the major challenges regarding their application is the development of efficient cryopreservation protocols for hESC since current methods present poor recovery rates and/or technical difficulties which impair the development of effective processes that can handle bulk quantities of pluripotent cells. The main focus of this work was to compare different strategies for the cryopreservation of adherent hESC colonies. Slow-rate freezing protocols using intact hESC colonies was evaluated and compared with a surface-based vitrification approach. Entrapment within ultra-high viscous alginate was investigated as the main strategy to avoid the commonly observed loss of viability and colony fragmentation during slow-rate freezing. Our results indicate that entrapment beneath a layer of ultra-high viscous alginate does not provide further protection to hESC cryopreserved through slow-rate freezing, irrespectively of the cryomedium used. Vitrification of adherent hESC colonies on culture dishes yielded significantly higher recovery rates when compared to the slow-rate freezing approaches investigated. The pluripotency of hESC was not changed after a vitrification/thawing cycle and during further propagation in culture. In conclusion, from the cryopreservation methods investigated in this study, surface-based vitrification of hESC has proven to be the most efficient for the cryopreservation of intact hESC colonies, reducing the time required to amplify frozen stocks thus supporting the widespread use of these cells in research and clinical applications.
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20
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Turner RA, Mendel G, Wauthier E, Barbier C, Reid LM. Hyaluronan-supplemented buffers preserve adhesion mechanisms facilitating cryopreservation of human hepatic stem/progenitor cells. Cell Transplant 2012; 21:2257-66. [PMID: 22472355 DOI: 10.3727/096368912x637000] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
The supply of human hepatic stem cells (hHpSCs) and other hepatic progenitors has been constrained by the limited availability of liver tissues from surgical resections, the rejected organs from organ donation programs, and the need to use cells immediately. To facilitate accessibility to these precious tissue resources, we have established an effective method for serum-free cryopreservation of the cells, allowing them to be stockpiled and stored for use as an off-the-shelf product for experimental or clinical programs. The method involves use of buffers, some serum-free, designed for cryopreservation and further supplemented with hyaluronans (HA) that preserve adhesion mechanisms facilitating postthaw culturing of the cells and preservation of functions. Multiple cryopreservation buffers were found to yield high viabilities (80-90%) of cells on thawing of the progenitor cells. Serum-free CS10 supplemented with 0.05% hyaluronan proved the most effective, both in terms of viabilities of cells on thawing and in yielding cell attachment and formation of expanding colonies of cells that stably maintain the stem/progenitor cell phenotype. Buffers to which 0.05 or 0.1% HAs were added showed cells postthaw to be phenotypically stable as stem/progenitors, as well as having a high efficiency of attachment and expansion in culture. Success correlated with improved expression of adhesion molecules, particularly CD44, the hyaluronan receptor, E-cadherin, β4 integrin in hHpSCs, and β1 integrins in hepatoblasts. The improved methods in cryopreservation offer more efficient strategies for stem cell banking in both research and potential therapy applications.
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Affiliation(s)
- Rachael A Turner
- Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
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21
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Nicoud IB, Clarke DM, Taber G, Stolowski KM, Roberge SE, Song MK, Mathew AJ, Reems JA. Cryopreservation of umbilical cord blood with a novel freezing solution that mimics intracellular ionic composition. Transfusion 2012; 52:2055-62. [DOI: 10.1111/j.1537-2995.2011.03547.x] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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22
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Hayakawa J, Joyal EG, Gildner JF, Washington KN, Phang OA, Uchida N, Hsieh MM, Tisdale JF. 5% dimethyl sulfoxide (DMSO) and pentastarch improves cryopreservation of cord blood cells over 10% DMSO. Transfusion 2010; 50:2158-66. [PMID: 20492608 DOI: 10.1111/j.1537-2995.2010.02684.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
BACKGROUND Cell number and viability are important in cord blood (CB) transplantation. While 10% dimethyl sulfoxide (DMSO) is the standard medium, adding a starch to freezing medium is increasingly utilized as a cytoprotectant for the thawing process. Similar to hetastarch, pentastarch has the advantages of faster renal clearance and less effect on the coagulation system. STUDY DESIGN AND METHODS We compared a lower DMSO concentration (5%) containing pentastarch with 10% DMSO and performed cell viability assay, colony-forming units (CFUs), and transplantation of CB cells in NOD/SCID IL2Rγ(null) mice. RESULTS CB cells in 5% DMSO/pentastarch had similar CD34+, CD3+, and CD19+ cell percentages after thawing as fresh CB cells. CB cells in 5% DMSO/pentastarch had higher viability (83.3±9.23%) than those frozen in 10% DMSO (75.3±11.0%, p<0.05). We monitored cell viability postthaw every 30 minutes. The mean loss in the first 30 minutes was less in the 5% DMSO/pentastarch group. At the end of 3 hours, the viability decreased by a mean of 7.75% for the 5% DMSO/pentastarch and 17.5% for the 10% DMSO groups. CFUs were similar between the two cryopreserved groups. Frozen CB cells engrafted equally well in IL2Rγ(null) mice compared to fresh CB cells up to 24 weeks, and CB cells frozen in 5% DMSO/pentastarch engrafted better than those in 10% DMSO. CONCLUSION Our data indicate that the lower DMSO concentration with pentastarch represents an improvement in the CB cryopreservation process and could have wider clinical application as an alternate freezing medium over 10% DMSO.
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Affiliation(s)
- Jun Hayakawa
- Molecular and Clinical Hematology Branch (MCHB), National Institutes of Diabetes and Digestive and Kidney Disorders (NIDDK), National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health, Bethesda, Maryland, USA
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23
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Malpique R, Osório LM, Ferreira DS, Ehrhart F, Brito C, Zimmermann H, Alves PM. Alginate Encapsulation as a Novel Strategy for the Cryopreservation of Neurospheres. Tissue Eng Part C Methods 2010; 16:965-77. [DOI: 10.1089/ten.tec.2009.0660] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Affiliation(s)
- Rita Malpique
- Instituto de Biologia Experimental e Tecnológica, Animal Cell Technology Unit, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Luísa M. Osório
- Instituto de Biologia Experimental e Tecnológica, Animal Cell Technology Unit, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Daniela S. Ferreira
- Instituto de Biologia Experimental e Tecnológica, Animal Cell Technology Unit, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Friederike Ehrhart
- Instituto de Biologia Experimental e Tecnológica, Animal Cell Technology Unit, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Catarina Brito
- Kryobiophysik & Kryotechnologie, Fraunhofer-Institut for Biomedical Engineering, Universität des Saarlandes, St. Ingbert, Germany
| | - Heiko Zimmermann
- Kryobiophysik & Kryotechnologie, Fraunhofer-Institut for Biomedical Engineering, Universität des Saarlandes, St. Ingbert, Germany
| | - Paula M. Alves
- Instituto de Biologia Experimental e Tecnológica, Animal Cell Technology Unit, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
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Hebert TL, Wu X, Yu G, Goh BC, Halvorsen YDC, Wang Z, Moro C, Gimble JM. Culture effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on cryopreserved human adipose-derived stromal/stem cell proliferation and adipogenesis. J Tissue Eng Regen Med 2010; 3:553-61. [PMID: 19670348 DOI: 10.1002/term.198] [Citation(s) in RCA: 84] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Previous studies have demonstrated that EGF and bFGF maintain the stem cell properties of proliferating human adipose-derived stromal/stem cells (hASCs) in vitro. While the expansion and cryogenic preservation of isolated hASCs are routine, these manipulations can impact their proliferative and differentiation potential. This study examined cryogenically preserved hASCs (n = 4 donors), with respect to these functions, after culture with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) at varying concentrations (0-10 ng/ml). Relative to the control, cells supplemented with EGF and bFGF significantly increased proliferation by up to three-fold over 7-8 days. Furthermore, cryopreserved hASCs expanded in the presence of EGF and bFGF displayed increased oil red O staining following adipogenic induction. This was accompanied by significantly increased levels of several adipogenesis-related mRNAs: aP2, C/EBPalpha, lipoprotein lipase (LPL), PPARgamma and PPARgamma co-activator-1 (PGC1). Adipocytes derived from EGF- and bFGF-cultured hASCs exhibited more robust functionality based on insulin-stimulated glucose uptake and atrial natriuretic peptide (ANP)-stimulated lipolysis. These findings indicate that bFGF and EGF can be used as culture supplements to optimize the proliferative capacity of cryopreserved human ASCs and their adipogenic differentiation potential.
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Affiliation(s)
- Teddi L Hebert
- Stem Cell Biology Laboratory, Pennington Biomedical Research Center, Baton Rouge, LA, USA
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25
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Malpique R, Ehrhart F, Katsen-Globa A, Zimmermann H, Alves PM. Cryopreservation of adherent cells: strategies to improve cell viability and function after thawing. Tissue Eng Part C Methods 2009; 15:373-86. [PMID: 19196129 DOI: 10.1089/ten.tec.2008.0410] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
The commonly applied cryopreservation protocols routinely used in laboratories worldwide were developed for simple cell suspensions, and their application to complex systems, such as cell monolayers, tissues, or biosynthetic constructs, is not straightforward. In particular for monolayer cultures, cell detachment and membrane damage are often observed after cryopreservation. In this work, combined strategies for the cryopreservation of cells attached to Matrigel-coated well plate's surfaces were investigated based on cell entrapment in clinicalgrade, ultra-high viscosity alginate using two cell lines, neuroblastoma N2a and colon adenocarcinoma Caco-2, with distinct structural and functional characteristics. As the cryopreservation medium, serum-free CryoStor solution was compared with serum-supplemented culture medium, both containing 10% DMSO. Using culture medium, entrapment beneath an alginate layer was needed to improve cell recovery by minimizing membrane damage and cell detachment after thawing; nevertheless, up to 50% cell death still occurred within 24 h after thawing. The use of CryoStor solution represented a considerable improvement of the cryopreservation process for both cell lines, allowing the maintenance of high postthaw membrane integrity as well as full recovery of metabolic activity and differentiation capacity within 24 h postthawing; in this case, entrapment beneath an alginate layer did not confer further protection to cryopreserved Caco-2 cells, but was crucial for maintenance of attachment and integrity of N2a neuronal networks.
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Affiliation(s)
- Rita Malpique
- Animal Cell Technology, IBET/ITQB-UNL, 27801-901 Oeiras, Portugal
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Clarke DM, Yadock DJ, Nicoud IB, Mathew AJ, Heimfeld S. Improved post-thaw recovery of peripheral blood stem/progenitor cells using a novel intracellular-like cryopreservation solution. Cytotherapy 2009; 11:472-9. [PMID: 19499402 DOI: 10.1080/14653240902887242] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND AIMS Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products, which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate-freezing and liquid nitrogen storage have become 'routine' practice in many cell-processing facilities, there is clearly room for improvement as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment, and can also expose the patients to relatively undefined serum components and larger volumes of dimethylsulfoxide (DMSO) that can contribute to the morbidity and mortality of the transplant therapy. METHODS This study compared cryopreservation of PBSC in a novel intracellular-like, fully defined, serum- and protein-free preservation solution, CryoStor (BioLife Solutions Inc.), with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly, human PBSC apheresis specimens were collected and 5 x 10(7) cells/1 mL sample vial were prepared for cryopreservation in the following solutions: (a) FHCRC standard, Normosol-R, 5% human serum albumin (HAS) and 10% DMSO; and (b) CryoStor CS10 (final diluted concentration of 5% DMSO). A standard controlled-rate freezing program was employed, and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of 1 week. Vials were then thawed and evaluated for total nucleated cell count (TNC), viability, CD34 and granulocytes by flow cytometry, along with colony-forming activity in methylcellulose. RESULTS The PBSC samples frozen in CryoStor CS10 yielded significantly improved post-thaw recoveries for total viable CD34(+), colony-forming units (CFU) and granulocytes. Specifically, relative to the FHCRC standard formulation, cryopreservation with CS10 resulted in an average 1.8-fold increased recovery of viable CD34(+) cells (P=0.005), a 1.5-fold increase in CFU-granulocyte-macrophage (GM) numbers (P=0.030) and a 2.3-fold increase in granulocyte recovery (P=0.045). CONCLUSIONS This study indicates that use of CryoStor for cryopreservation can yield significantly improved recovery and in vitro functionality of stem/progenitor cells in PBSC products. In addition, it is important to note that these improved recoveries were obtained while not introducing any extra serum or serum-derived proteins, and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary; however, these findings imply use of CryoStor for cryopreservation could result in improved engraftment for those patients with a lower content of CD34(+) cells in their PBSC collections, along with reducing the requirement for additional apheresis collections and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO.
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Katsares V, Petsa A, Felesakis A, Paparidis Z, Nikolaidou E, Gargani S, Karvounidou I, Ardelean KA, Grigoriadis N, Grigoriadis J. A Rapid and Accurate Method for the Stem Cell Viability Evaluation: The Case of the Thawed Umbilical Cord Blood. Lab Med 2009. [DOI: 10.1309/lmle8bvhywct82cl] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022] Open
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Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (−20 °C). ZYGOTE 2009; 17:341-51. [DOI: 10.1017/s0967199409005474] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
SummaryUsually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 °C/min in a low-temperature (−80 °C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; −20 °C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type.
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Abstract
PURPOSE OF REVIEW To describe the response of prostate cancer to thermal therapies with an emphasis on cryoablative techniques. RECENT FINDINGS Long-term follow-up studies demonstrate clearly the effectiveness of the use of modern cryoablative techniques in the management of prostate cancer. Recently published American Urology Association Best Practice Guidelines identify prostate cryoablation as both primary and salvage therapies. Recent findings demonstrate the effectiveness of -40 degrees C exposure as lethal to prostate cancer genotypes following a double freeze-thaw encounter. In addition, the use of adjunctive agents to sensitize the cancer to freezing is reported. SUMMARY Thermal therapeutic options, especially cryoablation, are of growing interest for the treatment of prostatic and renal cancers. The methods of application of cryoablative therapy and the mechanisms of cell death that are attendant to the freezing-thaw encounter are clearly understood. Research focused on the development of freeze sensitizing agents that work adjunctively is of central interest in furthering the efficacy of this therapy.
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van Heeckeren WJ, Fanning LR, Meyerson HJ, Fu P, Lazarus HM, Cooper BW, Tse WW, Kindwall-Keller TL, Jaroscak J, Finney MR, Fox RM, Solchaga L, Forster M, Creger RJ, Laughlin MJ. Influence of human leucocyte antigen disparity and graft lymphocytes on allogeneic engraftment and survival after umbilical cord blood transplant in adults. Br J Haematol 2008; 139:464-74. [PMID: 17910637 DOI: 10.1111/j.1365-2141.2007.06824.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
The dose of graft-nucleated cells and CD34(+) haematopoietic progenitor cells are predictors of allogeneic engraftment and survival in umbilical cord blood (UCB) recipients. In this single institution prospective phase II trial, flow cytometric analyses of CD34(+) progenitor and lymphocyte populations in unmodified single unit human leucocyte antigen (HLA)-disparate UCB grafts infused into 31 consecutive adults (median age 41 years, range 20-64) receiving myeloablative conditioning were compared with clinical outcomes. Median infused UCB graft-nucleated cells and CD34(+) dose was 2.2 x 10(7)/kg and 1.2 x 10(5)/kg respectively. Day to absolute neutrophil count >/=0.5 x 10(9)/l with full donor chimerism averaged 27 d (range 12-41). Univariate analyses demonstrated that UCB graft-infused cell doses of CD34(+) (P = 0.015), CD3(+) (P = 0.024) and CD34(+)HLADR(+)CD38(+) progenitors (P = 0.043) correlated with neutrophil engraftment. This same analysis did not demonstrate a correlation between CD34(+) (P = 0.11), CD3(+) (P = 0.28) or CD34(+)HLADR(+)CD38(+) (P = 0.108) cell dose and event-free survival (EFS). High-resolution matching for HLA-class II (DRB1) resulted in improved EFS (P = 0.02) and decreased risk for acute graft-versus-host disease (GVHD) (P = 0.004). Early mortality (prior to post-transplant day +28) occurred in three patients, while 26 patients achieved myeloid engraftment. These results suggest that UCB graft matching at DRB1 is an important risk factor for acute GVHD and survival, while higher UCB graft cell doses of CD34(+), committed CD34(+) progenitors and CD3(+) T cells favourably influence UCB allogeneic engraftment.
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Derivation, maintenance and cryostorage of human embryonic stem cells. DRUG DISCOVERY TODAY. TECHNOLOGIES 2008; 5:e105-48. [PMID: 24125545 DOI: 10.1016/j.ddtec.2010.04.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
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Emmrich F. Abstracts of the 3rd World Congress on Regenerative Medicine, October 18-20, 2007, Leipzig, Germany. Regen Med 2007; 2:485-740. [PMID: 17941763 DOI: 10.2217/17460751.2.5.485] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Affiliation(s)
- Frank Emmrich
- Congress President Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany
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