Methods in Molecular Biology 1590 Jeremy M Crook Tenneille E Ludwig Editors Stem Cell Banking Concepts and Protocols METHODS IN MOLECULAR BIOLOGY Series Editor John M Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Stem Cell Banking Concepts and Protocols Edited by Jeremy M Crook ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, AIIM Facility, Innovation Campus, University of Wollongong, Fairy Meadow, NSW, Australia Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia Department of Surgery, St Vincent’s Hospital, The University of Melbourne, Fitzroy, VIC, Australia Tenneille E Ludwig WiCell Research Institute, Madison, WI, USA Editors Jeremy M Crook ARC Centre of Excellence for Electromaterials Science Intelligent Polymer Research Institute AIIM Facility, Innovation Campus University of Wollongong Fairy Meadow, NSW, Australia Tenneille E Ludwig WiCell Research Institute Madison, WI, USA Illawarra Health and Medical Research Institute University of Wollongong Wollongong, NSW, Australia Department of Surgery St Vincent’s Hospital The University of Melbourne Fitzroy, VIC, Australia ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6919-7 ISBN 978-1-4939-6921-0 (eBook) DOI 10.1007/978-1-4939-6921-0 Library of Congress Control Number: 2017934046 © Springer Science+Business Media LLC 2017 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or 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U.S.A Preface Stem cell banking has a critical role to play for supporting high quality research and transcending the clinical potential of stem cells to actual medicine Ideally, this is achieved by operating within a regulatory framework of good laboratory practice (GLP) or good manufacturing practice (GMP) for standardized, optimized, and controlled cell line production, storage, and distribution Among other benefits, creating repositories of quality “seed stock” is a most immediate way to circumvent the problems associated with extended cell culture, including susceptibility to genetic and phenotypic drift during propagation, loss of cells due to crosscontamination with microorganisms or other cell lines, and stem cell differentiation In recognizing the need for modern banking systems, major developed nations including the US, UK, and Japan have invested significantly in stem cell banking to prepare for the next major phase in researching and commercializing stem cells and producing clinical treatments Importantly, stem cell banking need not entail setting up large and expensive standalone facilities that operate on a national or international scale, but can involve smaller initiatives to support the activities of individual universities, research institutes, or laboratories Whatever the scale, a bank should align with global “best practice” for handling stem cells, ideally endorsed by leading stem cell organizations, networks, and consortia around the world Moreover, a bank should ensure the management and distribution of cell lines in the most efficient and cost-effective way For example, the succession of commercial and clinical aspirations could be facilitated by having low-cost quality-controlled GLP cells for research that are also available as more expensive clinical-grade GMP lines In addition, research and clinical-grade variants of the same cell lines/banks will provide consistency between laboratory and clinical activities for more predictable and better translational application Given the recent upsurge in stem cell research and development (R&D), including technological breakthroughs in creating new types of stem cells such as induced pluripotent stem cells (iPSCs), as well as clinical trials of human stem cell-based therapies, the publication of this book on Stem Cell Banking is timely This volume brings together contributions from experts in the field to guide stem cell banking, and in turn champion quality stem cell R&D and facilitate the translation of stem cells to clinical practice The book covers concepts and protocols relating to the banking of both pluripotent and somatic stem cells, from the ethical procurement of tissues and cells for the provision of “seed stock,” standardized methods for deriving hESCs and iPSCs, isolating mesenchymal stem cells, cell culture and cryopreservation, in addition to quality assurance (including cell line characterization) and information management As a volume in the highly successful Methods in Molecular Biology™ series, it aims to contribute to the development of competence in the subject by providing advice that is crucial to establishing a bona fide stem cell bank By proffering Stem Cell Banking, we hope to strengthen and maximize the use of existing and future stem cell resources Finally, the volume should serve as a valuable resource for established stem cell scientists and those new to the field Wollongong, NSW, Australia Jeremy M Crook v Contents Preface Contributors PART I GENERIC THEMES IN STEM CELL BANKING Stem Cell Banking: A Global View Glyn Stacey Quality Assurance in Stem Cell Banking: Emphasis on Embryonic and Induced Pluripotent Stem Cell Banking Therése Kallur, Pontus Blomberg, Sonya Stenfelt, Kristian Tryggvason, and Outi Hovatta Acquisition and Reception of Primary Tissues, Cells, or Other Biological Specimens Lyn E Healy Information Management Alberto Labarga, Izaskun Beloqui, and Angel G Martin Cryopreservation: Vitrification and Controlled Rate Cooling Charles J Hunt Quality Assured Characterization of Stem Cells for Safety in Banking for Clinical Application Kevin W Bruce, John D.M Campbell, and Paul De Sousa Ethics and Governance of Stem Cell Banks Donald Chalmers, Peter Rathjen, Joy Rathjen, and Dianne Nicol PART II v ix 11 17 29 41 79 99 PROTOCOLS FOR PLURIPOTENT STEM CELL BANKING Derivation of Human Embryonic Stem Cells Jeremy M Crook, Lucy Kravets, Teija Peura, and Meri T Firpo Derivation of Human-Induced Pluripotent Stem Cells in Chemically Defined Medium Guokai Chen and Mahendra Rao 10 Culture, Adaptation, and Expansion of Pluripotent Stem Cells Jennifer L Brehm and Tenneille E Ludwig 11 Cryobanking Pluripotent Stem Cells Jeremy M Crook, Eva Tomaskovic-Crook, and Tenneille E Ludwig 12 Genome Editing in Human Pluripotent Stem Cells Jared Carlson-Stevermer and Krishanu Saha vii 115 131 139 151 165 viii Contents PART III PROTOCOLS FOR MESENCHYMAL STEM CELL BANKING 13 Isolation, Culture, and Expansion of Mesenchymal Stem Cells Izaskun Ferrin, Izaskun Beloqui, Lorea Zabaleta, Juan M Salcedo, Cesar Trigueros, and Angel G Martin 14 Cryobanking Mesenchymal Stem Cells Andrés Pavón, Izaskun Beloqui, Juan M Salcedo, and Angel G Martin PART IV 177 191 PROTOCOLS FOR HUMAN NEURAL STEM CELL BANKING 15 Culturing and Cryobanking Human Neural Stem Cells Jeremy M Crook and Eva Tomaskovic-Crook 199 Index 207 Contributors IZASKUN BELOQUI • StemTek Therapeutics, Derio, Spain PONTUS BLOMBERG • Vecura, Karolinska University Hospital, Stockholm, Sweden JENNIFER L BREHM • WiCell Research Institute, Madison, WI, USA KEVIN W BRUCE • Censo Biotechnologies Ltd and Roslin Cell Sciences Ltd, Midlothian, UK JOHN D.M CAMPBELL • Scottish Blood Transfusion Service, Edinburgh, UK JARED CARLSON-STEVERMER • Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, USA DONALD CHALMERS • Centre for Law and Genetics, Faculty of Law, University of Tasmania, Hobart, TAS, Australia GUOKAI CHEN • Faculty of Health Sciences, University of Macau, Taipa, Macau, China; Center for Molecular Medicine, National Heart, Lung and Blood Institute, Bethesda, MD, USA JEREMY M CROOK • ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, AIIM Facility, Innovation Campus, University of Wollongong, Fairy Meadow, NSW, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia; Department of Surgery, St Vincent’s Hospital, The University of Melbourne, Fitzroy, VIC, Australia IZASKUN FERRIN • StemTek Therapeutics, Derio, Spain MERI T FIRPO • Division of Endocrinology and Stem Cell Institute, Department of Medicine, McGuire Translational Research Facility, University of Minnesota, Minneapolis, MN, USA LYN E HEALY • The Francis Crick Institute, London, UK OUTI HOVATTA • CLINTEC, Karolinska Institute, Flemingsberg, Sweden CHARLES J HUNT • UK Stem Cell Bank, National Institute for Biological Standards and Control, Hertfordshire, UK THERÉSE KALLUR • BioLamina, Stockholm, Sweden LUCY KRAVETS • Centre for Blood Cell Therapies, Peter MacCallum Cancer Centre, East Melbourne, Australia ALBERTO LABARGA • Department of Computer Science and Artificial Intelligence, University of Granada, Gardana, Spain TENNEILLE E LUDWIG • WiCell Research Institute, Madison, WI, USA ANGEL G MARTIN • StemTek Therapeutics, Derio, Spain DIANNE NICOL • Centre for Law and Genetics, Faculty of Law, University of Tasmania, Hobart, TAS, Australia ANDRÉS PAVÓN • StemTek Therapeutics, Derio, Spain TEIJA PEURA • Genea Biomedx, Sydney, NSW, Australia MAHENDRA RAO • New York Stem Cell Foundation Research Institute, New York, NY, USA; Q Therapeutics, Salt Lake City, UT, USA; Wake Forest Institute for Regenerative Medicine, Wake Forest University, Winston-Salem, NC, USA ix x Contributors JOY RATHJEN • School of Medicine, University of Tasmania, Hobart, TAS, Australia PETER RATHJEN • The Menzies Institute of Medical Research, University of Tasmania, Hobart, TAS, Australia KRISHANU SAHA • Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, USA JUAN M SALCEDO • StemTek Therapeutics, Derio, Spain PAUL DE SOUSA • Roslin Cell Sciences Ltd., Midlothian, UK; Censo Biotechnologies Ltd., Midlothian, UK; Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK GLYN STACEY • UK Stem Cell Bank, National Institute for Biological Standards and Control, Hertfordshire, UK SONYA STENFELT • Department of Neuroscience, Uppsala University, Uppsala, Sweden EVA TOMASKOVIC-CROOK • AIIM Facility, ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, University of Wollongong, Fairy Meadow, NSW, Australia; Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia CESAR TRIGUEROS • StemTek Therapeutics, Derio, Spain KRISTIAN TRYGGVASON • BioLamina, Stockholm, Sweden LOREA ZABALETA • StemTek Therapeutics, Derio, Spain Part I Generic Themes in Stem Cell Banking Cryobanking Mesenchymal Stem Cells 195 Fig Schematic of a typical freezing curve from a BioCool control rate freezer set from +10 °C to −80 °C X axis represents temperature (°C) while Y axis represents time (min) Note the hold step at −10 °C to allow for ice crystal seeding Freezing containers (such as the Thermo Scientific “Mr Frosty” -ref.: 5100-0001-) provide a simple-to-use system designed to achieve a rate of cooling very close to −1 °C/min, the optimal rate for cell preservation Freeze cells in tubes from to mL depending on the size of container used Store at room temperature when not in use Replace alcohol after every fifth use Do not manipulate two samples or lines at the same time Clean and sterilize the hood between samples or lines Add 4–6 mL 1× PBS to a T25 flask, 13–18 mL to a T-75 flask, or 20–25 mL to a T175 flask Add mL trypsin to a T25 flask, mL to a T-75 flask, and mL to a T175 flask It is important to observe the flask by microscope during detachment procedure All the cells have to be detached and well disaggregated, without clumps If the cells are not detached after min, tap the side of flask Ensure clumps of cells are dispersed 196 Andrés Pavón et al The cell resuspension step is tricky and requires slow methodical mixing Initially, the pipette or tip will be full of medium Release half of the volume and suck back up Repeat three to five times until no visible cell clumps can be seen Then release all liquid from the pipet tip Suck up from the bottom and release liquid at the top Try to minimize air bubble formation during mixing, as bubbles can destroy cells Freeze MSCs at a highest possible concentration (106 cells/vial for a working cell stock or 0.5 × 106 cells/vial for a master cell stock), making sure the cells are at least 90% viable at the time of freezing If the quantity of cells being frozen requires more than 15 vials, it is recommended that the cells be frozen in multiple batches to reduce DMSO contact time The time that cells are in contact with DMSO should be minimized until frozen In order to have a consistent freezing process it is important to change the methanol frequently, as it will become contaminated with water vapor 10 Control rate freezers can be programmed to maintain a desired temperature for prolonged periods of time once the target temperature is achieved It is recommended to set this parameter as “forever” (or equivalent), so the cells will be kept to temperature until transferred to a LN2 tank or freezer; preventing unexpected thawing if this operation cannot be performed on schedule References Nicol A, Nieda M, Donaldson C et al (1996) Cryopreserved human bone marrow stroma is fully functional in vitro Br J Haematol 94: 258–265 Haack-Sorensen M, Bindslev L, Mortensen S et al (2007) The influence of freezing and storage on the characteristics and functions of human mesenchymal stromal cells isolated for clinical use Cytotherapy 9:328–337 Ware CB, Baran SW (2007) A controlledcooling protocol for cryopreservation of human and non-human primate embryonic stem cells Methods Mol Biol 407:43–49 Part IV Protocols for Human Neural Stem Cell Banking Chapter 15 Culturing and Cryobanking Human Neural Stem Cells Jeremy M Crook and Eva Tomaskovic-Crook Abstract The discovery and study of human neural stem cells has advanced our understanding of human neurogenesis, and the development of novel therapeutics based on neural cell replacement Here, we describe methods to culture and cryopreserve human neural stem cells (hNSCs) for expansion and banking Importantly, the protocols ensure that the multipotency of hNSCs is preserved to enable differentiation to neurons and supporting neuroglia Key words Human neural stem cells, Culture, Expansion, Passaging, Freezing, Cryopreservation Introduction Human neural stem cells (hNSCs) hold great promise for both medical application and as a research tool for addressing fundamental questions in development and disease [1] While sourcing tissues for primary hNSC isolation and line derivation can be difficult, off-the-shelf ready-to-use hNSCs are available from a number of suppliers for research and clinical use Here, we describe in detail methods to culture hNSCs for expansion, and freezing for cell banking The protocols are derived from previously published work that can be referred to for additional information about working with hNSCs (i.e., to characterize hNSCs and their progeny) beyond the scope of the present chapter [2] Similar to other stem cell types described in this volume, a desired level of quality assurance can be applied for cell culture, freezing, and storage In any case, standardized and reliable protocols are necessary for basic science through to applied research and clinical product development This chapter details methods that are suitable for routine application, high-quality research and adaptable for clinical-compliance Jeremy M Crook and Tenneille E Ludwig (eds.), Stem Cell Banking: Concepts and Protocols, Methods in Molecular Biology, vol 1590, DOI 10.1007/978-1-4939-6921-0_15, © Springer Science+Business Media LLC 2017 199 200 Jeremy M Crook and Eva Tomaskovic-Crook Materials 2.1 hNSC Culture and Passaging hNSCs (eg., Millipore: SCC007) hNSC proliferation medium: NeuroCult® NS-A Proliferation Kit, human (STEMCELL Technologies, Cat no 05751) (see Note 1) Penicillin Streptomycin (10,000 U/mL, Life Technologies, Cat no 15140122) EGF (Cat no AF-100-15) and basic-FGF (Cat no AF-10018B; Peprotech, Lonza) Tris–HCl (Sigma, Cat no T5941) Human serum albumin (Sigma, Cat no A9511) TrypLE™ Select (Gibco, Life Technologies, Cat no 12563-029) Heparin (Sigma, Cat no H3149) DMEM/F12 (Gibco, Life Technologies, Cat no 11330-057) 10 Phosphate buffered saline (Sigma, Cat no P5368) 11 Ultra-low attachment 6-well plates (Corning Costar®, Cat no CLS3471, Sigma) 12 37 °C water bath 13 CO2 incubator 14 Benchtop centrifuge 15 Pipetman and tips 16 Pipet-Aid (motorized pipette) and serological pipettes 17 15 mL centrifuge tubes (Corning, Cat no 430052) 18 Class Biological Safety Cabinet (biosafety cabinet) 19 Sterilized Schott glass bottle, 100 mL 2.2 hNSC Cryopreservation and Thawing hNSC proliferation medium: NeuroCult® NS-A Proliferation Kit, human; STEMCELL Technologies, Cat no 05751) (see Note 1) TrypLE™ Select (Gibco, Life Technologies, Cat no 12563-029) DMEM/F-12 (Gibco Life Technologies, Cat no 11330-057) Isopropyl Alcohol (Chem-Supply, Cat no 323-4) 95% Ethanol CryoStor® CS10 (BioLife Solutions, Cat no 99-610-DV; STEMCELL Technologies) 15 mL centrifuge tubes (Corning, Cat no 430052) 50 mL centrifuge tubes (Corning, Cat no 43029) Culturing and Cryobanking Human Neural Stem Cells 201 1.5 mL Cryovials (Nunc, Cat no 5000-1020) 10 Ultra-low attachment 6-well plates (Corning Costar®, Cat no CLS3471; Sigma) 11 −80 °C freezer 12 Benchtop centrifuge 13 37 °C water bath 14 CO2 incubator 15 Pipet-Aid (motorized pipette) 16 Ice bucket 17 LN2 storage tank 18 Isopropanol freezing units (Nalgene®, Cat no 5100-0001, Cryo °C “Mr Frosty” Freezing Container, ThermoFisher Scientific) or equivalent non-isopropanol freezing units (CoolCell: Corning, Cat no 432001) 19 Cryovial racks 20 Metal forceps 21 Sterilized Schott glass bottle, 100 mL Methods Reagent preparation and cell culture work should be performed in a biosafety cabinet unless otherwise specified All media and reagents should remain sterile Incubations and culturing should be performed in a 37 °C incubator with a humidified atmosphere of 5% CO2 in air Centrifugation steps are performed at room temperature (RT) 3.1 hNSC Culture and Passaging hNSC can be cultured as neurosphere suspension cultures or as adherent monolayer cultures The method outlined below details neurosphere suspension culture Prepare hNSC culture medium (e.g., ~500 mL): (a) Neurocult media: 450 mL (b) Neurocult supplement: 50 mL (c) EGF (200 μg/mL stock): 50 μL (for 20 ng/mL working solution) To prepare 200 μg/mL EGF stock solution, dissolve 500 μg vial of EGF in 2.5 mL filter sterilized mM Tris–HCl containing 0.5% human serum albumin, pH 7.6 Store 50 μL aliquots of 200 μg/mL EGF stock solution at −80 °C Do not freeze/thaw each vial more than twice Add 50 μL to 500 mL Complete hNSC Proliferation Medium If preparing 100 mL Complete hNSC Proliferation Medium, add 10 μL to 100 mL Complete hNSC Proliferation Medium Aliquot unused portion to 202 Jeremy M Crook and Eva Tomaskovic-Crook × 10 μL aliquots of 200 μg/mL EGF stock solution at −80 °C for later use (d) bFGF (200 μg/mL stock): 50 μL (to get 20 ng/mL working solution) To prepare 200 μg/mL bFGF stock solution, dissolve 500 μg vial of bFGF in 2.5 mL filter sterilized mM Tris–HCl containing 0.5% human serum albumin, pH 7.6 Store 50 μL aliquots of 200 μg/mL bFGF stock solution at −80 °C Do not freeze/thaw each vial more than twice Add 50 μL to 500 mL Complete hNSC Proliferation Medium If preparing 100 mL Complete hNSC Proliferation Medium, add 10 μL to 100 mL Complete hNSC Proliferation Medium Aliquot unused portion to × 10 μL aliquots of 200 μg/mL bFGF stock solution at −80 °C for later use (e) Heparin (2 mg/mL stock): 500 μL (to get μg/mL working solution) (see Note 2) Store prepared 500 μL aliquots of mg/mL heparin stock solution in PBS at −20 °C Add 500 μL to 500 mL Complete hNSC Proliferation Medium If preparing 100 mL Complete hNSC Proliferation Medium, add 100 μL to 100 mL Complete hNSC Proliferation Medium Aliquot unused portion to × 100 μL aliquots of mg/mL Heparin stock solution at −20 °C for later use (f) To prepare hNSC Proliferation Medium with cytokines for expansion of hNSCs requires addition of heparin (2 μg/ mL; Sigma), epidermal growth factor (EGF, 20 ng/mL; Peprotech), and basic fibroblast growth factor (bFGF, 20 ng/mL; Peprotech) (g) To ensure stability of components, prepare sufficient working volume of Complete hNSC Proliferation Medium (with additives) for 2–3 weeks expansion of hNSCs at any one time To prepare a 100 mL volume of Complete hNSC Proliferation Medium (without additives), thaw 50 mL bottle of NeuroCult NS-A Proliferation Supplement at RT for 3–4 h until just thawed Mix well Aliquot NeuroCult NS-A Proliferation Supplement into 10 mL volumes in × 15 mL tubes for storage at −20 °C until required Add remaining 10 mL contents to 90 mL NeuroCult NS-A Basal Medium in sterile 100 mL bottle Mix well Complete hNSC Proliferation Medium can be stored without cytokines for up to weeks To prepare Complete hNSC Proliferation Medium with cytokines requires addition of heparin (2 μg/mL; Sigma), epidermal growth factor (EGF, 20 ng/mL; Peprotech), and basic fibroblast growth factor (bFGF, 20 ng/mL; Peprotech) Complete hNSC Proliferation Medium (with additives; ADDS) can be stored at °C for 2–3 weeks Culturing and Cryobanking Human Neural Stem Cells 203 Seed hNSCs at a density of 2–3 × 106 cells in mL Complete hNSC Proliferation Medium (with ADDS) per well of an ultralow attachment 6-well plate Label plates with cell line, passage number, date, and initials Return cultures to 37 °C incubator Agitate the plates daily to help prevent neurospheres from fusing together Monitor cultures daily and inspect morphology (semitransparent, microspikes on outer surface of neurosphere), neurosphere size (growth to less than 100–150 μm in diameter) and acidity of media (change media before reaching orange/ yellow color) Feed cultures every 3–4 days with a half media change To replenish media, collect medium containing neurospheres into a 15 mL tube and centrifuge at 190 × g for Aspirate the half the volume of the medium Replace with equal volume of pre-warmed Complete hNSC Proliferation Medium (with ADDS) Gently agitate the pellet with a mL serological pipette to separate the cells Return medium containing neurospheres to same wells Passage cells approximately every 7–10 days so as not to allow neurospheres to exceed 100–150 μm diameter Collect medium containing neurospheres into a 15 mL tube and centrifuge at 190 × g for Aspirate the medium without disturbing the pellet (see Note 3) Add mL pre-warmed TrypLE to the tube and gently mix tube contents by flicking tube Place the tube in a water bath at 37 °C for 10 Gently agitate the pellet with a mL pipette to separate the cells (see Note 4) Add mL Complete hNSC Proliferation Medium (without ADDS) (or aspirate from step 4, if available) to stop the digestion Centrifuge at 190 × g for 11 Aspirate the supernatant and resuspend the cells with fresh pre-warmed Complete hNSC Proliferation Medium (with ADDS) Count number of viable cells with a hemacytometer using Trypan Blue exclusion assay 12 Seed the cells by repeating step 13 Label plates with cell line, passage number, date, and initials Return cultures to 37 °C incubator 3.2 hNSC Cryopreservation Neurospheres cultures that have been grown for at least 3–4 days and not exceed 100–150 μm diameter can been frozen down as whole neurospheres, or single-cell populations The method outlined below details the cryopreservation of single-cell populations 204 Jeremy M Crook and Eva Tomaskovic-Crook Determine number of vials to be cryopreserved (1 cryovial for each 6-well plate of confluent cells), and label vials appropriately Minimally including cell line name, passage number, and date frozen (see Note 5) Collect medium containing neurospheres from wells of each 6-well culture plate and combine contents into separate 15 mL tubes (one plate of cells per tube) Centrifuge at 190 × g for Aspirate the medium without disturbing the pellet Add mL pre-warmed TrypLE to each tube and gently mix tube contents by flicking tube Place the tube(s) in a water bath at 37 °C for Gently agitate the pellet(s) with a mL pipette to separate the cells (see Note 4) Add mL Complete hNSC Proliferation Medium (without ADDS) (or aspirate from step 4, if available) to stop the digestion Centrifuge at 190 × g for Aspirate the supernatant and gently resuspend the cells with mL cold (stored at °C, keep on ice) CryoStor CS10 solution (cryopreservation media) At this point cells in cryopreservation media prepared from different plates can be pooled into an appropriate sized tube (e.g., 50 mL centrifuge tube) 10 Using an appropriately sized serological pipette, transfer mL volume of cells in cryopreservation medium to a prepared cryovial, being careful not to contact the pipette tip with the cryovial Repeat until the pooled cell suspension is exhausted 11 Cap each cryovial securely, quickly place them into the room temperature Mr Frosty or CoolCell container, and transfer the container into the −80 °C freezer overnight 12 24 h later, remove the frozen cryovials from containers in the −80 °C freezer, and place into LN2 storage 3.3 Thawing hNSCs It is important that safety glasses be worn during this procedure, as while it is a rare occurrence, sealed vials can explode during the thaw process Pre-warm mL Complete hNSC Proliferation Medium (with ADDS) per well of an ultra-low attachment 6-well plate In general, a single frozen vial can be thawed into wells of a 6-well plate Add mL pre-warmed Complete hNSC Proliferation Medium (without ADDS) to a 15 mL tube for each cryovial to be thawed Retrieve the vial to be thawed from LN2 storage and transport to the lab in a dewar filled with LN2 or dry ice Culturing and Cryobanking Human Neural Stem Cells 205 Confirm the cap is tightly closed, and using metal forceps, immerse the vial into a 37 °C water bath without submerging the cap Swirl the vial gently in the water When only a small ice crystal remains, remove the vial from the water bath Confirm again that the vial cap is tightened, and immerse the vial into 95% ethanol In the biosafety cabinet, open the vial being careful not to touch the rim of the vial Using a sterile mL pipette, remove the cell suspension and gently transfer into a sterile 15 mL conical tube contained prewarmed hNSC media Slowly add contents of cyrovial dropwise to the hNSC media, gently moving the tube back and forth to mix It is critical to add the cells slowly to avoid osmotic chock to the cells Centrifuge at 190 × g for Aspirate the supernatant, being careful not to disturb the cell pellet (see Note 6) 10 Resuspend the cell pellet in Complete hNSC Proliferation Medium (with ADDS) per cryovial so that there will be a final volume of mL per well total in the prepared culture plate after adding the cells (i.e., if plates were prepared with mL of media per well, and the vial will be thawed into wells, resuspend in mL medium, ultimately adding mL of suspension to the mL in the plate for a total of mL) 11 Slowly add the cell suspension dropwise to the prepared plate for recovery and expansion 12 Place plate gently into the incubator Distribute the cells/neurospheres by gently shaking the plate front to back, rest a few seconds, then side to side, and close the incubator door gently 13 Passage cells after 5–7 days of recovery post-thawing Notes NeuroCult® NS-A Proliferation Kit, human; STEMCELL Technologies, 05751) consists of two components (NeuroCult NS-A Basal Medium (Stem Cell STEMCELL Technologies, Human; 05750; 450 mL) and NeuroCult NS-A Proliferation Supplement (STEMCELL Technologies, Human; 05753; 50 mL)) which are mixed to prepare Complete hNSC Proliferation Medium Complete hNSC Proliferation Medium can be stored without cytokines for up to weeks Heparin increases the affinity of bFGF for its receptors 206 Jeremy M Crook and Eva Tomaskovic-Crook The aspirated medium can be collected for use in step to inactivate TrypLE Do not flux the cells by pipetting more than seven times The labeled passage number should be increased by one compared to the harvested culture to be frozen Therefore, the culture vessel to be labeled at thaw will match the vial being thawed Vacuum aspiration should be used with extreme caution, as there is a significant risk of aspirating the cell pellet If the pellet is smaller than anticipated, or if the cells are particularly valuable or rare, use a sterile serological pipette to remove the supernatant Acknowledgment The authors wish to acknowledge funding from the Australian Research Council (ARC) Centre of Excellence Scheme (CE140100012) References Crook JM, Kobayashi NR (2008) Human stem cells for modeling neurological disorders: accelerating the drug discovery pipeline J Cell Biochem 105:1361–1366 Kobayashi N, Sui L, Tan PSL et al (2010) Modelling disrupted-inschizophrenia loss of function in human neural progenitor cells: tools for molecular studies of human neurodevelopment and neuropsychiatric disorders Mol Psychiatry 15:672–675 INDEX A Acquisition 17–23 Adaptation 15, 92, 139–150, 186, 189 Adipogenic 183, 187, 188 Adipose 177, 178, 183 Adult stem cells 101, 102, 107 Allogeneic 100, 177, 178 Anonymized 19, 82 Apoptosis 53, 62, 64–66 Australia 14, 102–104 Autologous 20, 101, 177 B Banking 3–9, 11–14, 17–23, 43, 58, 66, 69, 79, 84–87, 89, 93, 101, 104, 109, 110, 141, 151, 156, 161, 199 Batch 61, 131, 187, 193, 194, 196 Beloqui, I 29, 36, 177–189, 191–196 Best practice 4, 15, 18, 20, 21, 23, 67, 79, 81, 103–106, 108, 109 Bio-specimen 17, 23, 30, 36 Bisection 116, 123–125, 128 Blastocyst .4, 58, 115–127, 154 Blomberg, P .11–14 Bone marrow 45, 46, 48, 177, 178, 180–181, 184, 186, 188, 189 Brehm, J.L 139–150 Bruce, K .51, 79, 85, 87, 89, 93 C Campbell, J.D.M 79, 85, 87, 89, 93 Carlson-Stevermer, J .165–173 Cell banking 43, 58, 59, 84–86, 88, 92, 110, 141, 161, 199 Cell identity .18, 20, 42, 81, 115 Cell line history 7, 86 Cell potency 81, 90–91 Cell therapy .9, 42, 81, 101, 104, 177 Chain of custody 18, 31, 32 Chalmers, D 99 Characterization .8, 9, 13, 15, 20, 23, 79–97, 99, 104, 108, 115, 133 Chemically defined 15, 131–137 Chen, G 131–137 Clinical application 4, 8, 17–20, 23, 51, 58, 59, 68, 69, 79, 85, 87, 89, 93, 99–101, 103–106, 108, 110, 115 Clinical-grade 4, 7, 12, 13, 68, 101, 102, 104, 107, 116, 127, 140 Clinically-compliant 32, 51, 67–69 Clinical translation 104, 107, 108 Clinical trials 100, 178 Clonal 135, 169–170, 172 Code 31, 82, 83, 103–105, 108 Collagenase 140, 152, 157–159, 161, 163, 164, 179, 183, 185 Colonies 47, 56–59, 63, 67, 92, 134, 136, 137, 144, 149, 158, 159, 161, 163, 170, 172 Colony 21, 56, 57, 59, 62, 133, 134, 136, 149, 155, 156, 161, 163, 172 Comparative genome hybridization (CGH) 15, 94, 141 Conflicts of interest 108 Consent 7, 9, 13, 14, 18–20, 23, 30, 81–84, 103, 105, 106, 109, 110, 177 Contamination .3, 15, 20, 42, 57–59, 66–68, 80, 82, 85, 86, 91, 94–96, 145, 148, 154, 161, 172, 183, 185, 196 Controlled-rate freezing (CRF) 38, 61–62, 120, 152–154, 161–163 Cooling rate 44–52, 55, 57–62 Crook, J.M 115, 121, 122, 125, 151–164, 199–206 Cryopreservation 38, 41–68, 80, 86, 87, 91, 92, 95, 119, 127, 135, 151, 155, 157, 159, 160, 163, 193–194, 200–201, 203–204 Cryoprotectant 46, 51–57, 60, 63–64, 68, 69, 191 Cryoprotection 64 Cryovial 31, 57–59, 66, 153–156, 159, 160, 193, 201, 204, 205 Culture 3, 12, 18, 30, 42, 80, 101, 115, 131, 139, 152, 166, 178, 191, 199 D Defined .15, 35, 45, 49, 80, 84–86, 128, 129, 131–137, 140, 151, 152 Dental pulp 178, 184–186, 188 Depositors 104–107 de Sousa, P 79, 85, 87, 89, 93 Derivation 19, 20, 41, 68, 69, 80, 94, 99, 101–102, 115–129, 131–137, 140 Jeremy M Crook and Tenneille E Ludwig (eds.), Stem Cell Banking: Concepts and Protocols, Methods in Molecular Biology, vol 1590, DOI 10.1007/978-1-4939-6921-0, © Springer Science+Business Media LLC 2017 207 STEM CELL BANKING: CONCEPTS AND PROTOCOLS 208 Index Differentiation 8, 9, 42, 53, 56, 57, 60, 62, 65, 66, 90, 92, 107, 144, 145, 147, 149, 157, 172, 177–179, 184, 186–189, 191 Dimethyl sulfoxide (DMSO) 51–54, 56, 59–64, 66–68, 152, 155, 157, 159, 163, 191–194, 196 Dispase .135, 140, 146, 157, 158, 163, 179, 185 Dissociation 56, 65, 133, 135, 169, 171, 173 Distribute .20, 88, 105, 106, 144, 145, 149, 161, 205 Documentation 12, 22, 23, 82, 86, 100, 108, 109 Donor 4, 9, 12, 14–16, 19, 20, 23, 30, 31, 37, 81–83, 86–88, 94, 95, 100, 110, 165–167, 171, 177 Dulbecco’s Modified Eagle Medium (DMEM) .117, 118, 122, 132–134, 141–143, 146, 148, 149, 152, 155, 157, 158, 163, 167, 168, 178, 179, 183, 185, 192, 193, 200 E Efficiency 8, 131, 132, 134–136, 139, 150, 159, 166, 168–171, 177 Electroporation .166–172 Embryo 5, 8, 9, 20, 47, 56–58, 67, 80, 95, 99, 101–103, 115–129, 151 Embryoid body 64, 87, 90 Embryonic stem cells (ESCs) colony picking .56 culture 123, 158, 159 lines 4, 5, 8, 20, 41, 58–60, 68, 89, 100, 104, 107, 108, 115, 116, 119, 126, 128, 172 medium 117, 118, 123–126 thawing 42, 62, 119, 126, 151 transfection 166, 167, 169 Embryo transfer 122, 124, 126, 128 Engineering 165 Equilibrium 43, 44, 46, 48, 49, 53, 55 Ethical conduct 103, 105, 108 Ethics 7, 9, 13, 14, 19, 99–110 Ethylenediaminetetraacetic acid (EDTA) 133–137, 140, 147, 157, 163, 179, 181, 182, 184, 185, 192, 193 European Union (EU) 15, 103 Expansion .9, 15, 21, 56, 68, 80, 84, 85, 90, 91, 107, 115, 135, 139–150, 156, 161, 163, 177–189, 199, 202, 205 F Feeder cells See Human foreskin fibroblast; Mouse embryonic fibroblasts Feeder-free culture 144, 150 Ferrin, I 177–189 Fibroblasts 20, 21, 47, 68, 117, 118, 123, 131, 133–136, 157, 183, 188, 202 Firpo, M.T 115, 121, 122, 125 Flow cytometry 15, 87–89, 91, 92, 133, 168, 170, 187 Framework 7, 12, 14, 17, 18, 20, 21, 36, 68, 80, 100, 103–105 Freezing 12, 13, 38, 42–56, 61 , 63, 65, 119, 120, 151, 152, 154–163, 173, 191–196, 199, 201 G G-banding 87, 92–94 Genetic identity 86–88 Genetic stability 16, 92–94, 141 Genetically-modified 21, 66, 107 Genome editing 165–173 Genotyping 5, 21, 30, 87 Germ layers 4, 87, 90, 91, 141 Giemsa staining 93 Good laboratory practice (GLP) 12, 15, 18, 30 Good manufacturing practice (GMP) 12, 13, 15, 18, 30, 51, 67–69, 132 Governance 7, 18–20, 99 Growth profiling 88, 91–92 Guardianship 105–106 H Harmonization 23, 109 Healy, L 17 Hermetically sealed 161 Hovatta, O .11–14 Human leukocyte antigens (HLAs) 87, 88, 187 Hunt, C.J 41–68 I Immunogenicity .178 Immunosurgery 117–118, 123–125 In vitro fertilization (IVF) 116–120, 122, 124 Induced pluripotent stem cell (iPSCs) 4, 5, 8, 9, 11–14, 17, 18, 20, 21, 42, 81, 84, 91, 93, 101, 131–137, 151, 172 Information flow 30–31 Inner cell mass (ICM) 58, 115, 116, 118, 120, 122–125, 128, 129 Internal identification 37 International Society for Biological and Environmental Repositories (ISBER) 106 International Society for Stem Cell Research (ISSCR) .12, 14, 103, 104, 107, 108 International Standards Organization (ISO) 12, 13, 30 International Stem Cell Banking Initiative (ISCBI) 4, 12, 20, 100, 103, 104, 109 International Stem Cell Forum (ISCF) 9, 20, 100, 103 Intracellular ice 44, 46–50, 55, 62, 193–194 Irradiated 117, 118 J Japan 61, 102 Jurisdictions 100, 103, 109 STEM CELL BANKING: CONCEPTS AND PROTOCOLS 209 Index K Kallur, T 11–14 Karyotype 15, 92–94, 140, 141, 149 Kravets, L 115, 121, 122, 125 L Labarga, A .29, 36 Laboratory information managing systems (LIMS) .31–39 Laboratory workflows .37 Legal 13, 14, 17, 31, 81, 105 Legislation 5, 14, 18, 19, 21, 102, 103 Lipoaspirate 183, 186, 188 LN2 38, 57, 58, 61, 62, 66–68, 116, 120–122, 127, 143, 152–157, 160, 162, 170, 192–194, 196, 201, 204 Ludwig, T 151–164 M Management 5, 7, 11–13, 18, 21, 23, 29–39, 80, 105 Martin, A.G .29, 36, 177–189, 191–196 Master cell bank (MCB) 15, 84, 85, 92, 181, 183–185, 187, 188 Material Deposit and Distribution Agreement (MDDA) 106 Material transfer agreement (MTA) 22, 23, 106, 110 Matrigel 63, 134, 136, 137, 140–144, 146–148, 157, 163, 167 Media 8, 15, 67, 68, 86, 90, 92, 94, 116, 123, 126, 127, 132–135, 140, 142, 155–157, 160–163, 166–170, 172, 173, 201, 203–205 Medium 53, 96, 120, 131, 140, 152, 168, 179, 191, 200 Mesenchymal stem cells (MSCs) .48, 56, 64, 66, 100, 101, 177–189, 191–196 Microbiological tests 81, 94–97 Mouse embryonic fibroblasts medium .57 passaging 56 preparation 57 mTeSR1 140–147, 149, 163, 166–168, 170 Mycoplasma 3, 20, 86, 87, 96 Osmotic equilibrium 43, 44 Osteogenic 179, 183, 187–189 Outgrowth 80, 115, 116, 119, 125–126, 128, 129 P Passaging 65, 85, 91, 92, 119, 125–126, 133, 136, 140, 144, 145, 147, 149, 150, 186, 189, 200–203 Patient-specific 100, 101, 131 Pavon, A 191–196 Performance metrics .37, 38 Peura, T 115, 121, 122, 125 Phenotype 15, 42, 87–92, 166, 187, 188 Pluripotent .4, 11, 17, 42, 79, 99, 115, 131, 140 Pluripotent stem cells 4–5, 7–8, 20–21, 42, 47, 48, 59–69, 79, 81, 88, 91, 99, 101, 104, 131–137, 139–173 Polymerase chain reaction (PCR) 86, 87, 89, 91, 96, 169, 172, 173 Pre-master cell bank .84, 85 Primary cultures 146, 180–186, 189 Prions 83, 97 Protocol 7, 8, 13, 42, 53, 55, 56, 59, 60, 62, 63, 65, 66, 68, 69, 86, 90, 116, 119, 132, 136, 140, 141, 143, 147, 151, 157, 163, 167–171, 187, 194, 199 Purity 13, 81, 91 Q Quality assurance (QA) 7, 11–16, 79, 85, 87, 89, 93, 105, 107–108, 110, 161, 199 Quality control (QC) 4, 8, 12, 15, 18–21, 32, 37, 38, 42, 80, 83, 85, 100, 102, 109, 110 Quality standards 7, 29, 104 R National Institute for Biological Standards and Control (NIBSC) Neural stem cells 199–206 Nicol, D 99 Nucleation 43, 44, 46, 47, 49–51, 53, 54, 56, 61–63, 69, 162 Rao, M 131–137 Rathjen, J .99 Rathjen, P 99 Reagents 7, 15, 68, 86, 95–97, 116, 122, 131, 140, 142, 152, 157, 166, 167, 172, 178–179, 189, 192, 201 Regenerative medicine .6, 68, 99, 100, 139, 177 Regulations 19, 22, 81, 99, 102–110, 180 Report 4, 8, 31, 32, 37, 38, 41, 46–48, 56, 58–66, 68, 80, 105, 107, 116, 154, 184 Reprogramming 20, 91, 131–136 Requestors 104–106 Research 3, 11, 17, 29, 42, 79, 99, 115, 131, 139, 151, 177, 191, 199 Rewarming rate 58 Rho-associated kinase (ROCK) inhibitors 65–66, 134, 136, 137, 146, 167–170, 172 O S Off-the shelf 100, 199 Oocytes 45, 101 Operators .57, 92, 93, 104, 128, 129 Safety 4, 7, 12, 15, 16, 18, 21, 22, 32, 42, 79, 85, 87, 89, 93, 101, 103, 107–108, 110, 127, 142, 148, 154, 160, 161, 179, 180, 189, 194, 200, 204 N STEM CELL BANKING: CONCEPTS AND PROTOCOLS 210 Index Saha, K 165–173 Salcedo, J.M 177–189 Sample cession 37 handling 37 movement .31 processing 30, 180–185 procurement 30 shipping 31 storage 31 Sendai virus 131–136 Serum-free 124, 125, 135 Short tandem repeats (STRs) 9, 20, 86, 87 Single nucleotide polymorphisms (SNPs) 15, 87, 88, 94, 166 Slow-rate freezing 191 Source 3, 8, 17, 18, 20, 21, 58, 61, 68, 83, 86, 94, 96, 97, 99–101, 103, 107, 116, 127, 131, 163, 167 Spain 6, 31, 36, 102 Spectral karyotyping (SKY ) 87, 93, 94 Stacey, G .3–4, Standardization 23, 79, 80, 93, 99, 106, 110 Stem cell banking 3, 6, 11–14, 17–22, 104, 109 Stenfelt, S 11–14 Storage 13, 14, 18–20, 23, 29–31, 33, 38, 42, 50, 57, 58, 66–69, 84, 86, 101, 104, 106, 107, 109, 119, 143, 147, 148, 153, 154, 156, 157, 160, 162, 191, 193, 194, 199, 201, 202, 204 Straw 57–59, 61, 66, 120, 121, 127, 152–156, 161–163 T Teratoma 8, 15, 59, 87, 90, 107, 141 Thawing cells 42, 49, 50, 62, 156, 162 embryos 116–117, 119–122, 127 Therapy 5, 9, 42, 95, 177, 191 Tissue 4, 5, 7, 9, 12, 13, 17, 29, 36, 45, 46, 55, 56, 58, 62, 68, 80–84, 87, 90, 94, 95, 97, 100–103, 105, 106, 121–123, 167, 177, 178, 180, 181, 183–186, 189, 194, 199 Tomaskovic-Crook, E 151–164, 199–206 Transfection 166–170 Transfer .121, 122, 124–128, 169 Transgene 166, 169 Transgenesis .166 Transportation 22, 57, 66–69, 184 Treatment 8, 15, 31, 47, 82, 83, 100, 101, 123, 140, 183, 184 Trigueros, C 177–189 Trophectoderm 115, 116, 120, 123, 125 Tryggvason, K .11–14 Trypsin 92, 96, 135, 136, 163, 179, 181, 182, 184, 185, 192, 193, 195 U UK Stem Cell Bank 4, 6, 68, 102, 105–108 Umbilical cord 101, 177, 178, 181–183, 186, 188, 189 Undifferentiated 56, 128, 140, 147, 149 United Kingdom (UK) 4–6, 19, 36, 59, 61, 68, 83, 102, 103, 105–108 United States of America (USA) 6, 14, 36, 102, 127, 141, 142, 147 V Vectors 91, 167, 171 Viability 37, 38, 42, 45, 80, 81, 87, 88, 91–92, 128, 140, 151, 154, 161, 172, 189, 193, 194 Viral testing 86, 87 Viruses 20, 22, 96, 133 Vitrification 41–68, 120, 152–156, 194 W Warming rate 49, 55, 56 Working cell bank (WCB) .42, 67, 84, 85, 92, 181, 183–187, 189 X Xeno 68 Z Zabaleta, L 177–189 zona pellucida 123, 124, 128 ... nations including the US, UK, and Japan have invested significantly in stem cell banking to prepare for the next major phase in researching and commercializing stem cells and producing clinical... IN STEM CELL BANKING Stem Cell Banking: A Global View Glyn Stacey Quality Assurance in Stem Cell Banking: Emphasis on Embryonic and Induced Pluripotent Stem Cell. .. practice A network of stem cell banking centers and individuals and organizations committed to supporting formalized stem cell banking called the International Stem Cell Banking Initiative (ISCBI) has