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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Helps those that use cell preservation to develop new protocols or improve existing protocols This book provides readers with the tools needed to develop or debug a preservation protocol for cells. The core structure and content of the text grew from a professional short course that has been offered at the Biopreservation Core Resource for the last 10 years. This comprehensive text describes, step by step, the individual elements of a protocol, including the relevant scientific principles for each phase of the protocol. It can be used by anyone who is involved in cell preservation--even by those who are not experts in freezing of cells--because it provides the scientific basis for those that want to understand the basis for the protocol. Preservation of Cells: A Practical Manual begins by first introducing readers to the subject of preserving cells. It then goes on to cover Pre-freeze Processing and Characterization; Formulation and Introduction of Cryopreservation Solutions; Freezing Protocols; Storage and Shipping of Frozen Cells; Thawing and Post Thaw Processing; Post-thaw Assessment; and Algorithm-driven Protocol Optimization. * Clearly explains the reasons behind every step in the development of a preservation protocol and the scientific principles behind them * Provides alternative modes of preservation for when conventional methods of cryopreservation are not appropriate for a given cell type or application * Enables more organization to achieve improved post thaw recoveries and process consistency Preservation of Cells: A Practical Manual is an important book for researchers, laboratory technicians and students in cell biology, stem cell biology, tissue engineering, and regenerative medicine. It is also useful to cell bankers, regenerative medicine, biomarker discovery or precision medicine companies, and cell therapy labs, blood bankers, biobankers, and biotechnology companies.
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Table of Contents
Cover
Title Page
Preface
Acknowledgments
Nomenclature
1 Introduction
Mammalian Cells: Modern Workhorses
Bridging the Gap
The Preservation Toolkit
Fit‐for‐Purpose
One Size Does Not Fit All
The Process is the Product
Reproducibility
Safety
Dispelling the Myth of the Cold Black Box
References
2 Pre‐freeze Processing and Characterization
Pre‐freeze Processing
Pre‐freeze Characterization
Annotation of Pre‐freeze Processing
Scientific Principles
Putting Principles into Action
References
3 Formulation and Introduction of Cryopreservation Solutions
Importance of Cryoprotective Agents
Mechanisms of Cryoprotection
Formulating a Cryopreservation Solution
Toxicity of CPAs
Developing a Protocol for Introducing CPA Solutions
Cell Concentration
Safety Considerations for Cryopreservation Solutions
Cryopreservation Containers
Labeling
Sample Annotation
Scientific Principles
Putting Principles into Practice
References
4 Freezing Protocols
Importance of Cooling Rate
Controlled‐rate Freezing
Passive Freezing
Transfer to Storage
Vitrification
Independent Temperature Measurement
Scientific Principles
Putting Principles into Practice
References
5 Storage and Shipping of Frozen Cells
Scientific Basis for Selection of a Storage Temperature
Standards, Guidelines, and Best Practices
Monitoring Systems
Safety
Inventory Management System
Stability in Storage
Fit‐for‐Purpose Storage Practices
Risk Mitigation in Long‐Term Storage
Shipping or Transport of Cells
Sample Annotation
Scientific Principles
Putting Principles into Practice
References
6 Thawing and Post‐Thaw Processing
Thawing Equipment
Estimating Your Thawing Rate
Safety Considerations for Thawing
Post‐Thaw Processing
Removal of Vitrification Solutions
Scientific Principles
Putting Principles into Practice
References
7 Post‐Thaw Assessment
Common Measures Used in Post‐Thaw Assessment
Strategies to Improve the Accuracy and Reproducibility of Post‐Thaw Assessment
Optical Methods of Post‐Thaw Assessment
Release Criteria
Scientific Principles
Putting Principles into Practice
References
8 Algorithm‐Driven Protocol Optimization
Small Cell Number/High Throughput Approach
Practical Notes
Modeling in Cryobiology
References
Introduction
Protocol Contributors
Cryopreservation of Endothelial Cells in Suspension
Principle
Equipment and Supplies
Safety
Procedure
Expected Results
References
Cryopreservation of Peripheral Blood Mononuclear Cells from Whole Blood
Principle
Equipment
Materials
Reagents
Procedure
Equipment
Materials
Reagents
Procedure
APPENDIX A
Human Serum AB Freezing Media
Cryopreservation of Human Adipose Stem Cells
Principle
Equipment and Supplies
Reagents and Media
Procedure
Cryopreservation
Reference
Cryopreservation of Red Blood Cells
Method I: High Glycerol/Slow Cooling Technique (Meryman and Hornblower 1972)
Method II: A Low Glycerol/Rapid Cooling Technique (Rowe, Eyster, and Kellner 1968)
Method III: Hydroxyethylstarch/Rapid Cooling Technique (Sputtek 2007)
References
Cryopreservation of Oocytes by Slow Freezing
Principle
Specimen Requirements
Equipment and Supplies Needed
Procedure
Safety
Calculations
Reporting Results
Procedure Notes
Limitations of Procedure
Oocyte Vitrification and Warming
Principle
Equipment and Supplies
Procedure
Quality Control
Safety
Transportation of Hematopoietic Progenitor Cells and Other Cellular Products
Principle/Rationale
Specimen
Equipment/Reagents
Quality Control
Procedure
Additional Information
Further Reading
Cryopreservation of Hematopoietic Progenitor Cells
Principle/Rationale
Protocol/Processing Schema
Specimen
Equipment/Reagents
Quality Control
Procedure
Appendix A Alternate Cryopreservation Harness Set‐2 or 4 Bags
Further Reading
Thawing of Hematopoietic Progenitor Cells
Principle/Rationale
Equipment/Reagents
Quality Control
Procedure
Further Reading
Processing and Cryopreservation of T‐Cells
Principle/Rationale
Protocol/Processing Schema: N/A
Specimens
Equipment/Reagents
Quality Control
Procedure
Further Reading
Thawing and Reinfusion of Cryopreserved T‐Cells
Principle/Rationale
Protocol/Processing Schema
Specimen
Equipment/Reagents
Quality Control
Procedure
Further Reading
Index
End User License Agreement
List of Tables
Chapter 03
Table 3.1 Cryoprotective molecules.
Table 3.2 Common cryopreservation solution compositions for
in vitro
, cell therapy, and cell‐banking applications.
Chapter 07
Table 7.1 Summary of different categories of post‐thaw assessments for cells: Multiple measures of post‐thaw assessment should be used to characterize the cells post‐thaw.
Cryopreservation of Hematopoietic Progenitor Cells
Table 1 Preparation of DMSO freezing solution for a final concentration of 10% vol/vol.
Processing and Cryopreservation of T‐Cells
Table 1 Proper volume of cryopreservation and plasma‐lyte/HSA solutions for different bag capacities.
Thawing and Reinfusion of Cryopreserved T‐Cells
Table 1 Preparation of thawing media.
List of Illustrations
Chapter 01
Figure 1.1 Minding the gap: preservation is used to maintain the critical biological properties of the cells when they are needed at a later time or in a different location.
Figure 1.2 Preservation toolkit for cells. Preservation methods should be fit‐for‐purpose. The mode and duration of storage should be appropriate for the given application.
Figure 1.3 The different steps of the preservation process. Each step of the process can be designed based on scientific principles. Each step contributes to the overall quality of the end product.
Figure 1.4 The reagents, starting material, and processes determine the quality of the product at the end of preservation.
Chapter 03
Figure 3.1 Volumetric response of cells with introduction of a (a) penetrating cryoprotective agent; (b) non‐penetrating cryoprotective agent, and (c) upon removal of the cryoprotective agent after thawing Vo is the initial volume of the cells and V is the volume of the cell at time, t.
Chapter 04
Figure 4.1 (a) Variation in survival with cooling rate for a given cell type. (b) Variation in survival with cooling rate for a specific cell type but varying composition of cryopreservation medium.
V
c1
and
V
c2
are the post‐thaw viabilities associated with compositions 1 and 2, respectively. and are the optimum cooling rates associated with compositions 1 and 2, respectively.
Figure 4.2 Temperature as a function of time for the freezing chamber during a controlled‐rate protocol. The programmed elements of a controlled‐rate protocol include Segment 1 (S1), initial hold; Segment 2 (S2), controlled cooling rate; Segment 2a (S2a), nucleation step (optional); and Segment 3 (S3), higher cooling rate (optional) to final temperature where sample is removed and placed in storage. “B” is the cooling rate for the sample, which is typically specified in the protocol.
Figure 4.3 (a) Segment 1 (S1) as part of the overall protocol. (b) Expanded view of Segment 1. The chamber temperature is given as a solid line. If the time for equilibration is sufficient and the sample equilibrates with the surrounding chamber, then it will track with the initial portion of Segment 2 (S2) (dotted line). If the time of equilibration is insufficient, the sample temperature will not track with the chamber temperature (dotted and dashed lines).
Figure 4.4 The cell remains in the unfrozen liquid between adjacent ice crystals. As the temperature decreases, the size of the gap between adjacent ice crystals decreases and the concentration of the unfrozen solution increases.
Figure 4.5 Expanded views of Segments 1 and 2 for the controlled‐rate protocol demonstrating the three different methods of nucleation: (a) uncontrolled nucleation, (b) manual nucleation, and (c) automatic nucleation. The solid line represents the temperature of the cooling chamber. The dashed line represents the temperature of the sample.
Figure 4.6 Identifying the temperature at which ice forms in the extracellular solution. The sample cools to a temperature below the melting temperature. The release of the latent heat of fusion results in an increase in the temperature. The point at which the temperature increase starts can be considered the
T
nuc
.
Chapter 05
Figure 5.1 (a) Schematic representation of the transfer of a sample from a storage Dewar to a transport carrier; (b) temperature excursion of the sample (picked vial) being removed from the storage unit and placed in the transit carrier (solid line) as well as the “innocent vial” that was in the same rack and box as the picked vial (dashed line).
Chapter 06
Figure 6.1 Typical thawing curve for a sample. Initial warming rates will be high and the warming rate will diminish as the sample approaches the melting temperature of the solution. The duration of the plateau associated with the latent enthalpy of melting will vary with the volume of the solution and the heat transfer rate of the sample.
Figure 6.2 (a) Normalized cell volume as a function of time for cells washed in an isotonic saline solution. Water enters the cell to balance the high chemical potential followed by slower efflux of penetrating cryoprotective agent; (b) normalized cell volume as a function of time for cells washed in an engineered wash solution. The solution is slightly hypertonic and contains molecules that exert an osmotic force but are too large to penetrate the cell. Water influx is diminished when compared to part (a). Changes in cell volume are reduced.
Chapter 07
Figure 7.1 Pre‐freeze and post‐thaw assessment of a given sample. In this diagram, cells that are clear are viable and cells that are shaded grey are nonviable. The total number of cells in panel (a) is 100 and 93% of the cells are viable pre‐freeze. The total number of cells in panel (b) is 71 and 93% of the cells are viable post‐thaw. If cell losses are not included, the recovery of cells is artificially biased upward.
Figure 7.2 Variation in cell number with time post‐thaw. Cells capable of proliferation will experience an increase in cell number after the rate of proliferation exceeds the rate of cell death. Cells that do not proliferate will initially die off and then cell numbers will plateau (dashed line).
Chapter 08
Figure 8.1 Flow chart of the DE algorithm. Black boxes represent DE algorithm steps and grey boxes represent experimental steps. The algorithm produces a vector in generation 0 that randomly spans the parameter space and a trial population generation 1 that is based on mutation of generation 0. Both of these vectors are tested experimentally and the corresponding live cell recovery is input into the DE algorithm, resulting in an emergent population (pop) and the process is repeated.
Oocyte Vitrification and Warming
Figure 1 Vitrification freezing diagram.
Figure 2 Vitrification thawing diagram.
Guide
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Preservation of Cells
A Practical Manual
Allison Hubel
This edition first published 2018© 2018 John Wiley & Sons, Inc.
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Library of Congress Cataloguing‐in‐Publication Data
Names: Hubel, Allison, author.Title: Preservation of cells : a practical manual / by Allison Hubel.Description: Hoboken, NJ : John Wiley & Sons, 2018. | Includes index.Identifiers: LCCN 2017042796 | ISBN 9781118989845 (cloth) | ISBN 9781118989876 (epub) | ISBN 9781118989852 (epdf)Subjects: LCSH: Cells–Cryopreservation–Handbooks, manuals, etc. | Cells–Preservation–Handbooks, manuals, etc.Classification: LCC QH324.9.C7 H83 2018 | DDC 571.6/34–dc23LC record available at https://lccn.loc.gov/2017042796
Cover Design and Image: Created by Alex Brown
Preface
Preservation of cells is performed thousands of times every day by technicians across the world. For the vast majority of those people, the process is shrouded in mystery. The reasons for specific steps in the protocol are not clear. If there are problems with the protocol, the manner by which the problems occur is also unclear. There are numerous books that contain cryopreservation protocols for specific cell types or describe scientific understanding of the field or current research in the field. I could not find a book, which helped many, that uses preservation to develop new protocols or improve existing protocols. The objective of this book is to describe step by step the development of a preservation protocol and the scientific principles behind these steps. At the end of every chapter (with the exception of Chapter 8), specific links are made between scientific principles and the manner by which those principles are put into action.
Cells are being used for an increasing number of downstream uses. The applications of cells include the production of therapeutic proteins, viral vaccines, and antibodies. Cells are being used as biomarkers for health and disease and even for the treatment of disease. These applications are described in Chapter 1 and the role of preservation in the clinical and commercial applications of cells is also described. Different modes of preservation are also described. Different applications of cells may involve hypothermic storage of cells, cryopreservation, or vitrification.
Cells undergo a variety of processes prior to cryopreservation. These processes can include digestion from a tissue, selection of subpopulations, genetic modification, culture, and so forth. Chapter 2 describes these processes in more detail and the resulting nutrient deprivation, shear, or other sublethal stresses that can influence post‐thaw recovery of cells. Strategies to minimize stress or cell losses from pre‐freezing processes are described. Newly developed gene‐editing technologies are described. The ability to edit cells may lead to both new challenges and opportunities for cell preservation. It is likely that insertion or deletion of specific genes may influence the ability of a cell to survive the stresses of freezing and thawing. Gene editing may also enable us to understand the role of specific genes in enhancing survival of certain cells.
Characterization of the cells being cryopreserved is also critical. Chapter 2 also describes standard testing of cells prior to cryopreservation, including identification of cells, testing for adventitious agents, and other types of testing such as genetic stability. Misidentification of cells is a serious concern in the area of life science research, and there is increasing emphasis on proper identification of both primary cells and cell lines being used.
Cryopreservation uses specialized solutions designed to help the cells survive the stresses of freezing and thawing. These solutions are not physiological. Chapter 3 describes formulation of a solution and development of methods to introduce the solution. A listing of molecules that have been used to stabilize cells during freezing is given in the chapter. The development of new solutions is an ongoing area of research, but these solutions will still, more than likely, need to be introduced and removed prior to downstream use.
The influence of cooling rate on the post‐thaw survival of cells has been known for almost 50 years. Chapter 4 describes the cooling process and the manner by which cells are typically frozen (i.e., controlled‐rate freezing, passive freezing, or vitrification). Designing a cooling protocol and methods of verifying the protocol are also described. The importance of temperature and its variation in time during freezing also suggests that the method of measuring temperature independently during freezing is valuable, in particular during the development of methods.
Cells that have been cryopreserved may be stored for weeks, months, or even decades. Chapter 5 describes the scientific basis for storage of cells in liquid nitrogen, fundamentals of repository design, safe operation of a repository, and shipping of samples from the site of storage to the site of use. The factors that influence stability of samples in storage are also discussed. Transient warming events (TWEs) are being documented for a wide variety of biospecimens in storage and our understanding of the influence of TWEs on sample quality continues to grow. It is likely that new technologies can be used to eliminate this issue and improve stability of samples in storage.
The purpose of preservation is to maintain the critical biological properties for downstream use of the cells; downstream use of the cells requires thawing of the sample. The thawing process and the manner by which you can characterize your average thawing rate and improve the thawing process are described in Chapter 6. Newly developed controlled‐thawing technology will provide the opportunity to improve the consistency of thawing. In addition, new types of thawing protocols may be developed in the future, which will improve overall outcome.
It is common for cells to be washed post‐thaw and prior to downstream applications. Methods and technologies for washing cells post‐thaw are also described in Chapter 7. For vitrification solutions or cells that are sensitive to osmotic stress, strategies for improved methods of washing are described.
Effective methods of preserving cells cannot take place without effective methods of characterizing post‐thaw recovery. Post‐thaw assessment of cells is a very common area for errors and poor practices. The need for post‐thaw function of cells, in particular for cells used therapeutically, implies that post‐thaw function is critical and methods of assessment must be meaningful. Different methods of post‐thaw assessment are described. Specific recommendations are given to reduce bias and errors.
The traditional method of optimizing a preservation protocol typically involves empirical testing (i.e., varying composition and cooling rate and measuring post‐thaw viability). Chapter 8 describes the use of a differential evolution algorithm to reduce the experimentation required to optimize composition, cooling rate, and other processing parameters for cells. As there is pressure to develop fit‐for‐purpose protocols, new methods to streamline the cost and time required for optimization are critical and this approach has the potential to be transformative.
The growth in clinical and commercial applications of preservation brings with it the need for consistency and reproducibility. Chapter 1 describes common errors in preservation practices that lead to poor reproducibility (both poor outcome and high variability). Subsequent chapters describe common pitfalls that can negatively affect reproducibility of the preservation process and strategies to avoid those pitfalls and improve reproducibility.
For all the reasons listed previously, conventional methods of cryopreservation may no longer be appropriate for a given cell type or a given application. Drift in preservation protocols is also common. As director of the Biopreservation Core Resource, I get phone calls and emails from organizations that start having problems with existing protocols. The overall goal of this book is to help both groups. The process of developing a new protocol or understanding problems with an existing protocol can be approached logically and systematically based on scientific principles. It is my hope that this book will enable more organization to achieve improved post‐thaw recoveries and consistency.
Acknowledgments
This book grew out of a short course, “Preservation of Cellular Therapies,” offered at the University of Minnesota for well over a decade. Dave McKenna, Fran Rabe, and Diane Kadidlo from the Molecular Cellular Therapy Program at the University of Minnesota helped me understand the complexities of preserving cells in a clinical context, regulatory issues, and the importance of quality systems in preservation.
Ian Pope from Brooks Life Sciences helped structure the chapter on storage and his critical reading of the manuscript helped me understand the importance of directly linking the scientific principles to actual practice. Amy Skubitz brought her decades of experience in biobanking and her critical eye to the manuscript as well. Her insights made the book far better and for that I am grateful. Alex Brown contributed the wonderful illustrations and his artistic eye to the project.
I would also like to thank all of the protocol contributors: Leah A. Marquez‐Curtis, A. Billal Sultani, Locksley E. McGann, and Janet A. W. Elliott from the University of Alberta; Rohit Gupta and Holden Maeker from Stanford University; Melany Lopez and Ali Eroglu from the Medical College of Georgia; Andreas Sputtek from Medical Laboratory Bremen; Jeffrey Boldt from Community Health Network; and Jerome Ritz, Sara Nikiforow; and Mary Ann Kelley from Dana Farber Cancer Institute, Boston, MA, USA. All of these protocols are excellent examples of putting the scientific principles described in the book into practice.
Finally, thank you to John Martin Hansen, my husband, for his patience and support through this process.
Nomenclature
∆
T
Undercooling of the cells
AABB
American Association of Blood Banks
AATB
American Association of Tissue Banks
ALP
Alkaline phosphatase
B
Cooling rate
CR
Crossover rate
DMSO
Dimethylsulfoxide
DOT
Department of Transportation
DSC
Differential Scanning Calorimetry
ES cells
Embryonic stem cells
F
Weighting
FACT
Foundation for the Accreditation of Cellular Therapy
FDA
Food and Drug Administration
GMP
Good Manufacturing Practices
HIV
Human immunodeficiency virus
HSC
Hematopoietic stem cells
IATA
International Air Transport Association
ICAO
International Civil Aviation Organization
iPS cells
Induced pluripotent stem cells
ISBER
International Society for Biological and Environmental Repositories
k
Interaction parameter
LN
2
Liquid nitrogen
MSC
Mesenchymal stromal cells
NP
Generation size
PBPC
Peripheral blood progenitor cells
R
Rate constant
RBCs
Red blood cells
RNA A
Ribonuclease A
T
ext
Temperature at which ice forms in the extracellular solution
T
final
Final temperature
t
final
Final time
T
g
Glass transition temperature
T
initial
Initial temperature
t
initial
Initial time
T
m
Melting temperature
T
nuc
Nucleation temperature
UCB
Umbilical cord blood
x
Weight fraction
1Introduction
Mammalian Cells: Modern Workhorses
Mammalian cells have become modern workhorses capable of a variety of applications:
Production of therapeutic proteins, viral vaccines, and antibodies
Therapeutic agents (cell therapy or regenerative medicine applications)
Biomarkers for health or disease
In vitro
models (i.e., replacement for animal testing)
These applications represent significant economic sectors and have a major impact on human health.
Products from Cells
The production of human tissue plasminogen activator (tPA) in the mid‐1980s became the first therapeutic protein derived from mammalian cells to be made available commercially (see Wurm (2004) for review). Erythropoietin, human growth hormone, interferon, human insulin, and a variety of other proteins are produced from mammalian cells and are used therapeutically. Since the production of tPA, roughly 100 recombinant protein therapeutics have been approved by the FDA (Lai, Yang, and Ng 2013).
In addition to therapeutic proteins, vaccines are commonly produced from mammalian cells. For example, polio, hepatitis B, measles, and mumps vaccines are all produced via mammalian cell culture. New vaccines currently under development (human immunodeficiency virus (HIV), Ebola, new influenza strains) are also based on mammalian cell cultures.
Antibodies are used for a wide range of application (both in vitro and in vivo) (Waldmann 1991). The diagnosis of disease using antibodies is an extremely common application. Enzyme‐linked immunoabsorbent assays, flow cytometry, immunohistochemistry, and radioimmunoassays all use monoclonal antibodies produced by mammalian cells. Clinical applications of antibodies have historically included treatment of viral infections. Immunotherapy for the treatment of cancer using antibodies has grown rapidly (Weiner, Surana, and Wang 2010). Antibodies are now being used to selectively target tumors. The ability to accomplish targeting of tumors in humans resulted directly from advances in antibody engineering that enabled production of chimeric, humanized, or fully human monoclonal antibodies. Antibodies have also been conjugated to drugs or radioactive isotopes and used as target therapies. Currently, more than 10 different antibodies are approved for the treatment of cancer. All of these antibodies are produced using mammalian cells.
Cells as Therapeutic Agents
Cell therapy began in the 1970s with bone marrow transplantation for the treatment of blood and immune disorders. The uses of hematopoietic stem cells (HSCs) have grown since then, and clinical studies are expanding the use of HSCs to include a wider range of diseases and indications. Over 430 clinical trials using HSCs are underway, targeting the immune system, cardiovascular diseases, neurological disorders, vascular disease, lung disease, and HIV, to name a few (Li, Atkins, and Bubela 2014). The discovery that HSCs can be found in the peripheral blood (if a patient has been given a drug to mobilize HSCs to circulate in the peripheral blood), and umbilical cord blood (UCB) has enabled growth in the use of this cell type therapeutically because these cells can be harvested using nonsurgical methods.
Stromal cells present in the bone marrow microenvironment have also been studied for therapeutic uses. Mesenchymal stromal cells (MSCs) provide important support for hematopoiesis in the bone marrow microenvironment. MSCs can also be isolated from adipose tissue and UCB. Initial studies using MSCs focused on regenerative medicine applications and the use of these cells to form bone or cartilage. Subsequent studies demonstrated that the principal actions of MSCs are immunomodulatory and trophic (Caplan and Correa 2011). The diverse capabilities of this cell type and the ability to access the cells easily (from bone marrow aspirate, UCB, or small biopsy of adipose tissue) have facilitated clinical use of these cells. Clinical trials use MSCs to treat orthopedic disorders, cardiovascular disease, autoimmune disease, neurological disorders, and more (Sharma et al. 2014). MSCs are immune privileged, and as a result cells from allogeneic donors can be given therapeutically.
Biomarkers for Health or Disease
Most people have had a vial of blood drawn at the doctor’s office. Blood counts are performed and can indicate the presence of anemia, infection, or other medical conditions. The cells in whole blood are typically not stored for an extended period of time but counted shortly after collection. Other cell‐based assays include quantification of circulating tumor cells as a marker of tumor burden in cancer (Plaks, Koopman, and Werb 2013). Flow cytometry of lymphocyte subsets is also used to monitor immune status for AIDs patients and others with immune disorders (Shapiro 2005).
Mass cytometry is a recently developed experimental technique in which heavy metals are tagged to antibodies and those labels are attached to cells. The cells are then analyzed using a time‐of‐flight mass spectrometer. This approach avoids the limitations intrinsic to conventional flow cytometry. This capability enables labeling of heterogeneity in a cell population as well as single cell analysis of multiple markers (Spitzer and Nolan 2016). Other single cell “omic” (genomic, proteomic, and metabolomic) technologies are in development and could represent powerful new diagnostics. It is likely that cells will continue to grow in importance for diagnostics.
In Vitro Models
For many years, isolated hepatocytes have been used for screening of drugs. The development of induced pluripotent stem cells (iPS cells) (Yu et al. 2007) and the ability of these cells to differentiate into a variety of cell types have enabled the testing of drugs in a wider variety of cell types. For example, the cardiotoxicity of a drug can be evaluated using cardiomyocytes differentiated from iPS cells (Avior, Sagi, and Benvenisty 2016).
Three‐dimensional cultures of multiple cell types in a microfluidic environment that permits continuous perfusion of the cells can be used to model the physiological function of an organ or tissue. Also known as “Organ‐on‐a‐chip,” these cultures are also being used to screen drugs and understand the effect of specific drugs on organ systems (Bhatia and Ingber 2014).
Organ‐on‐a‐chip and iPS cells are also used for modeling of disease. Cells from donors with a given disorder can be transformed into iPS cells and then differentiated into disease‐specific cells that can be used for understanding disease development as well as drug/treatment screening (Avior, Sagi, and Benvenisty 2016). Patient‐derived iPS cells have been developed for a wide range of diseases, including neurological disorders and cardiovascular disease.
Clearly, mammalian cells are critical for biomedical research, diagnosis of disease and its treatment. These cells must be functional and available at the site and at the time of downstream use.
Bridging the Gap
It is common for cells to be collected or cultured in one location and used at a later time and in another location (Figure 1.1). The critical biological properties of the cells must be preserved in order for the cells to be useful for the downstream applications.
Figure 1.1 Minding the gap: preservation is used to maintain the critical biological properties of the cells when they are needed at a later time or in a different location.
Cells that are to be used therapeutically must be properly stored to meet the safety and quality control testing prior to release (and use) of the cells. Preserving cells permits coordination of the therapy with patient‐care regimes (i.e., the cells are ready when the patient is ready). Cells used therapeutically are produced in specialized facilities. The ability to preserve cells will help manage staffing requirements for cell‐processing facilities (i.e., the cells can be processed independent of patient availability) and control the inventory of the therapy.
UCB banking is a good example of the need for preservation. Babies are born at unpredictable times and at a variety of locations. The UCB must be collected immediately after birth. The UCB is typically shipped directly to a cord blood–processing facility where the sample is depleted of red blood cells, cryopreserved, and stored. The unit is stored until it is needed (typically years later), and it is common for the unit to be used in a third location. The UCB unit is useful only if the critical biological properties have been preserved. The genetic diversity of births implies that the preservation of UCB, in particular, can improve the genetic diversity of cells available for therapeutic applications.
One option for preserving a cell may be keeping the cells in culture until they are ready for use. Certain cell types do not retain their critical biological characteristics if they are cultured outside the body for extended periods of time. Other cell types that have defined genetics (e.g., mammalian cells used for the production of recombinant proteins) may experience genetic drift with long‐term culture. Finally, long‐term culture can be very expensive. For many of the cells described above, the downstream use of the cells may be months or weeks after the cells are collected. As a result, cryopreservation is a useful tool for preserving the critical biological properties of the cell for extended periods of time.
The Preservation Toolkit
There are a variety of methods that can be used to preserve the cell depending upon its downstream application (Figure 1.2). Multiple modes of preservation may also be used in a given protocol. Using the example given above, UCB is collected in the delivery room, but it is processed in specialized facilities for cord blood banking. The cells are shipped using short‐term liquid storage to a centralized facility where they are cryopreserved. Therefore, this particular application uses liquid storage followed by cryopreservation. Each mode has its advantages and limitations.
Figure 1.2 Preservation toolkit for cells. Preservation methods should be fit‐for‐purpose. The mode and duration of storage should be appropriate for the given application.
Hypothermic Storage
Hypothermic storage is commonly used for short‐term (hours to days) storage of cells. Cells are taken as collected or resuspended in a storage solution and typically refrigerated or placed on ice (hence the term hypothermic storage). Reducing the temperature of cells reduces their metabolic activity, enabling the cells to be shipped or transported. It is noteworthy that when refrigerated, the cells are still consuming oxygen and other nutrients. Storage conditions (e.g., temperature, time, and duration) must be chosen such that the cells are functional at the completion of the liquid storage.
Red blood cells (RBCs) are the most common cell type that is stored using this method. Red blood cells are separated from whole blood and resuspended into a specially designed short‐term storage solution (e.g., AS‐3) and refrigerated. RBCs can be stored up to 42 days in this solution. Since they do not replicate, the ability of RBCs to be stored for this period of time in liquid storage reflects the unique biology of this cell type. Most nucleated cells cannot be stored for this period of time, even when refrigerated.
At reduced temperatures, ion pumps in the cell membrane do not function properly and there is a change in ionic concentration inside the cell. Low temperatures also influence mitochondrial activity (e.g., reduced ATP production and diminished free radical scavenging). It has been hypothesized that damage during hypothermic storage results from reactive oxidative species (Rauen and de Groot 2002). Even with specially designed solutions, liquid storage of nucleated cells before significant losses occur is typically limited to short periods of time (less than 72 h).
Liquid storage can be used in combination with a cryopreservation protocol. For example, UCB is typically collected in the labor and delivery room and shipped to a cord blood bank. It is common for the cells to be chilled (on ice) but not frozen and then processed at the UCB bank within a short (approximately 24 h) period of time after collection. Studies have shown that improper liquid storage conditions can result in poor post‐thaw recovery of cord blood (Hubel et al. 2004).
Cryopreservation
The use of freezing to stabilize biological cells results from the need to control or stop degradative processes. Specifically, during freezing, liquid water is removed from the sample in the form of ice. Liquid water is a critical component in a variety of metabolic functions of the cell. Freezing of the water in the sample reduces the mobility of water molecules and therefore their ability to participate in reactions that could potentially degrade the cell.
All cells contain degradative enzymes (e.g., DNAses, proteases, etc.). The activity of these enzymes is a function of temperature. As the temperature is reduced, the activity of the enzymes decreases and there is a threshold at which the enzyme is no longer active and therefore cannot participate in degradation of the cell. Activity of a limited number of enzymes has been measured at freezing temperatures, and these studies suggest that the threshold temperatures for activity may be approximately −90°C (Hubel, Spindler, and Skubitz 2014).
Cells that have been successfully frozen can be stored for longer periods of time (years to decades), thereby extending the shelf‐life of the product. The process requires maintaining a cold chain (a temperature controlled supply chain that keeps the product at a desired low temperature) during freezing, storage, and transport.
Vitrification
As described above, water is removed from the sample in the form of ice during conventional cryopreservation. As a result, significant changes in the chemical and mechanical environment of the cells take place. When water is removed in the form of ice, the remaining unfrozen solution contains high concentration of solutes. The cells are sequestered in gaps between adjacent ice crystals and are therefore subjected to high concentrations and mechanical forces as freezing progresses.
One approach to preserving the cells at low temperatures, known as vitrification, involves avoiding the formation of ice. Typically, high concentrations of cryoprotective agents are used to suppress ice formation during freezing and, in fact, concentrations can be considerably higher than used in conventional cryopreservation. These solutions are not physiological and, typically, the process of adding these solutions to a biological system is called introduction of the solution. Introduction and removal of these high‐concentration solutions may be elaborate and require multiple steps for introduction. For example, if the final concentration of the solution is 4 M and the cell cannot tolerate introduction of the solution in a single step, the cell may be introduced to a solution whose concentration is intermediate (say 1.2 M) followed by introduction to the solution at its final concentration (4 M). The process is designed to reduce cell losses resulting from osmotic stress (see Chapter 2
