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Cell bank

A cell bank is a repository of standardized, reproducible cell lines—such as primary cells, immortalized cell lines, or stem cells—derived from a single source and preserved under controlled conditions, typically through cryopreservation, to maintain their viability, genetic stability, and functionality for applications in biomedical research and biopharmaceutical production.^[1]^[2]^[3] Cell banks play a critical role in biotechnology by providing a consistent, reliable source of cells that ensures reproducibility in experiments, supports large-scale manufacturing of biologics like vaccines and monoclonal antibodies, and facilitates advancements in drug discovery, regenerative medicine, cancer research, virology, and tissue engineering.^[1]^[2]^[4] The process begins with the establishment of a master cell bank (MCB), which consists of a well-characterized initial stock of cells frozen in multiple aliquots after rigorous testing for identity, purity, potency, and contaminants such as mycoplasma or adventitious agents.^[1]^[2] From the MCB, a working cell bank (WCB) is derived under defined conditions to generate genetically identical cells for routine use in production or assays, minimizing variability and extending the lifespan of the original cell line.^[1]^[2]^[5] Cryopreservation is the cornerstone of cell banking, involving the suspension of cells in a protective medium with cryoprotectants like dimethyl sulfoxide (DMSO) before rapid freezing to -196°C in liquid nitrogen vapor phase storage systems, which prevents ice crystal formation and cellular damage.^[1]^[4] Storage occurs in secure, monitored biorepositories compliant with good manufacturing practices (GMP), with regular viability assessments and quality controls to ensure long-term stability, often spanning decades.^[2]^[4] Regulatory standards, such as the ICH Q5D guideline for cell line derivation and ISO 20387 for biobanking, along with region-specific regulations like the FDA's points to consider for cell substrates and the EU Directive 2004/23/EC for human cells, govern cell banking to uphold safety, traceability, and ethical considerations, such as material transfer agreements (MTAs) for proprietary lines.^[1]^[6]^[7]^[8] These frameworks are essential for clinical and commercial applications, where cell banks from authoritative institutions like the American Type Culture Collection (ATCC) provide authenticated materials that underpin global research and therapeutic development.^[2]

Overview

Definition and Purpose

A cell bank is a systematically organized collection of cryopreserved viable cells derived from a single cell line or primary cell source, maintained under controlled conditions to preserve their long-term stability and availability.^[3] This repository ensures that the cells retain their genetic and functional integrity, serving as a foundational resource in biotechnology.^[4] The primary purpose of a cell bank is to provide a reliable, consistent supply of well-characterized cells for reproducible scientific experiments, biopharmaceutical manufacturing, and therapeutic development, thereby minimizing variability and contamination risks.^[9] By maintaining genetic stability over extended periods, cell banks support applications such as vaccine production, gene therapy, and regenerative medicine, where uniformity in cell populations is critical for safety and efficacy.^[10] This approach also facilitates the prevention of genetic drift or adventitious agent introduction that could occur in continuous cultures. Basic components of a cell bank include comprehensive documentation of the cells' origin, such as derivation history and donor screening if applicable, alongside characterization data encompassing viability assessments, genetic profiling, and purity testing.^[8] Storage protocols, tailored to the specific cell line, are also integral to ensure traceability and compliance with regulatory standards.^[11]

Importance and Applications

Cell banks play a pivotal role in biotechnology by enabling standardization through the provision of uniform cell populations that ensure consistent experimental and production conditions across studies and manufacturing runs. This uniformity is essential for reproducible results in research, as it minimizes discrepancies arising from differences in cell sources or handling protocols. ^[1] By preserving cells at defined passage levels, cell banks reduce variability in research outcomes, allowing scientists to focus on biological insights rather than artifacts from inconsistent materials. ^[12] Furthermore, they support scalability in biopharmaceutical production by serving as a reliable reservoir for expanding cell cultures to meet large-scale demands, such as in therapeutic manufacturing. ^[13] In addition to standardization and scalability, cell banks act as a critical backup mechanism against the loss of valuable cell lines due to contamination, equipment failure, or genetic drift over time. This preservation strategy facilitates rapid recovery and continuity in operations, mitigating disruptions that could otherwise delay research or production timelines. ^[14] Cell banks also address key challenges in biotechnology, such as batch-to-batch variability, by enabling the use of well-characterized, frozen aliquots that maintain genetic and phenotypic stability. ^[15] They further promote ethical sourcing by documenting the origins of cells from donors or immortalized lines, ensuring compliance with consent and transparency standards that uphold scientific integrity. ^[1] Applications of cell banks span diverse fields, particularly in vaccine development, where they provide stable substrates for propagating viruses like influenza, allowing for efficient seasonal and pandemic vaccine production. ^[16] For instance, during the COVID-19 pandemic, cell banks supported the rapid scaling of vaccine manufacturing using established cell lines to meet global demands. ^[17] In monoclonal antibody production, cell banks derived from optimized clones, such as Chinese hamster ovary (CHO) cells, ensure consistent high-yield expression for therapeutics targeting diseases like cancer and autoimmune disorders. ^[18] Cell banks are equally vital in regenerative medicine, where they store stem cell lines for therapies aimed at repairing damaged tissues, such as in cardiac or neurological conditions. ^[19] In genetic engineering for CAR-T cell therapies, banked T cells from healthy donors enable the creation of off-the-shelf products that enhance accessibility and reduce manufacturing time for treating refractory cancers. ^[20] Overall, these applications underscore the role of cell banks in bolstering supply chain reliability for clinical trials, ensuring a steady supply of quality-controlled cells to advance therapeutic development without interruptions. ^[8]

Types

Master Cell Bank

The master cell bank (MCB) is defined as an aliquot of a single pool of cells derived from a selected cell clone or population, prepared under controlled conditions, dispensed into multiple containers, and stored under specified conditions to serve as the primary source for deriving all subsequent working cell banks (WCBs).^[21] It acts as the foundational, fully characterized repository ensuring consistency in biotechnological and biological product manufacturing, such as vaccines and cell therapies, by providing a uniform starting material for production lots.^[10] This role minimizes variability and supports regulatory compliance in processes like virus propagation and biopharmaceutical development.^[21] The creation of an MCB involves initial cell selection from a research or preliminary bank, followed by controlled expansion in culture, pooling of cells, and cryopreservation using cryoprotectants in sealed containers, typically stored in liquid nitrogen vapor phase.^[21] At establishment, extensive testing is conducted to verify identity through methods like DNA analysis or isoenzyme assays, purity via bioburden, mycoplasma, and adventitious agent screening, potency by assessing growth and production consistency, and sterility according to pharmacopeial standards.^[11] To reduce handling risks and contamination potential, only a limited number of aliquots—often exceeding 200 vials—are produced, with each vial containing sufficient cells for downstream applications.^[11] Designed for long-term archival storage at temperatures below -130°C, the MCB is not intended for direct use in routine production but rather to generate WCBs, thereby preserving the original cell line's integrity over decades.^[21] Comprehensive genetic and phenotypic profiling, including karyotyping, short tandem repeat analysis, and stability studies, accompanies the MCB to document its characteristics and enable traceability.^[10] In contrast to WCBs, which are derived from the MCB for operational use, the MCB emphasizes initial, exhaustive characterization to safeguard the production lineage.^[21]

Working Cell Bank

The working cell bank (WCB) is a collection of cryopreserved cells derived from the master cell bank (MCB) through controlled expansion and serves as the primary source for initiating routine production processes in biomanufacturing, thereby preserving the MCB for reference and replacement purposes.^[21] Unlike the MCB, the WCB is designed for practical, expendable use in generating working cultures for direct manufacturing of biologics, such as vaccines or recombinant proteins, ensuring a consistent and stable cell population without depleting the original stock.^[8] This derivative role allows the WCB to support repeated experimental or production runs while maintaining genetic and phenotypic uniformity inherited from the validated MCB.^[21] The creation of a WCB involves thawing one or more vials from the MCB, expanding the cells through limited serial subcultures under defined conditions to achieve the desired volume, pooling the expanded cells, and then aliquoting them into multiple sterile vials with a cryopreservation medium before freezing, typically in liquid nitrogen vapor phase.^[8] This process emphasizes controlled propagation to produce sufficient vials—often hundreds—for operational demands, with fewer characterization tests required compared to the MCB, as it inherits most validated properties; testing is limited to confirming identity, purity (e.g., absence of adventitious agents introduced during expansion), and basic viability, performed once per WCB batch.^[21] Such abbreviated qualification ensures efficiency while relying on the MCB's extensive prior validation to mitigate risks.^[8] In usage, the WCB supports ongoing bioprocessing by providing cells to inoculate production bioreactors for generating drug substance lots, as seen in the manufacture of monoclonal antibodies or viral vectors, where multiple vials may be pooled to start a single run.^[21] To monitor stability, passage numbers or population doubling levels are tracked from the initial MCB thaw through WCB-derived cultures, ensuring they do not exceed predefined limits that could compromise product consistency or yield.^[8] This tracking is critical for maintaining process reliability across production campaigns, with new WCBs generated from the MCB as needed to replenish stocks.^[21]

Preparation

Cell Selection and Expansion

Cell selection for banking begins with evaluating the origin and type of the cell line, which may include primary cells derived directly from tissues, immortalized cell lines engineered for indefinite propagation, or stem cells such as mesenchymal or induced pluripotent stem cells, depending on the intended therapeutic or research application.^[22] Criteria emphasize high post-thaw viability, typically targeting greater than 90% to ensure reliable recovery and functionality upon use, alongside confirmation of suitability through assessments of growth characteristics like in vitro proliferation rates and adherence to monolayer or suspension culture formats.^[23] Essential authentication methods include short tandem repeat (STR) profiling for genetic identity verification in human cell lines and karyotyping or cytogenetic analysis to detect chromosomal abnormalities, ensuring the cells match the documented source and maintain genetic stability.^[24]^[8] Absence of contaminants is a core prerequisite, with rigorous testing required for sterility, mycoplasma, and adventitious agents such as viruses, achieved through documented sourcing from screened donors or tissues and comprehensive qualification of the master cell bank (MCB).^[8] These selections prioritize genetic homogeneity via low-passage cultures and scalability potential, minimizing risks of phenotypic drift or heterogeneity that could compromise downstream applications.^[22] Once selected, cell expansion involves controlled culturing to generate sufficient biomass, often aiming for 10^8 to 10^9 cells total across multiple vials in the MCB, using static flasks for small-scale initial growth or dynamic bioreactors like stirred-tank or hollow-fiber systems for larger volumes to support uniform nutrient distribution and oxygenation.^[25] Protocols optimize culture media composition, incorporating basal nutrients, sera or serum alternatives, and specific growth factors (e.g., fibroblast growth factor for stem cells) to sustain proliferation while preserving phenotypic markers such as marker expression and differentiation potential.^[26] Passage limits are strictly enforced, typically 10-20 subcultures from the original source, to avoid genetic instability or loss of functionality, with monitoring of doubling times and morphology throughout to confirm homogeneity before proceeding to banking.^[27] This process ensures the expanded population remains representative of the selected cells, facilitating scalable production without introducing variability.^[28]

Freezing Methods

Cryopreservation is a critical step in establishing cell banks, where expanded cell populations are preserved in a viable state for long-term storage following initial culturing and selection. This process involves the controlled dehydration and cooling of cells to prevent damage from ice formation, ensuring high recovery rates upon future use. Standard methods emphasize the use of cryoprotective agents and precise temperature control to maintain cellular integrity.^[29] Cryoprotectants are essential additives that mitigate intracellular ice crystal formation by altering water's physical properties, such as depressing the freezing point and promoting glass-like vitrification. Dimethyl sulfoxide (DMSO) is widely used at concentrations of 5-10% (v/v), while glycerol serves as an alternative at 5-15% (v/v), with selection depending on cell type sensitivity. These agents are introduced stepwise or gradually, often at 0-4°C over 10-30 minutes, to minimize osmotic shock and toxicity from rapid exposure.^[30]^[29]^[31] Freezing protocols typically begin with equilibration of the cell suspension in cryoprotectant-supplemented medium under sterile conditions, followed by dispensing into pre-labeled cryogenic vials (e.g., 1-2 mL volumes in 2-mL polypropylene vials) within a biosafety cabinet to prevent contamination. The vials are then subjected to slow-rate freezing using programmable freezers, cooling at -1 to -3°C per minute from room temperature down to -80°C, which allows extracellular ice formation and solute concentration without excessive intracellular damage. This is often followed by immediate transfer to liquid nitrogen vapor phase for deeper cooling.^[30]^[29]^[31] Optimization of these methods focuses on parameters like cell density, typically 10^6 to 10^7 viable cells per mL, to balance packing efficiency and post-thaw recovery, with tailored cooling curves monitored via temperature probes to achieve greater than 80% viability. Factors such as serum or albumin supplementation in the freezing medium further enhance protection, and protocols are validated for specific cell lines to ensure consistent outcomes.^[30]^[29]^[31]

Storage and Maintenance

Storage Conditions

Cell banks are typically stored in the vapor phase of liquid nitrogen at -196°C to maintain ultra-low temperatures while minimizing the risk of cross-contamination, which can occur in the liquid phase due to microbial transfer through the nitrogen.^[8] Alternatively, mechanical ultra-low temperature freezers operating between -130°C and -150°C provide a reliable option for long-term preservation, ensuring cell viability without the hazards associated with liquid nitrogen immersion.^[31] These conditions halt metabolic activity, preserving cellular integrity by preventing ice crystal reformation or enzymatic degradation.^[30] Storage containers consist of sterile, cryogenic-grade cryovials or ampoules, which are sealed to withstand extreme temperatures and placed in organized racks within the storage units for efficient access and tracking.^[31] Barcoding systems, often including 2D codes etched on vials, facilitate inventory management and traceability, ensuring precise identification during retrieval.^[8] To enhance security, cell banks are distributed across multiple backup sites, providing redundancy against equipment failure or environmental disasters.^[6] These storage protocols are designed for long-term stability, with studies demonstrating viability retention for up to 20 years or more in certain cryopreserved biological materials.^[32] Regular validation confirms that cells retain functionality upon thawing, supporting their use in manufacturing without degradation over extended periods.^[6]

Quality Control and Monitoring

Quality control and monitoring of cell banks during storage are essential to maintain cell integrity, prevent degradation, and ensure compliance with regulatory standards. These protocols involve ongoing oversight to detect any deviations that could compromise the bank's usability for biomanufacturing. Applied to master and working cell banks stored under controlled cryogenic conditions, such measures help preserve viability and purity over extended periods.^[8] Routine checks form the foundation of storage monitoring, encompassing periodic viability testing on retained vials to assess recovery rates post-thaw, typically using trypan blue exclusion or automated cell counters. Temperature logging is conducted continuously via data loggers in storage units like liquid nitrogen vapor-phase systems, with alarm systems triggered for deviations exceeding set thresholds (e.g., above -130°C) to enable immediate corrective actions. Additionally, mycoplasma screening is performed routinely on representative samples using methods such as agar and broth culture or PCR assays, while endotoxin testing via limulus amebocyte lysate ensures absence of bacterial contaminants that could arise from storage breaches.^[21]^[8]^[33] Stability assessments evaluate long-term cell bank performance through structured studies that track genetic and phenotypic consistency. These include genetic stability monitoring via next-generation sequencing to detect mutations or insertions in expression constructs, or flow cytometry to analyze marker expression and apoptosis levels in thawed samples. Such evaluations are conducted at intervals determined by risk assessment and stability data.^[34]^[35] Documentation is meticulously maintained to support traceability and regulatory audits, including certificates of analysis that detail viability, sterility, and identity results from each monitoring event. Chain-of-custody records track vial inventory, storage locations, and handling history, ensuring accountability from deposition to retrieval. These records are required in regulatory submissions and updated with each check to provide a verifiable history of bank integrity.^[8]^[21]

Usage

Thawing Procedures

Thawing procedures for cryopreserved cell banks involve retrieving vials from liquid nitrogen storage and rapidly warming them to revive cellular metabolism while minimizing damage from ice recrystallization and cryoprotectant toxicity.^[29] The standard method employs a 37°C water bath for quick thawing, typically lasting 1 to 2 minutes with gentle swirling until only a small ice crystal remains, to achieve warming rates of approximately 45–70°C per minute and limit exposure to potentially harmful intracellular ice formation.^[36]^[29] Immediately following thawing, the cell suspension is diluted in pre-warmed recovery medium to reduce osmotic shock from cryoprotectants like DMSO, which can otherwise cause cellular stress if exposure persists.^[37]^[29] Post-thaw handling prioritizes swift removal of cryoprotectants to further decrease toxicity risks. The diluted suspension is transferred dropwise into a centrifuge tube containing 9–10 mL of complete growth medium, then centrifuged at 125–200 × g for 5–10 minutes to pellet the cells and discard the supernatant containing the cryoprotectant.^[36]^[37] The cell pellet is gently resuspended in 1–2 mL of fresh growth medium and transferred to a culture vessel for incubation, with the entire process from thaw initiation to medium addition targeted to complete in under 5 minutes to optimize recovery.^[37] Best practices emphasize maintaining sterility throughout to prevent contamination, including performing all steps within a biosafety cabinet (laminar flow hood) under aseptic conditions, decontaminating the vial exterior with 70% ethanol prior to opening, and using pre-warmed media and equipment to avoid temperature shocks.^[36]^[37] Personal protective equipment, such as gloves and eye protection, is recommended due to the explosion risk from pressure buildup in cryovials stored in liquid phase.^[37]

Viability and Recovery Assessment

Viability assessment of thawed cells from a cell bank is a critical step to ensure the integrity and functionality of the cell population prior to use in manufacturing or research. This evaluation typically occurs immediately after thawing and focuses on quantifying the proportion of live cells using established assays. The trypan blue exclusion method, considered a gold standard, involves staining cells with trypan blue dye, which is excluded by intact cell membranes of viable cells, allowing manual or automated counting under a microscope to determine the percentage of live cells.^[38] Alternative assays, such as the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric test, measure metabolic activity in viable cells by assessing the reduction of MTT to formazan, providing a complementary readout of cell health that correlates with proliferation potential.^[39] For more detailed analysis, flow cytometry can detect apoptosis markers, such as annexin V binding to phosphatidylserine on early apoptotic cells or propidium iodide uptake in late apoptotic or necrotic cells, enabling precise quantification of cell death mechanisms.^[40] A common acceptance threshold for viability is greater than 70%, as lower levels may compromise downstream applications unless supported by additional data demonstrating no impact on safety or efficacy.^[41] Beyond initial viability, recovery assessment evaluates the cells' ability to resume normal function post-thaw, typically over a short culture period. Growth rate monitoring involves seeding thawed cells and measuring population doubling times or total cell yields over 24-48 hours using techniques like cell counting or automated imaging to confirm proliferative capacity comparable to pre-freeze conditions.^[42] For adherent cell types, attachment efficiency is assessed by calculating the percentage of cells that adhere to the culture substrate within 1-24 hours post-seeding, often via microscopy or dye-based quantification, as this metric strongly correlates with long-term colony formation and expansion success.^[43] Functional assays, such as generating proliferation curves through serial passaging or specific activity tests (e.g., marker expression via flow cytometry), further verify that recovered cells maintain their intended biological properties.^[44] Acceptance criteria for viability and recovery ensure that thawed cells from the master or working cell bank exhibit characteristics matching the original bank, including viability above 70%, stable growth kinetics, and absence of aberrant phenotypes, thereby validating the cryopreservation process's efficacy before proceeding to production or experimentation.^[8] This post-thaw evaluation directly follows the thawing procedure to confirm cell bank stability under storage conditions.^[41]

Regulatory Framework

Standards and Guidelines

Cell banks are governed by several key international and industry frameworks to ensure safety, quality, and consistency in their use for biotechnological and medicinal products. In the United States, the Food and Drug Administration (FDA) regulates biological products, including cell banks, through 21 CFR Part 610, which outlines general standards for potency, sterility, purity, and safety to prevent risks such as contamination or loss of viability.^[45] In the European Union, the European Medicines Agency (EMA) issues guidelines on human cell-based medicinal products, covering aspects of development, manufacturing, and quality control to support the authorization of advanced therapy medicinal products derived from cells.^[46] Additionally, the International Council for Harmonisation (ICH) Q5D guideline provides harmonized standards for the derivation and characterization of cell substrates, including requirements for genetic stability, identity verification, and adventitious agent testing in cell lines used for biotechnological production. Core requirements across these frameworks prioritize Good Manufacturing Practice (GMP) compliance to maintain controlled conditions during cell banking processes, ensuring reproducibility and minimizing variability.^[45] Traceability is mandated from the origin of the cells through all stages of banking, including documentation of donor information, processing steps, and storage details, to facilitate accountability and risk management.^[46] Risk-based approaches to contamination control are emphasized, involving assessments for microbial, viral, and genetic contaminants, with measures such as validated cleaning procedures and environmental monitoring integrated into operations. Global variations exist to address specific applications, such as the World Health Organization (WHO) recommendations for vaccine seed lots, which require the establishment of master and working seed lots with rigorous testing for identity, purity, and stability to support consistent vaccine manufacturing worldwide.^[47] For stem cell banks, the International Society for Cell & Gene Therapy (ISCT) endorses standards like ISO/TS 22859 and ISO 24651, focusing on biobanking protocols for mesenchymal stromal cells from sources such as Wharton's jelly and bone marrow, including detailed characterization for potency, immunophenotype, and sterility to enable therapeutic applications.^[48] These standards apply broadly to all cell bank types, promoting interoperability in global research and clinical use.

Validation and Testing Requirements

Validation and testing requirements for cell banks ensure the integrity, safety, and consistency of cells used in biopharmaceutical production and therapeutic applications. These processes are governed by international standards such as the International Council for Harmonisation (ICH) Q5D guideline, which outlines comprehensive testing to qualify master cell banks (MCBs) and working cell banks (WCBs). Testing is typically performed on aliquots of the bank or derived cultures, with the goal of verifying that the cells meet predefined specifications before release for use.^[21] The testing suite encompasses several key categories to assess the bank's quality attributes. Identity testing confirms the cells' origin and uniqueness, often employing DNA fingerprinting techniques such as restriction fragment length polymorphism (RFLP) or variable number tandem repeats (VNTR) analysis for metazoan cells, alongside cytogenetic methods like karyotyping to detect genetic stability.^[8]^[21] Purity evaluations address contamination risks, including sterility tests for bacteria and fungi compliant with pharmacopeial standards (e.g., Ph. Eur., USP), mycoplasma detection via PCR or culture methods, and screening for adventitious agents such as viruses through in vitro/in vivo assays and molecular techniques like PCR.^[8]^[21] Potency assessments verify the cells' functional capability, utilizing assays to confirm consistent product expression or recombinant DNA stability, particularly at the limits of in vitro cell age.^[21] Safety testing includes genotoxicity evaluations, such as limits on residual DNA (e.g., <10 ng per dose for parenteral products), and tumorigenicity studies in animal models like nude mice for continuous cell lines to rule out oncogenic potential.^[8] Validation steps are integral to establishing the reliability of cell bank processes and long-term usability. Process validation for freezing and thawing involves viability assessments post-reconstitution to ensure recovery rates meet acceptance criteria, which vary depending on cell type.^[8] Stability studies evaluate the bank's integrity over its shelf-life under defined storage conditions, testing at multiple time points to confirm no degradation in identity, purity, or potency.^[21] Comparability protocols are required for bank extensions or modifications, comparing the new bank's characteristics to the original via side-by-side testing to demonstrate equivalence in performance and safety.^[21] Documentation is a critical component to support traceability and regulatory compliance. Comprehensive batch records detail the cell history, banking procedures, and all testing outcomes, while audit trails maintain electronic or paper logs of all manipulations and decisions.^[8] Release criteria are predefined specifications for each quality attribute, such as viability thresholds and absence of contaminants, ensuring only qualified banks are approved for downstream use; failure to meet these triggers rejection and investigation per good manufacturing practices (GMP).^[21]

History

Early Development

The concept of cell banking emerged in the mid-20th century, driven by advances in cryopreservation techniques that enabled the long-term storage of viable cells for research and production purposes. In 1949, researchers Christopher Polge, Audrey U. Smith, and Alan S. Parkes accidentally discovered the cryoprotective properties of glycerol while attempting to preserve fowl spermatozoa, allowing revival after freezing to -79°C. This breakthrough was quickly extended to mammalian cells; by 1950, Polge and colleagues successfully cryopreserved bull spermatozoa using glycerol, demonstrating its efficacy for preserving cellular integrity during freeze-thaw cycles.^[49] Smith further applied these methods to human red blood cells, confirming glycerol's role in preventing haemolysis at low temperatures.^[50] These early cryopreservation innovations were pivotal for virus propagation in cell cultures, as they addressed the challenge of maintaining consistent cell lines for virological studies. In the 1950s, as tissue culture techniques advanced, researchers began storing frozen aliquots of cell lines to ensure reproducibility in experiments, marking the initial shift toward formalized cell banking systems.^[51] This was particularly relevant in virology, where stable cell substrates were needed to grow viruses without repeated isolation from animal tissues, reducing variability and contamination risks. The World Health Organization (WHO) formalized early guidelines for cell substrates in 1959, specifically for inactivated polio vaccine production using primary monkey kidney cells, emphasizing the need for standardized, testable cell sources.^[52] Initial applications of cell banking focused on virology for vaccine production, with short-term storage in research laboratories to support consistent virus yields. In the 1960s, the development of human diploid cell strains, such as WI-38 by Leonard Hayflick in 1962, revolutionized this field by providing safer alternatives to primary animal cells for polio virus propagation.^[53] WI-38 cells were banked in frozen vials to serve as a reliable source for vaccine manufacturing, enabling the production of polio vaccines free from simian viruses like SV40 that had contaminated earlier monkey kidney-based lots.^[54] These seed banks of cell lines facilitated the global rollout of polio vaccines, prioritizing controlled storage over ad hoc culturing. Foundational techniques relied on glycerol as the primary cryoprotectant, added to cell suspensions at concentrations of 10-15% before slow freezing to -70°C in mechanical freezers, followed by transfer to liquid nitrogen for indefinite storage.^[50] This basic protocol minimized ice crystal formation and osmotic damage, achieving post-thaw viabilities of 50-80% in early mammalian cell experiments. These methods laid the groundwork for broader applications in biotechnology.

Key Milestones and Advances

In the 1980s, the advent of recombinant DNA technology prompted the World Health Organization (WHO) to introduce the master cell bank (MCB) and working cell bank (WCB) system as a standardized framework for ensuring the consistency and safety of cell substrates used in producing biologicals, particularly recombinant proteins and vaccines.^[55] This two-tiered banking approach, formalized in WHO's 1987 requirements for animal cell substrates, minimized variability by deriving the WCB from the MCB and requiring extensive characterization for contaminants, genetic stability, and productivity prior to use.^[56] By the 1990s, the U.S. Food and Drug Administration (FDA) extended these principles through Good Manufacturing Practice (GMP) guidelines tailored to gene therapy, emphasizing rigorous validation of cell banks to support viral vector production and somatic cell modifications in clinical applications. The 2000s marked significant technological progress in cell banking, with the widespread adoption of automated controlled-rate freezing systems to enhance cryopreservation reproducibility and cell viability during large-scale biopharmaceutical production. These systems, such as programmable freezers from manufacturers like Planer and Thermo Fisher, allowed precise cooling rates (typically 1–2°C/min) to prevent ice crystal formation, reducing batch-to-batch variability in master and working banks for mammalian cell lines like CHO.^[35] Concurrently, advances in genomic stability assessment tools, including fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH), became integral to bank qualification, enabling detection of chromosomal aberrations and ensuring long-term genetic integrity over multiple passages.^[57] From the 2010s onward, cell banking evolved to support regenerative medicine, with the establishment of induced pluripotent stem cell (iPSC) banks facilitating personalized therapies by providing HLA-matched lines for disease modeling and transplantation. Institutions like Japan's Center for iPS Cell Research and Application (CiRA) developed GMP-compliant iPSC banks in the mid-2010s, storing thousands of lines to enable off-the-shelf applications in conditions such as Parkinson's disease.^[58] This period also saw the integration of CRISPR-Cas9-edited cell lines into banking protocols, allowing precise genomic modifications for therapeutic enhancement, such as knockouts for improved safety in CAR-T cells, with banks now routinely incorporating next-generation sequencing (NGS) for edit verification and stability monitoring.^[59] Cell banks played a pivotal role in the rapid response to the COVID-19 pandemic, exemplified by the accelerated establishment of bacterial cell banks (e.g., E. coli) for plasmid DNA production in mRNA vaccine manufacturing. In 2020, companies like Moderna and Pfizer/BioNTech quickly generated and qualified MCBs and WCBs under emergency GMP protocols to scale up plasmid yields, enabling billions of doses within months while maintaining quality through expedited sterility and sequence integrity testing.^[60]

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Cryopreservation: An Overview of Principles and Cell-Specific ... - NIH
Mar 24, 2021 · Glycerol or DMSO can be used as CPAs for long-term storage of islet cells at subzero temperatures (typically less than −100°C). Chandravanshi et ...
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Cryogenic Storage of Animal Cells - ATCC
DMSO (ATCC 4-X, 5 × 5 mL) is most often used at a concentration of 5 to 10% (v/v) in freezing media; the optimum concentration varies with the cell line.
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[PDF] gc-1044-cryopreservation-of-cells.pdf - US Pharmacopeia (USP)
Sep 27, 2018 · This chapter presents best practices for cryopreservation, maintenance, and use of a wide range of cells, cell therapy products, and cell banks ...
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[PDF] Q5D Guideline - ICH
Cell banks used to prepare gene therapy products or vaccines should follow the recommendations presented in this document. Some biological products, such as ...<|control11|><|separator|>
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https://www.atcc.org/resources/technical-documents/cryogenic-storage-of-animal-cells
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Genetic Stability Testing Using NGS and MAM: Optimizing CHO Cell ...
Sep 11, 2025 · Genetic stability testing is the process of verifying that a cell line retains its genetic composition over time, without accumulating mutations ...Missing: flow cytometry
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Use of flow-cytometric analysis to optimize cell banking strategies for ...
Here, we describe a novel strategy employing flow-cytometric (FC) analysis of Annexin V-stained cells for high-throughput characterization of cell banks. Our ...Missing: sequencing | Show results with:sequencing
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ATCC Animal Cell Culture Guide
The recovery of cryopreserved cells is straightforward: Cells are thawed rapidly in a water bath at 37°C, removed from the freeze-medium by gentle ...
[34]
Thawing Cells | Thermo Fisher Scientific - US
Remove the cryovial containing frozen cells from the liquid nitrogen storage and immediately place it into a 37°C water bath. · Quickly thaw the cells (<1 minute) ...
[35]
Guidelines for the use of flow cytometry and cell sorting in ...
Testing for cell viability. The “gold standard” of viability determination is the Trypan blue staining technique, which is carried out with a hemocytometer ...
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a comparison with MTT, NR, Trypan blue and cloning efficiency assays
In this paper we compared high-throughput real-time measurements through Electric Cell-substrate Impedance-Sensing (ECIS®) with endpoint measurements of ...
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Viable Mononuclear Cell Stability Study for Implementation in a ...
Our data suggest flow cytometry as an analytical method of choice, because of its higher specificity relative to Trypan Blue assessment and to CASY cell ...Missing: MTT | Show results with:MTT
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[PDF] Guidance for FDA Reviewers and Sponsors
You should establish minimum release criteria for viability. For somatic cellular therapies, the minimum acceptable viability specification is generally set at ...
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Post-Thaw Culture and Measurement of Total Cell Recovery Is ...
Jun 5, 2020 · We critically study post-thaw cellular outcomes upon addition of macromolecular cryoprotectants to provide unambiguous evidence that post-thaw culturing time ...Scheme 1 · Cryopreservation Of Cell... · Apoptosis Assay
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The effect of Me2SO overexposure during cryopreservation on HOS ...
Notably, a correlation of attachment to long term growth of 0.9704 was observed (Fig. 2E), indicating that a 1 h attachment efficiency assay reflected the ...The Effect Of Meso... · 2. Materials And Methods · 3. Results And Discussion
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Efficient and reproducible generation of human iPSC-derived ...
Jul 15, 2024 · Our freeze/thaw protocol yielded ~94% viable cells (trypan blue negative) after cryo-recovery, with plating efficiencies of ~51% for bCMs and ~ ...
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21 CFR Part 610 -- General Biological Products Standards - eCFR
No lot of any licensed product shall be released by the manufacturer prior to the completion of tests for conformity with standards applicable to such product.Title 21 · 21 CFR 610.12 -- Sterility. · 21 CFR 610.18 -- Cultures. · 610.39 – 610.48
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May 30, 2008 · This guideline replaces the Points to Consider on the Manufacture and Quality Control of Human. Somatic Cell Therapy Medicinal Products ...
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[PDF] Annex 2 - World Health Organization (WHO)
Annex 2 provides guidelines for independent lot release of vaccines, involving assessment before market release, and confirmation each lot meets specifications.
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Christopher Polge and Lionel Edward Aston Rowson's Experiments ...
Dec 19, 2017 · In 1950, Polge and Rowson began a series of two experiments in which they attempted to freeze mammalian sperm that can be used to successfully ...Missing: origins banking
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Cryopreservation: An emerging paradigm change - PMC
In 1949 Polge, Parks and Smith reported on the “chance” discovery of glycerol's cryoprotective function during their efforts to preserve avian spermatozoa in ...Missing: origins | Show results with:origins
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Cell bank system, establishment, and application in the virus ...
The two primary types of cell banks in biotechnology manufacturing are the master cell bank and the working cell bank, each of which serves different ...<|control11|><|separator|>
[48]
Cell substrates - World Health Organization (WHO)
The first WHO requirements for cell cultures used in the production of biologicals were formulated in 1959 as part of the production of inactivated ...
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The Role of the WI-38 Cell Strain in Saving Lives and Reducing ...
Mar 2, 2017 · Here we illustrate the unique contribution of the WI-38 cell strain to all human virus vaccines administered globally since the strains' origin ...
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The controversial cells that saved 10 million lives - BBC
Nov 4, 2020 · For decades, the polio vaccine had been made in cells taken from monkey kidneys, some of which – it was later discovered – were infected ...
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[PDF] Annex 3 - World Health Organization (WHO)
The concept of a master cell bank (MCB) and working cell bank. (WCB) system and the characterization of the cell substrate were introduced during that period ...
[52]
WHO requirements for the use of animal cells as in vitro substrates ...
WHO requirements for the use of animal cells as in vitro substrates for the production of biologicals (Requirements for biological susbstances no. 50).
[53]
[PDF] Recommendations for the evaluation of animal cell cultures as ...
The first requirements for cell substrates were published by WHO in 1959 for the production of inactivated poliomyelitis vaccine in PCCs derived from the ...
[54]
Human iPSC banking: barriers and opportunities
Oct 28, 2019 · The introduction of induced pluripotent stem cells (iPSCs) has opened up the potential for personalized cell therapies and ushered in new ...
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Induced pluripotent stem cells (iPSCs): molecular mechanisms of ...
Apr 26, 2024 · iPSCs have been widely applied to model human development and diseases, perform drug screening, and develop cell therapies.
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[PDF] assessment report: COVID-19 vaccine Moderna
Jan 4, 2021 · The applicant requested the active substance CX-024414 (single-stranded, 5'-capped messenger RNA. (mRNA) produced using a cell-free in vitro ...