Cell Line Development
Stable cell lines are widely used in a number of important applications including biologics (e.g. recombinant protein and monoclonal antibody) production, drug screening, and gene functional studies. The process of developing stable cell lines often starts with transfecting selected host cells, typically CHO or HEK 293 cells, with desired plasmids. After transfection, researchers then screen and quantify high-expressing clones. Once these high producers are identified, the cell lines and/or the proteins produced by the cells are validated.
The manual screening methods traditionally used for cell line development are time-consuming and labor-intensive, creating a great demand for high throughput, automated solutions for such efforts. The general workflow below helps identify the systems that can aid in your research.
Cell line development process:
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Transfection is the process of introducing a foreign DNA (encoding the recombinant protein of interest) into a host cell. A small population of cells with the foreign DNA integrated into their genome that maintain their ability to express recombinant protein for long periods of time are referred to as stably transfected cells.
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Antibodies screening/titer ranking – Discovery and selection of high-value clones from a transfected pool of cells. To screen large populations by quantifying cell surface expression of protein-of-interest or secreting antibodies (titer ranking) will increase the probability of finding rare high-affinity binder or high producer.
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Single-cell isolution and cell viability – Single, viable cells must be isolated and cloned in order to ensure that the cell population is genetically identical, significantly reducing the heterogeneity of expression
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Monoclonality assurance – When developing cell lines for biotherapeutics, it is crucial from a quality and regulatory perspective to ensure that the cell line originates from a single progenitor and is therefore monoclonal. Documentation of monoclonality (a regulatory metric for therapeutic cell lines) is typically image-based, whereby an image of a single cell is recorded and included in regulatory filings.
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Clone productivity screening and titer – This is a test that detects the amount of recombinant protein or antibodies produced from the clonally-derived cell line.
Applications and methods for the development of cell lines
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Cell surface expression screening
Many proteins that express to the surface of cells are targets for the discovery and development of biopharmaceuticals. For instance, G-protein coupled receptors (GPCRs) are the largest class of cell-surface proteins and are targets for almost 40% of existing drugs. Discovery and selection of high value clones with elevated cell surface expression of GPCRs from a transfected pool of cells can be challenging. The ClonePix 2 System represents an automated method of screening large populations of cells that increases the probability of finding rare high-affinity binder or high producer.
- Rapid automated selection of mammalian cell colonies by cell surface protein expression
- Rapid selection and development of GPCR expressing mammalian cell lines using novel ClonePix Technology
- Rapid and efficient selection of high producing mammalian cells secreting non-mAb proteins
- Brochure: ClonePix2
Cell surface expression screening
Many proteins that express to the surface of cells are targets for the discovery and development of biopharmaceuticals. For instance, G-protein coupled receptors (GPCRs) are the largest class of cell-surface proteins and are targets for almost 40% of existing drugs. Discovery and selection of high value clones with elevated cell surface expression of GPCRs from a transfected pool of cells can be challenging. The ClonePix 2 System represents an automated method of screening large populations of cells that increases the probability of finding rare high-affinity binder or high producer.
- Rapid automated selection of mammalian cell colonies by cell surface protein expression
- Rapid selection and development of GPCR expressing mammalian cell lines using novel ClonePix Technology
- Rapid and efficient selection of high producing mammalian cells secreting non-mAb proteins
- Brochure: ClonePix2
Cell viability
Cell line development requires the discovery of single cell-derived clones that produce high and consistent levels of the target therapeutic protein. A critical first step in the process is the isolation of single, viable cells. Single cells proliferate to form colonies that can then be assessed for productivity of the target therapeutic protein. Viability and growth rates of single cell-derived clones can be characterized on the CloneSelect Imager and the SpectraMax i3x microplate reader and imaging cytometer.
Cell viability
Cell line development requires the discovery of single cell-derived clones that produce high and consistent levels of the target therapeutic protein. A critical first step in the process is the isolation of single, viable cells. Single cells proliferate to form colonies that can then be assessed for productivity of the target therapeutic protein. Viability and growth rates of single cell-derived clones can be characterized on the CloneSelect Imager and the SpectraMax i3x microplate reader and imaging cytometer.
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Clone productivity screening and titer
An important component in identifying high-value clones is determining productivity of single cell-derived colonies. Screening for productivity using traditional approaches is laborious and time consuming, generally consisting of a multistep process that involves isolating single cells from limiting dilution followed by assessment of titer using ELISA. The ClonePix 2 system combines phenotype selection, single-cell isolation and productivity screening into a single step, resulting in dramatically shorter screening times and increased number of candidates.
Clone productivity screening and titer
An important component in identifying high-value clones is determining productivity of single cell-derived colonies. Screening for productivity using traditional approaches is laborious and time consuming, generally consisting of a multistep process that involves isolating single cells from limiting dilution followed by assessment of titer using ELISA. The ClonePix 2 system combines phenotype selection, single-cell isolation and productivity screening into a single step, resulting in dramatically shorter screening times and increased number of candidates.
Comparison of traditional cloning methods vs. f.sight
As regulations for cell line development become increasingly more stringent, researchers will be required to perform single-cell cloning and provide evidence that a cell line is derived from a single cell (proof of clonality). In this study, we conducted six sets of experiments to demonstrate a combined f.sight and CSI workflow with high monoclonality assurance in a single round of cloning. We also compared the cloning eciency of two suspension CHO cell lines on a CloneSelect f.sight single cell printer versus two other accepted traditional cloning methods, flow cytometry and limiting dilution.
Comparison of traditional cloning methods vs. f.sight
As regulations for cell line development become increasingly more stringent, researchers will be required to perform single-cell cloning and provide evidence that a cell line is derived from a single cell (proof of clonality). In this study, we conducted six sets of experiments to demonstrate a combined f.sight and CSI workflow with high monoclonality assurance in a single round of cloning. We also compared the cloning eciency of two suspension CHO cell lines on a CloneSelect f.sight single cell printer versus two other accepted traditional cloning methods, flow cytometry and limiting dilution.
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Monoclonality assurance
Cell line development and assurance of monoclonality are critical steps in the process of generating biopharmaceutical molecules, such as monoclonal antibodies. A cell line can be established following the isolation of a single viable cell robustly expressing the protein of interest. A key milestone in this process is documenting evidence of clonality. Documentation of clonality is typically image-based, whereby an image of a single cell is produced and included in regulatory filings.
Monoclonality assurance
Cell line development and assurance of monoclonality are critical steps in the process of generating biopharmaceutical molecules, such as monoclonal antibodies. A cell line can be established following the isolation of a single viable cell robustly expressing the protein of interest. A key milestone in this process is documenting evidence of clonality. Documentation of clonality is typically image-based, whereby an image of a single cell is produced and included in regulatory filings.
Single cell sorting
Cell line development requires the discovery of single cell-derived clones that produce high and consistent levels of the target therapeutic protein. The first step in the process is the isolation of single, viable cells. Limiting dilution is a technique that relies on statistical probability but is time consuming. The CloneSelect Single-Cell Printer enables the gentle isolation of cells in a manner that maximizes cell viability, as well as providing direct evidence of clonality via a series of five images captured as cells dispense.
- A microfluidics-based, single cell printing and microplate imaging workflow optimized for efficiency, viability and monoclonality report generation
- A workflow combining single-cell isolation and microplate imaging improves cloning efficiency with higher assurance of clonality
- Brochure: CloneSelect Single-Cell Printer
Single cell sorting
Cell line development requires the discovery of single cell-derived clones that produce high and consistent levels of the target therapeutic protein. The first step in the process is the isolation of single, viable cells. Limiting dilution is a technique that relies on statistical probability but is time consuming. The CloneSelect Single-Cell Printer enables the gentle isolation of cells in a manner that maximizes cell viability, as well as providing direct evidence of clonality via a series of five images captured as cells dispense.
- A microfluidics-based, single cell printing and microplate imaging workflow optimized for efficiency, viability and monoclonality report generation
- A workflow combining single-cell isolation and microplate imaging improves cloning efficiency with higher assurance of clonality
- Brochure: CloneSelect Single-Cell Printer
Resources of Cell Line Development
Application Note
A microfluidics-based, single cell printing and microplate imaging workflow optimized for efficiency, viability and monoclonality report generation
A microfluidics-based, single cell printing and microplate imaging workflow optimized for efficiency, viability and monoclonality report generation
In this app note, we demonstrate a unified workflow that combines single cell deposition and clonality verification. We couple the use of the SCP and CSI to deposit single cells into a…
Brochure
ClonePix 2
ClonePix 2
Explore ClonePix 2 from Molecular Devices that screens more clones in less time with its accurate, automatic colony picking features.
Data Sheet
CloneSelect Single-Cell Printer Series
CloneSelect Single-Cell Printer Series
The CloneSelect™ Single-Cell Printer™ Series deposit single cells gently and with high efficiency using a patented, inkjet-like disposable, oneway dispensing cartridge.
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Brochure
CloneSelect Imager Monoclonality Report Feature
CloneSelect Imager Monoclonality Report Feature
Providing image evidence of monoclonality in the cell line development process is not as simple as exporting an image of a single cell. For example, high-resolution images of the entire w…
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Application Note
Measuring cell viability and cytotoxicity with the EarlyTox Cell Integrity Kit
Measuring cell viability and cytotoxicity with the EarlyTox Cell Integrity Kit
The EarlyTox™ Cell Integrity Kit is an optimized set of reagents that simplifies the identification of live and dead cells. It can be used to measure the effects of different treatments on…
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Application Note
Comparison of traditional cloning methods vs. CloneSelect Single-Cell Printer f.sight using CHO cell lines commonly used for monoclonal antibody production
Comparison of traditional cloning methods vs. CloneSelect Single-Cell Printer f.sight using CHO cell lines commonly used for monoclonal antibody production
As regulations for cell line development become increasingly more stringent, researchers will be required to perform single-cell cloning and provide evidence that a cell line is derived…
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Application Note
A workflow combining single-cell isolation and microplate imaging improves cloning efficiency with higher assurance of clonality
A workflow combining single-cell isolation and microplate imaging improves cloning efficiency with higher assurance of clonality
The isolation of single cells has a broad spectrum of applications, from single-cell genomics to antibody discovery and cell line development.
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Application Note
Confident assurance of clonality using calcein AM with minimal effect on viability
Confident assurance of clonality using calcein AM with minimal effect on viability
The development of cell lines that express a specified protein of interest is critical to the generation of biopharmaceutical molecules such as monoclonal antibodies. To establish a cell…
Scientific Poster
Rapid Screening and Selection of Stable High Producing Clones
Rapid Screening and Selection of Stable High Producing Clones
Screening cell populations for stable, high producing clones is a key rate-limiting step in the development and production of new protein therapeutics.
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Scientific Poster
Rapid Monoclonality Verification Methods to Boost Cell Line Development
Rapid Monoclonality Verification Methods to Boost Cell Line Development
Here we present a fluorescent method for identifying monoclonal CHO-S cells using CellTracker Green CMDFA.
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Application Note
Rapid and efficient selection of high producing mammalian cells secreting non-mAb proteins
Rapid and efficient selection of high producing mammalian cells secreting non-mAb proteins
The technology in ClonePix Systems removes the need for serial dilution or cell-sorting techniques. It allows monoclonal populations of high-secreting cells to be established in a much…
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Application Note
Automated Optimization of IgG Production in CHO Cells
Automated Optimization of IgG Production in CHO Cells
Monoclonal antibodies have long been an essential tool for cell and molecular assays and have moved into the clinic as part of a shift from small molecules into biologic treatments for…
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Application Note
Confident Identification of Monoclonal CHO-S Cells Grown in Semi-Solid Media using the CloneSelect Imager
Confident Identification of Monoclonal CHO-S Cells Grown in Semi-Solid Media using the CloneSelect Imager
Regulatory criteria to obtain approval of biological drugs are constantly evolving. One of the regulatory metrics for biological cell lines, the digital documentation of monoclonality, has…
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Application Note
Confluence and growth curve generation on the CloneSelect Imager
Confluence and growth curve generation on the CloneSelect Imager
The ability to monitor cell proliferation has become increasingly important, particularly with the widespread use of in vitro cell lines as model systems. Proliferation rates can be used to…
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Application Note
Rapid automated selection of mammalian cell colonies by cell surface protein expression
Rapid automated selection of mammalian cell colonies by cell surface protein expression
The ability to select cells based on surface protein expression is of great use in establishing cell lines expressing the receptor(s) of interest for downstream phenotyping/genotyping,…
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Application Note
Rapid selection and development of GPCR expressing mammalian cell lines using novel ClonePix Technology
Rapid selection and development of GPCR expressing mammalian cell lines using novel ClonePix Technology
Discovery and selection of high expressing GPCR clones from a transfected pool of cells can be challenging with regards to cell-line development. The technology in ClonePix 2 Systems…
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Application Note
Enhanced development of virus-specific hybridomas using ClonePix and CloneSelect Imager Technologies
Enhanced development of virus-specific hybridomas using ClonePix and CloneSelect Imager Technologies
The ClonePix System was employed in the high-throughput screening of hundreds of sub-cloned colonies from parental hybridoma material (historically parental line yields were less than 1 mg/…
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Application Note
Luminescent cell viability and cytotoxicity assays on the SpectraMax i3x Multi-Mode Microplate Reader
Luminescent cell viability and cytotoxicity assays on the SpectraMax i3x Multi-Mode Microplate Reader
The CellTiter-Glo assay from Promega uses luciferase enzyme, which requires ATP in order to generate light. The luminescent signal produced in the assay depends on the amount of ATP in the…
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Brochure
SpectraMax i3x Multi-Mode Detection Platform
SpectraMax i3x Multi-Mode Detection Platform
The SpectraMax i3x Multi-mode microplate reader measures spectral-based Absorbance, Fluorescence, and Luminescence with added functaionlity of modular upgrades for Western Blot, Imaging,…
Videos & Webinars
The many and varied workflows of cell line development