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Cell Line Development

Improving the cell line selection reduces time to the clinical phase. Amplify your biopharmaceutical drug discovery efforts with high throughput cell line- and process development!

A Tiny Cell Makes All The Difference

single_cell_on_well_edge_no_shadow_no_edge_effect

Developing a new biopharmaceutical and the associated cell line development implies many steps between the starting-cell and the final production-process.

During cell line development the production clone is generated by integrating the gene of interest (GOI) and a selection marker into a host cell. The next step after transfection is the cell selection and the single cell cloning (SCC), followed by the clone selection, the clone- and product characterization and finally by the process optimization.

The productivity of the later recombinant cell line depends on many process parameters. And the establishment of a stable, monoclonal production cell line is overall a very costly and time-consuming process which may take up to one year on average.

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Transfection/Transduction Efficiency – A Very Efficient Method

[Confluence (1F) – Suspension Cell Count (1F)]

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Transfection is a common method in cell line development (stable transfection) and protein research (transient transfection). Despite the standard use of this method, it is associated with many difficulties. Everyone knows how challenging and time consuming it may be to establish an ideal transfection protocol. Even if the desired vector is designed and cloned, the way to a reliable transfection is still far!

A widely applied method is the expression of a selection marker in combination with a fluorescent marker gene (e.g. GFP) additionally to the target gene. The read out for the qualitative and quantitative analysis of transfection will be usually performed by user-dependent and time-consuming manual microscopy or, respectively, precise but invasive flow cytometry.

In contrast to these methods SYNENTEC's cell imagers quantify the transfection efficiency within minutes via software assisted image analysis in both adherent and suspension cells within a wide range of fluorescence spectra. Our YT-software® can analyze a whole microplate non-invasively and even track your culture over days to determine growth-curves and the productivity of your host cells.

Extract from the software readout for adherent cells (for further information look at ShortNote Confluence 1F)

  • % Confluence BF
  • % Confluence FL
  • % Ratio of Confluence BF/FL

Extract from the software readout for suspension cells (for further information look at ShortNote Suspension Cell Count 1F)

  • # of Cells in Brightfield
  • # of Cells in Fluorescence
  • # of Cells Brightfield AND Fluorescence

Monitoring Viability with Trypan Blue – The Evergreen In Each Lab

[Trypan Blue]

Trypan Blue Assay.

Cultivation of cells and cell based assays often require researchers to determine the number of cells they are preparing for these assays. Furthermore, in routine sub-culturing, cell line development or upscale bioreactor cultivation (harvesting), it is important to control the viability of the culture. The Trypan Blue viability test is a widely used technique to determine the cell number and the culture viability. In a lot of cases this is performed manually by counting cells with a hemocytometer which is tedious and leads to subjective and biased results. Automated or semi-automated cell counters are available for this purpose and each one has individual advantages and disadvantages e.g. consumption of sample volume and measurement time.

SYNENTEC has adopted the classical Trypan Blue staining method for its NyONE® and Cellavista® cell imagers, with which the Trypan Blue assay can be carried out as whole-well measurements in 96 well microplates with significantly increasing throughput (5 minutes per 96 samples), reducing the time-to-result and requiring smaller sample volumes (20 µl in total) than other existing assay formats.

Extract from the software readout (for further information look at ShortNote Trypan Blue)

  • Viability in %
  • VCD/mL
  • CD/mL
  • # of Aggregates/mL
  • Ratio of Aggregates in %
  • Avg. Cell Diameter in µm

Proof Of Monoclonality – Can You Afford Not To Be Sure?

[Single Cell Cloning – FASCC  - HighRes Cloning]

Clone Gallery for Report

Single cell cloning (SCC) represents a crucial step in cell line development. The aim of SCC is to identify and isolate wells where a single cell was seeded and to monitor its growth rate. Usually limited dilution or fluorescence activated cell sorting (FACS) is used for seeding single cells into microplates. For the proof of monoclonality for documentation and regulatory approval it is essential to have a robust and reliable imaging system which combines robustness and reliability with a high image resolution (below 1 micron per pixel) as well as using high contrast brightfield images and the power of fluorescence.

We are well established in the field of cell line development since a decade and our experience has shown that many roads lead to Rome! Each cell line behaves differently, cultivation conditions vary and the requirements of the clients of our customers are individually. Therefore we provide adapted methods with our automated high throughput imaging systems Cellavista® and NyONE® to determine the clonality of a cell line. All of these approaches work with both adherent and suspension cells within a wide range of fluorescence spectra.

1. SCC - The Conventional And Speedy Way

This application provides the opportunity to scan the whole well of the entire plate with one of our imaging systems in bright field at high resolution which is below 1 micron per pixel, on the first day, just after seeding the cells.

Benefit:

  • fast cloning approach
  • seamless stitching
  • no area in the well will be missed
  • automatable measurement day by day
  • all colonies are recognized bye our software
  • tracking back to the first day to demonstrate monoclonality
  • automatic generating of a clone gallery

This will give you a proof that your colony in fact grows from a single cell or - the undesired event: grew from a doublet.

SCC-Workflow
SCC-Workflow

2. FASCC – Overcome The Edge-Effect

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Ghost cells represent one of the main challenges in cell line development and drug discovery. Those undetected cells usually lie on the well edge and can be caused by the fact that most of the common imaging systems for CLD are equipped with a low resolution optic (2 microns per pixel), combined with a white light (brightfield) source only.

This lack of resolving power causes a lack of information within the image and therefore it is hardly possible to distinguish cells from debris, or well-edge artefacts. Because of this inaccuracy, a precise statement about the monoclonality is not unambiguously possible. Adding fluorescence information to your application simplifies to detect these cells in a reliable way, even at the well edge!

SYNENTEC has developed a unique approach (FASCC) to overcome all disadvantages of the classic white light single cell cloning. FASCC (fluorescence activated single cell cloning) provides a fully automated way of screening your micro titer plate by using the advantage of short term, non-toxic and animal-free cell staining. The proof of monoclonality is unambiguous and furthermore a faster way to perform full well scans for regulatory approval. This unique tool presents an unambiguous proof of monoclonality without guesses or doubts!

FASCC-Workflow

  1. a fast(1), low resolved (4x lens) high speed scanning mode (pre-scan) detect the fluorescence signal of e.g. Calcein-AM or Cell Tracker® stained cells(2)
  2. the embedded image analysis tool excludes all wells seeded with more than just one cell or any number of cell-count the user sets up
  3. in the highly resolved(3) Nanoview-scan all pre-scan results will be picked up and will be proceed by centering all single cells right in the middle of a sinlge image (benefit: no cells on stitching boarder and fast scan despite high resolution)
  4. over expansion-periode confluence measurement or suspension cell count with automated colony-detection can be performed to track cell proliferation

(1)The pre-scan will usually take less than 3 minutes for an entire 384-well-plate (Cellavista®). (2)Both dyes are non-toxic and will disappear within 3-4 hours for Calcein-AM and within days for CellTracker. The goal of Cell Tracker and Calcein-AM is an enzymatic reaction, which induces both dyes to emit fluorescence light to indicate a viable organism. (3)The images taken at the post-scan are now highly resolved (10x lens) and are offering a detailed view inside of each cell.

SCC - Automated Colony Detection in 96-well Plate.
SCC - Automated Colony Detection in 96-well Plate.

Apoptosis Monitoring – How Your Cells Prefer To Die

In cell line development and upscale bioreactor cultivation it is important to monitor the health of the culture to determine the growth rate of cells and to check the quality of the culture conditions (Media etc.). Also in the field of toxicity studies, cancer research and compound screening the investigation of apoptosis triggers and -mechanisms is essential.

With our automated microscopes Cellavista® and NyONE® we provide two different apoptosis assays. Both AnnexinV and JC-1 are well known as early apoptosis marker. With these assays SYNENTEC demonstrates very sensitive methods to get an overview of your cell status.

1. AnnexinV – Inside Out

[Virtual Cytoplasm 2F]

These images show the difference between viable, apoptotic and dead cells<br/>a) Shown are viable cells, the nuclei, stained with Hoechst. AnnexinV and PI could not bind. b) Shown are apoptotic cells with a green AnnexinV-FITC stained cell membrane and a blue Hoechst stained cell nucleus. c) Dead cells with PI (red) and Hoechst (blue) in the cell nucleus and a green membrane with AnnexinV-FITC.
These images show the difference between viable, apoptotic and dead cells
a) Shown are viable cells, the nuclei, stained with Hoechst. AnnexinV and PI could not bind. b) Shown are apoptotic cells with a green AnnexinV-FITC stained cell membrane and a blue Hoechst stained cell nucleus. c) Dead cells with PI (red) and Hoechst (blue) in the cell nucleus and a green membrane with AnnexinV-FITC.

The translocation of phosphatidylserine (PS) in the lipid bilayer of the cells is an early apoptosis marker. In healthy cells PS is usually located on the inner side of the cell membrane. At this early stage of apoptosis, AnnexinV conjugates have a direct access to the outer PS, even before the loss of membrane integrity. The fact that AnnexinV is a very early indicator of apoptosis allows an earlier detection of problems within the cell culture e.g. in fermenters than e.g. the analysis of apoptosis by DNA-based assays can provide. This possibility saves a lot of effort, money and time by achieving the best fermentation-results.

Assay Principle

Simplified scheme of the difference between viable, apoptotic and dead cells<br/>a) Shown is a viable cell, phosphatidylserine is only in the inner leaflet of the bilayer, AnnexinV-FITC cannot bind, PI stays outside the cell. b) Shown is a apoptotic cell, PS has moved to the outside of the membrane, AnnexinV binds, PI stays outside. c) Shown is a dead cell, the membrane is permeable, PI can get into the cell and stains the cell nucleus, AnnexinV-FITC binds to the inner and outer leaflet.

If AnnexinV is added to the cell culture it binds to all cells which have accessible PS. It binds to apoptotic cells when PS turns to the outside of the membrane. When a cell dies due to necrosis, the cell membrane becomes permeable whereby AnnexinV can get into the cell and also binds to the PS in the inner leaflet of the bilayer.

To distinguish between viable, apoptotic and dead cells we stained the cells with an AnnexinV-conjugate (here: FITC), Propidium iodide (PI) and Hoechst 33342 simultaneously. The Hoechst staining detects all cells in the blue channel, it stains all nuclei. Propidium iodide marks only the dead cells in the red channel and AnnexinV-FITC stains all apoptotic and dead cells in the green channel. Therefore, we have three different types of cells: The viable with a blue nucleus (Fig. 2 a), the apoptotic ones with a blue nucleus and a green membrane (Fig. 2 b) and the dead cells with a blue and red nucleus and a green membrane (Fig. 2 c). Due to this difference the NyONE® cell imager with its associated image analysis software differentiates highly sensitive and very precisely between live, dead and apoptotic cells.

Extract from the software readout (for further information refer to ShortNote Virtual Cytoplasm 2F)

  • # of only Hoechst33342-stained Cells (=viable Cells)
  • # of Hoechst33342 and AnxV stained Cells (=apoptotic Cells)
  • # of Propidium Iodide and AnxV stained Cells (=necrotic Cells)

2. JC-1 – The Question Of The Potential

[JC-1 mito potential]

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Another early characteristic of apoptosis is the loss of mitochondrial membrane potential (delta psim; ΔΨm) resulting from a disruption of the mitochondrial membrane. To indicate the mitochondrial health in a suspension cell culture treated with H2O2, we used the cationic, lipophilic dye 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1).

This dye has the property to change the emitted fluorescence via accumulation and aggregation. In healthy cells, JC-1 enters the negative charged mitochondrial matrix and enriches the mitochondria lumen where it builds J-aggregates after reaching a critical concentration. The J-aggregates emit red fluorescence. A collapse of ΔΨm, like in apoptotic cells, disables an accumulation of the JC-1 molecules or aggregates. In those cells JC-1 remains in a monomeric, green fluorescent form.

Therefore early apoptotic and healthy cells are easy to distinguish with fluorescence measurements by SYNENTEC's imaging systems.

Assay Principle

In healthy cells mitochondria are in an energized condition with a polarized mitochondrial membrane (Fig. 2a). Part of the apoptotic pathway is the uncoupling of the proton gradient of mitochondria and a loss of membrane integrity. This has the consequence that the cationic JC-1 dye gets no longer actively enriched in the mitochondrial lumen and therefore it cannot aggregate (Fig. 2b).

Schematic illustration of JC-1 monomers (green dots) and J-aggregates (red dots) depending on the mitochondrial membrane potential.<br/>In healthy cells mitochondria are in an energized condition with a polarized mitochondrial membrane (a). Part of the apoptotic pathway is the uncoupling of the proton gradient of mitochondria and a loss of membrane integrity. This has the consequence that the cationic JC-1 dye gets no longer actively enriched in the mitochondrial lumen and therefore it cannot aggregate (b).

Extract from the software readout (for further information refer to ShortNote Virtual Cytoplasm 2F)

  • total # of cells
  • % of Hoechst33342 and JC-green stained Cells (=Cells with loss of mitochondrial membrane potential
  • % of Hoechst33342 and JC-red stained Cells (=viable Cells)

IgG-Quantitation - Sometimes Less Means More

[PAIA]

PAIA Logo.

During the cell line development process hundreds to thousands of clones have to be screened to find the most promising, i.e. well growing clones that are also good producers for further development. The process of single cell cloning is usually performed in 96-well formats and increasingly also in 384-well plates. Thus, there is limited sample volume available for analysis and it is desirable to get the results as quickly as possible.

PAIA BIOTECH has taken on this challenge and their scientific team has developed a new ultra-fast-low-volume assay for IgG-quantification directly from the supernatant after single cell cloning!

This new technology gives SYNENTEC the opportunity, not only to determine the clonality and to support the process optimization with our high throughput imaging systems, but also to find in combination with the PAIAplate the high producer which also still grows fast and as quickly as possible. One full 384-well PAIAplate is processed in less than one hour and allows reliable identification of high producers in an automation-friendly workflow.

Assay Principle

Protein A coated capture beads, analyte and fluorescence markers are incubated in the wells of a 384-well PAIAplate. After sedimentation of the beads with the bound analyte-marker-complexes, the remaining unbound fluorescence marker is measured with the NyONE® or Cellavista®.

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Features

  • High throughput in 384 well plates
    • processing time for full plate < 1 hour
  • Low sample consumption
    • 2- 10 µL of crude sample
  • Simple protocols in single-use plates
    • no washing , no blocking, no regeneration
  • Cost-efficient (No Capex)
    • runs on fluorescence microscopes

Conclusion

Running PAIA IgG quantification assays on the Cellavista® and NyONE® imagers allow the reliable identification of high producers with a very limited amount of samples from cell culture supernatants and high throughput. The combination of PAIA assays and SYNENTEC's imaging systems enables to monitor monoclonality and cell growth plus the workflow is amenable to automation and offers a high throughput which can be increased to up to four 384-well plates per hour if several shakers are used in parallel.

Software Readout

PAIA Biotech has developed a software tool for the rapid generation of calibration curves, calculation of sample concentrations and result exports. The data generated with our YT-analysis software® can be exported as csv-files and can be imported into PAIAs software tool. In this software tool method templates facilitate the processing of recurrent plate layouts. They contain a predefined layout with the positions of standards, controls and samples as well as their concentration and dilution factors.

The measured fluorescence intensities are copied from the instrument and pasted into the tool, calibration curves are generated, and heat maps provide a quick result overview. Different report and export functions for QC data, results and graphs are implemented.

  •  IgG concentration curve.
    IgG concentration curve.
  • paia_analysis_software_1.png
    PAIA analysis software.