Cell Line Development - Proof of Monoclonality

The development of a new cell line with the characteristics of interest is a highly complex process consisting of many successive steps, mainly leading into the goal to produce specific biopharmaceuticals.

Especially single cell cloning (SCC) represents a critical stage. The aim of SCC is to identify and isolate the most productive monoclonal cell populations after transfection or hybridization. Our CLD-specified imaging systems greatly provide the determination of monoclonality in different customizable approaches.

The advantages of SYNENTEC's imaging and analysing capabilities can be used reliable in any section of the upstream process:

controlling your transfection efficiency
determining the cell numberand cell viability
FDA conform proof of monoclonality
FACS seeding control
apoptosis monitorig e.g. in fed-batch cultures
detecting and counting colonies
quantifying IgG levels

Detction of Aggregates of Trypan Blue Viability Test

Cultivation of cells and cell based assays often require researchers to determine the number of cells they for preparing for cellular 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, cost intensiv sample disposables, long measurement durations and no automated triplicate sample-scanning.

SYNENTEC has adopted the classical Trypan Blue staining method for its NYONE® and CELLAVISTA® cell images and solves many disadvantages of other viability testing methods.

whole-well measurements in standard 96-well microplates
significantly increasing throughput (5 minutes per 96 samples)
small sample volumes (20 µL in total)
exact reproducibility
simple triplicates in one measurement
automated image analysis and data management
batch processing capabilities

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

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

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 by 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.

2. FASCC – Overcome The Edge-Effect

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

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⁾
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
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)
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.

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 TechNote 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

The experiments performed using SYNENTEC’s imagers are a very potent tool to qualify and conduct single cell processes for the production of monoclonal cell lines and also to monitor user and process performance on a routine basis.

Seeding methods like fluorescent activated cell sorting, limited dilution, cell printing or other methods can also be qualified in the presented approach in a cell line development setting monitoring the complete process involving seeding performance, cloning efficiencies and ultimately the probability of monoclonality.

The advantage of using the fluorescence and brightfield capabilities of CELLAVISTA® and NYONE® for seeding control is that the taken images can be reviewed to detect undesirable seeding events.productivity of your host cells.

Extract from the software readout for suspension cells (for further information look at ShortNote Suspension Cell Count 1F + 2F)

# of Cells in Brightfield
# of Cells in Fluorescence1 (AND/OR Fluorescence2) only
# of Cells in Brightfield AND Fluorescence1 (AND/OR Fluorescence2)

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.

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.

An early marker of apoptosis is the translocation of phosphatidylserine (PS) in the lipid bilayer of apoptotic cells. In healthy cells PS is usually located on the inner side of the cell membrane. It is an active, enzyme dependent process to sustain this PS-asymmetry.

When the apoptosis cascade starts, the caspase-3 signal is given to stop the PS-asymmetry on the lipid bilayer, whereupon PS is translocated to the external membrane and serves as a recognition signal for phagocytes. On this early stage of apoptosis, AnnexinV conjugates have a direct access to the outer PS, even before loss of membrane integrity.
If AnnexinV is added to the cell culture it binds to all accessible PS-lipids. The FITC-labeled form of AnnexinV enables the fluorescent detection of apoptotic cells in the culture with SYNENTEC’s cell imager whereas the additional staining with Propidium Iodide helps to discriminate from dead/necrotic cells.

Extract from the software readout (for further information look at 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)
…

The programmed cell death (apoptosis) is a complex mechanism consisting of many particularly different meshing steps.
One early hallmark of apoptosis is the loss of mitochondrial membrane potential (delta psi_m; 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 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 are red fluorescent.
A collapse of ΔΨm, like in apoptotic cells, disables an accumulation of the JC-1 molecules. 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 the NYONE® or CELLAVISTA® System.

Extract from the software readout (for further information look at 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)
…

Apoptisis monitoring with multiple Caspase Assays

Caspases constitute a family of proteases responsible for the induction of controlled cell death. Upon activation, hydrolysis of the procaspase into active caspase occurs. Various caspases are among the critical enzymes in the apoptosis process and are well studied in the human organism. They are able to activate further procaseases and specifically hydrolyze cell substrates and structural proteins. The result of caspase activation is, among other things, the fragmentation of the cell, which ends in the formation of the 'apoptotic bodies'.

Due to the high degree of flexibility in channel selection, this assay can be carried out in a variety of ways with NYONE® and CELLAVISTA®. You can work with or without counter stain (Hoechst). You can use 2 differently fluorescent caspase detectors simultaneously. And the simultaneous use of propidium iodide or 7-AAD as necrosis detection is also possible.

Extract from the software readout (for further information look at ShortNote Virtual Cytoplasm 1F)

# of only Hoechst33342-stained cells (=viable cells)
# of Hoechst33342 and Caspase+ stained cells (=apoptotic cells)
% of Apoptotic Cells
…

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®.

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 capital expenditures)
- 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.

Cell Line Development - Proof of Monoclonality

Trypan Blue Viability Assay – The Evergreen In Each Lab

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

1. SCC - The Conventional And Speedy Way

2. FASCC – Overcome The Edge-Effect

FASCC-Workflow

Transfection/Transduction Efficiency and Genome Editing with CRISPR/Cas9 – A Very Efficient Method

FACS validation and seeding control – A Tiny Cell Makes All The Difference

AnnexinV – Inside Out

JC-1 – The Question Of The Potential

Caspase Assay - Speed Up with Image Cytometry

IgG-Quantitation - Sometimes Less Means More

Assay Principle

Features

Conclusion

Software Readout