Industrial cell line development needs mammalian cell lines derived from one progenitor cell. Therefore, regulatory agencies ask to evaluate the probability of clonality (PoC) to ensure safe commercial manufacturing.
The PoC estimates the chance that a production cell line is derivative of a single cell progenitor. Chen et al. vigorously compare cloning etiquette and assess their regulatory acceptance.1 They published their results in “Methods for Estimating the Probability of Clonality in Cell Line Development”.1 This work resulted in the creation of a gold standard assay now used by several companies to estimate the PoC of cell line development workflow.
- Two series of cloning are typically completed with limiting dilution cloning (LDC) to gain a high PoC, adding time to the process. Therefore, LDC needs to be replaced to shorten the timeline while preserving a high PoC.
- DispenCell™ is a streamlined alternative to LDC. It is a user-friendly, reliable, and less expensive single-cell dispenser built on impedance technology.2
- Below, the Chen et al. assay is used to estimate the PoC of clones isolated with the DispenCell in comparison to those isolated with the typical LDC (Fig.1).1
Benefits
- Very high Probability of Clonality (PoC): DispenCell comes through with an impressive 98.4% PoC, greater than the 76.1% with LDC. This guarantees robust cell line development and meets strict regulatory standards for safe commercial manufacturing.
- Efficient Early-Stage Cloning: DispenCell accelerates and streamlines the cloning, shrinking labor and resource costs.
- Enhanced Monoclonal Colony Yield: With DispenCell, a greater number of monoclonal colonies are gained than LDC. This greater yield is critical for success in cell line development, particularly in high-throughput industrial conditions.
- Integrated Visual Confirmation for Greater Confidence: The DispenCell – paired with the fluorescence imaging system – allows visual confirmation of monoclonality as soon as Day 0, improving the PoC, offering a sturdy and reliable method for IND (Investigational New Drug) application submissions, and guaranteeing greater confidence in the cell line development process.
Methods
CHO cells expressing GFP were combined with RFP-expressing cells and subsequently cloned with DispenCell and LDC as a control (N = 4 × 96-well plates, each). The PoC of the clones was estimated using Chen’s model.1
| Yg = Y × (1 - b) × a + Y × b × (1- (1 - a2)) |
(1) |
with these variances in parameters (Fig.1):
Y = wells with 1 cell by the DispenCell
Yg = wells among Y showing cell outgrowth
Ygm = wells among Yg with both green and red fluorescence
a = single-cell recovery rate
b = probability of 2 cells per well
(1) and (2) were combined and the experimental data were contributed to gain the variables a and b to calculate the PoC:
| PoC = (Y × (1 - b) × a + Y × b × 2 × a × (1- a)) / Yg |
(3) |
Figure 1. Experimental design of the PoC assay. Image Credit: Molecular Devices UK Ltd
Figure 2. PoC assessment schematics for each well condition. Image Credit: Molecular Devices UK Ltd
Results
- Cloning on Day 0: The clonality check completed by the DispenCell software allowed the selection of 316 wells that displayed a single-cell signature. In Fig. 3, these wells are noted in green, and the rejected ones are noted in red. For the plates completed by LDC, these options were impossible. The number of wells home to just one cell in LDC was estimated at 124 utilizing the Poisson distribution (0.6 cells/well).
- Fluorescence imaging of the colonies on Day 21: Of the 316 wells chosen by the DispenCell, 255 colonies were monitored; of these, only two were bi-colored. The calculated single-cell recovery rate (a) and the probability of developing two cells per well (b) were 80% and 2%. Of the 380 wells utilized by the LDC, 155 colonies were monitored, 23 of which were bi-colored. The calculated single-cell recovery rate (a) and the probability of two cells per well (b) were 90% and 26%.
- Based on the Chen et al. model, the PoC of these methods was computed considering the single-cell recovery rate (Fig. 4). The PoC computed for the DispenCell was 98.4%, while the PoC for the LDC was 76.1%.1
- A probability of clonality of 98.4% was found with DispenCell, as compared to 76.1% with LDC (Fig. 4)
- DispenCell offered a much greater PoC than LDC and twice the number of monoclonal colonies.
- Combining DispenCell’s impedance-based clonality evaluation with visual confirmation at Day 0 with a high-resolution imager (e.g., CloneSelect® Imager, Molecular Devices) would offer an even greater PoC and support a quick, streamlined, and confident IND filling.
Figure 3. Clonality check on Day 0 using the DispenCell analysis software (left panel) and imaging of colonies on Day 21 using fluorescent imaging (right panel). Image Credit: Molecular Devices UK Ltd
| |
DispenCell |
LDC |
| Total wells |
372 |
380 |
| Monoclonal wells–Day 0 |
316 (impedance-based) |
124* (estimated) |
| Total wells with colonies–Day 21 |
255 (observed) |
155 (observed) |
| Wells with mixed colonies–Day 21 |
2 (observed) |
23 (observed) |
| Probability of clonality (PoC) |
98.4% |
76.1%* |
| Monoclonal colonies |
251 |
118* (estimated) |
* Computed using the Poisson distribution-based model with 0.6 cells/well and taking into account cell survival statistics.
Figure 4. Experimental data analysis for PoC calculation. Image Credit: Molecular Devices UK Ltd
References and further references
- Chen et al., 2020. ‘Methods for Estimating the Probability of Clonality in Cell Line Development’, Biotechnol J. 15(2):e1900289.
- Hannart et al., 2022. Traceable Impedance-based single cell pipetting: from a research set-up to a robust and fast automated robot’, SLAS TECHNOL. 27(2):121–129.
About Molecular Devices UK Ltd 
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