November 1, 2017 (Vol. 37, No. 19)

Sarmad Al-Bassam Ph.D. Application Scientist Molecular Devices
Steve Wiltgen Ph.D. Product Manager Molecular Devices

Utilizing Microfluidics and Imaging to Improve the Cell-Line Development Workflow

Figure 1. The CloneSelect™ Single-Cell Printer™ offers increased throughput and ease-of-use to help meet the increasing demands in cell-line development.


The increasing adoption of large protein biologics such as monoclonal antibodies (mAbs) for the treatment of disease has heralded unprecedented growth in the R&D of novel therapeutics and biosimilars, bringing unique challenges to the biopharmaceutical industry.

In a typical workflow to cultivate cell lines, the gene that encodes the recombinant protein of interest is transfected into a cell line of choice (typically CHO), where copies of the gene are incorporated at multiple, random sites within the genome. Establishment and optimization of a cell line (referred to as cell-line development) entails finding the rare clones where random integration results in high, stable production of the therapeutic with desirable consistency and quality attributes.

In addition, this optimization must be accomplished under the scrutiny of regulatory agencies that require thorough demonstration of product safety and quality. In the process just described, the FDA specifically requires evidence that a given mAb is derived from a single cell and that necessary precautions are taken to minimize cross-contamination with potentially harmful agents (e.g., viruses, prions, etc.).

The successful isolation of single cells with high efficiency and viability is therefore critical to the cell-line development process, but traditional methods face significant challenges to meet both needs simultaneously. Limiting dilution utilizes a series of successive dilution steps to generate a low concentration of cells, which are then deposited into a microplate via manual pipetting or automated liquid handling. At such low concentrations, most wells will not contain a cell, but those that do will most likely contain a single cell (as determined statistically by a Poisson distribution). The process is simple, cost-effective, and gentle on the cells, resulting in high viability.

However, the statistical nature of the process significantly impacts efficiency because at best, 30% of wells will contain a single cell. Furthermore, one cannot assure the presence of a single cell per well, which often necessitates an additional round of cloning for regulatory purposes.

Another frequently used method is flow cytometry, which isolates single cells efficiently (up to 99% in some cases). Most systems also possess fluorescence sorting capabilities, which can be used to screen for productivity, but is limited in its application, because of the requirement of a membrane-bound fluorescence signal. The most significant limitation to flow-based systems, however, is that a portion of cells are nonviable due to the shear stress and pressures experienced within the flow cell.

Furthermore, cross-contamination is problematic because it can only be minimized by limiting access to an instrument or by replacing the flow cell, which is fairly expensive. Finally, the sophistication of the technique demands intricate knowledge of the instrument, considerably limiting accessibility to this workflow.


Adapting the Ink-jet Printer Principle

An emergent technology for single-cell isolation addresses a number of the shortcomings of existing techniques. The CloneSelect™ Single-Cell Printer™ (Figure 1 and Figure 3) has been adapted from the principle of ink-jet printers, whereby microscopic, cell-containing droplets are generated and ejected from a microfluidics-based cartridge. Cells are randomly encapsulated within the droplets as they are being dispensed into a microplate. Thus, a series of five images are captured in order to screen and select for single-cell containing droplets prior to deposition. If the software detects that zero or multiple cells are present within the droplet, the aforementioned cells are immediately siphoned off by a vacuum. However, when a single-cell event is detected, the vacuum is shut, allowing the droplet to fall into the well.

This method of prescreening droplets significantly improves the efficiency of single-cell deposit compared with limiting dilution. An average single-cell deposit efficiency of >80% is observed under typical experimental conditions with the single-cell printer (SCP). The likelihood of observing wells with multiple cells is also decreased compared with limiting dilution, as the method is not statistically based. With the SCP, only 6% of wells contained more than one cell, whereas 10% of wells had multiple cells using limiting dilution performed at a seeding density of 0.5 cells/well (Figure 2A).

Beyond improving single-cell deposition efficiency, use of the SCP may also improve viability. At the 2017 Cell Line Development conference, it was reported in one study that the use of the SCP delivered a cell viability >90%, whereas limiting dilution yielded roughly 70% viable clones.1 This study is preliminary, but it is supported by the authors’ findings (Figure 2B). The authors’ current hypothesis is that the gentle handling of cells within the cartridge, in combination with cell screening based on size and morphology, is a likely source for the increase in viability as it offers the ability to exclude unhealthy or otherwise asymmetrical cells. On average, the authors have observed 75% viability across a variety of commonly used cell lines (Figure 2C).


Figure 2. Three graphs compare the efficiency and viability of clones between use of a single-cell printer (SCP) and limiting dilution. (A) The efficiency of single-cell deposit on the SCP is 88% compared with 32% with limiting dilution. (B) The percent outgrowth (or viability) was 84% for SCP and 81.7% for limiting dilution. (C) The SCP is compatible with many cell types as shown by the percent viability of CHO-K1, HEK-293, and L-929.

The use of imaging on the SCP has several additional benefits beyond improving efficiency and viability. Images of single cells captured on the SCP provide high assurance of monoclonality through direct visual evidence (Figure 3). This has been validated by capturing images of the same cells post-printing with the CloneSelect Imager (an automated, label-free imager). A high degree of correlation (>98%) was observed between the single-cell images captured on both systems.

The CloneSelect Imager (CSI) adds significant value to this workflow by providing a second, independent measure of clonality, thereby substantially improving the probability of clonality. In addition, the CSI can also characterize the growth pattern and morphology of candidate clones, further complementing the cell-line development workflow downstream of single-cell printing.  An additional benefit to imaging on the SCP is that the nozzle is significantly smaller than the vast space of the microplate surface, allowing for quick confirmation of monoclonality. Not only is it much easier to identify a single cell in a label-free image, it is also much easier to confirm the absence of a second cell in the sample, further improving the assurance of monoclonality.

The improvements to efficiency, viability, and assurance of clonality shown with the CloneSelect Single-Cell Printer compared with traditional methods will have immediate and obvious impact on time and costs associated with cell-line development work flows. Expected clonal outgrowth using the SCP (combining efficiency and viability) is, at minimum, 60%; whereas flow cytometry and limiting dilution typically do not exceed 25%. Thus, fewer plates, less media, and less time are required to perform the same work using the SCP approach. Less obvious, but just as impactful, are the advantages SCP brings to downstream processes as well.

Due to the inefficiencies associated with current techniques, most researchers have to cherry-pick their clones prior to screening for titer. This process can be labor-intensive and error-prone if done manually, or alternatively, it may require an expensive liquid handler. With the high efficiencies achieved by the SCP, cherry-picking may no longer be required, simplifying the liquid-handling process, whether it is done manually or through automation. Taken together, these benefits allow for researchers to screen more clones, in less time, at reduced cost, and with higher confidence that a given cell line is monoclonal.


Figure 3. A sequence of five images at the nozzle portion of the cartridge provides evidence for single-cell deposition. The sequence illustrates the path of a single cell prior to and following ejection from the nozzle tip. Images 1–3 show the cell approaching the nozzle. Image 4 shows the detection of a single cell (inner circle) and the absence of any cells in the vicinity (outer circle). Finally, image 5 shows the nozzle after droplet ejection to provide evidence that the single cell was expelled from the nozzle.



























Sarmad Al-Bassam, Ph.D., serves as applications scientist and Steve Wiltgen, Ph.D. (steve.wiltgen@moldev.com), is product manager at Molecular Devices.

Reference
1.  A. Mayer-Bartschmid, “Evaluation of New Single Cell Deposition Technologies to Ensure Monoclonality,” Presentation at the meeting of Cell Line Development and Engineering Amsterdam, NL (April, 2017).

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