In cell line development, optimizing the outgrowth of clones from single cells is a challenging process. One step in this process is single-cell seeding. High seeding efficiencies of single cells per plate can be achieved with the Solentim VIPS™ (verified in-situ plate seeding) benchtop system. To ensure sterility, VIPS may be placed within a standard class II biological safety cabinet (Figure 1). All the tubes and other components that contact cells can be easily sterilized or replaced to avoid any reliance upon expensive proprietary consumables.

Figure 1. VIPS system

The system dispenses a single droplet from the cell reservoir, which contains an agitated cell suspension, into the bottom of an empty well in a 96-well plate followed by immediate imaging of the droplet. The single-cell image feedback loop works by assessing these images and determining if a cell was deposited or not. If a cell was deposited, the well will be filled with media immediately. If a cell is not detected, the system will deposit another drop to the side and image again. This process will repeat up to a maximum of 16 times per well. The system uses a stack of images to assess the presence of a cell in the droplet.

The VIPS seeding results are reported in real time so that the user can see which wells have been populated (single cells, more than one cell, or no cell), and VIPS achieves the entire dispensing of a 96-well plate in approximately 10 minutes.

Achieving good outgrowth of high-producing clones is also essential. Following single-cell cloning, the outgrowth of the cells is impacted by many factors. These include the pretreatment conditions, the choice of gene editing technology, the molecules expressed by the cells, and the media and reagents used. The process of cloning itself introduces stress on the cells as they are isolated.

Cell line optimization is therefore a vital part of any cell line development process to ensure good recovery of clones. Here we demonstrate the combination of the VIPS system, which increases seeding efficiency and reduces the number of plates required to screen the HD-BIOP3 GS Knockout CHO K1 cell line (Horizon Discovery), and InstiGRO CHO™ (SAL Scientific) reagents, which boost outgrowth.

Methods and materials

Cells were passaged 24 hours prior to seeding. On the day of seeding, cells were diluted to 9500 cells/mL in OptiCHO™ (Gibco). These cells were then used for VIPS seeding with the appropriate growth media dispensed following seeding. For manual limiting dilution (LD), the 9500 cells/mL in OptiCHO were further diluted to 2.5 cells/mL in the appropriate media (Gibco CD CHO or CD FortiCHO™ with conditioned media or InstiGRO CHO). Five 96-well plates for each condition were seeded.

The same plate type (Corning Costar 3300) was used for all conditions, and the final well volume for all was 200 µL. Cells were incubated in the same incubator at 37 °C and 5% CO2. Plates were imaged on the Solentim Cell Metric® 2 hours after seeding (day 0) and on days 1, 7, and 14. Images were assessed by eye to confirm clonality for manual LD and VIPS. Results displayed are the average of the five plates.

Comparison was carried out of InstiGRO CHO and InstiGRO CHOPLUS. Seeding conditions were identical to those described in the first experiment. Base media was FortiCHO supplemented with InstiGRO CHO or InstiGRO CHOPLUS.

Results and discussion

Comparisons were made of manual LD and VIPS seeding with different base media and conditioned media vs. InstiGRO CHO. VIPS seeding of the HD-BIOP3 cell line with InstiGRO for outgrowth resulted in approximately twofold increase in clonally derived colonies per plate when compared to manual LD (Figure 2). In this case, clonal outgrowth values are taken from a whole 96-well plate to allow comparison of manual LD to VIPS seeding.

Figure 2. Comparison of clonal outgrowth following manual LD and VIPS seeding with conditioned media and InstiGRO CHO.

The VIPS reports single-cell seeding; therefore, outgrowth can be calculated as a percentage of the known seeded, discounting empty wells (Figure 3).

Figure 3. Comparison of clonal outgrowth when using conditioned media and InstiGRO CHO reagent with VIPS seeding.

 

InstiGRO CHO resulted in clonal outgrowth of 32% when compared to the use of conditioned media alone (12–17%), thereby doubling the number of clonal colonies achieved per plate. VIPS seeding also resulted in reduction in the number of colonies derived from multiple cells when compared to plates seeded manually (Figure 4).

Figure 4. Cell Metric overview images for VIPS seeded plates and manual LD seeded plates. The red crosses represent wells where colonies arose from more than one cell.

The data in Figure 4 demonstrate that VIPS seeding results in a higher number of clonally derived colonies per plate and that InstiGRO CHO improves outgrowth twofold when compared to conditioned media for the HD-BIOP3 GS Knockout CHO K1 cell line, thereby reducing the number of plates requiring screening.

Further optimization of VIPS seeding for the HD-BIOP3 GS Knockout CHO K1 cell line was carried out comparing InstiGRO CHO to a new version of the supplement, InstiGRO CHOPLUS, using FortiCHO as the base media (Figure 5).

Figure 5. Comparison of InstiGRO CHO and InstiGRO CHOPLUS reagents with Horizon Discovery’s HD BIOP3 GS Knockout CHO K1 cell line.

 

InstiGRO CHOPLUS resulted in a 10% increase in clonal outgrowth when compared to InstiGRO CHO, resulting in 46% of single-seeded wells forming colonies. In order to test the conditions optimized on the parent cell line, the performance of InstiGRO CHOPLUS was then assessed with two pools expressing a relevant molecule (Figure 6).

Figure 6. Clonal outgrowth of two transfected pools expressing a molecule supplemented with InstiGRO CHO(PLUS).

 

As Figure 6 demonstrates, InstiGRO CHOPLUS supplementation results in 12–21% clonal outgrowth of pools expressing a relevant molecule. The results also demonstrate the impact of expression of a molecule on clonal outgrowth and highlights the importance of optimizing conditions in order to achieve clonal outgrowth that is as high as possible. It also demonstrates the performance difference between pools expressing the same molecule and derived from the same parent cell line.

Figure 7 demonstrates an example of one of the clones from seeding (day 0) through day 7 growth, as well as an example of VIPS identifying a well with multiple cells seeded within a drop (day 7).

Figure 7. (A–C) Example of a single seeded cell forming a colony. (A) VIPS seeding image. (B) Day 0 image on the Cell Metric. (C) Day 7 image. (D) Example of a well seeded with multiple cells identified by VIPS.

Conclusion

Overall, our data show that the combination of the VIPS for high seeding efficiency and the improved outgrowth conditions using the InstiGRO cloning supplement with Horizon Discovery’s HD-BIOP3 GS Knockout CHO K1 cell line is a powerful platform for cell line development. The increased number of single cells seeded per plate and the improved outgrowth using InstiGRO CHO and InstiGRO CHOPLUS enables the number of plates required for screening to be significantly reduced.

 

Camilla Domeneghetti and Amanda Holmes, PhD, are lab scientists, Andrea Gough is a product manager, Clare Richards, PhD, serves as head of biological sciences, and Ian Taylor, PhD (ian.taylor@solentim.com), is director of sales and marketing at Solentim.

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