In this article, Cell Microsystems explains the challenges that come with limiting dilution, and how they can be overcome with recent solutions, such as the CellRaft Technology for cell line development.
Sponsored Content by Cell MicrosystemsJul 12 2023Reviewed by Louis Castel Therapeutic proteins play a crucial role in the biological pharmaceutical sector, serving as treatment options for diseases such as diabetes, cancer, and anemia.
In contrast, the innovative CellRaft® Technology offers a streamlined and efficient solution for cell line development by combining the flask-like cell culture conditions of the CellRaft Array cell culture consumable with the automated isolation and imaging capabilities of the CellRaft AIR® System.
This approach proves to be time-consuming and requires a considerable amount of consumable resources. Plating cells at single-cell density often results in low efficiency, decreasing cell viability. Monoclonal cell line development using CellRaft technology The generation of monoclonal cell lines can be effectively accomplished using the innovative CellRaft AIR System in conjunction with the CellRaft Array. The initial step involves seeding cells onto the CellRaft Array, a disposable microwave substrate for culturing and imaging purposes.
The CellRaft AIR System comprises an optical system capable of performing bright field and three-channel fluorescent imaging. Conveniently, the software stores the scans in a centralized file for each array, enabling the analysis of phenotypic characteristics of cells of interest and facilitating the tracking of individual CellRafts and colony growth over time.
Overall, this workflow significantly reduces hands-on time and substantially enhances output, presenting a more time-efficient, labor-saving, and cost-effective alternative for cell line development.The wand picks up the microwell using a magnet.
Plates were incubated at 37 °C with 5% CO2 and monitored for clonal outgrowth. In the case of cell seeding on the CellRaft Arrays, two arrays were prepared: one with 100S CellRafts and another with 200S CellRafts. These arrays were seeded with 20,000 cells and 5,000 cells, respectively, using standard procedures .
Figure 3. Two-series serial dilution for limiting dilution plates usedto generate single cells/well. The region of expected single cell clones is identifi ed in yellow. Image Credit: Cell Microsystems Leveraging the proprietary software of the CellRaft AIR System, we successfully identified over 5,400 single cell-derived clones on the 100S array and 1,100 single cell-derived clones on the 200S array, all available for isolation into 96-well plates.
Of the total wells populated from the 100S and 200S arrays, 256 and 275 , respectively, exhibited continued outgrowth, vastly outnumbering the 27 colonies in the three LD plates that had yet to be genotypically confirmed as clonal. Seven days post-isolation into 96-well plates, colonies were dissociated for expansion using 0.25% trypsinEDTA to disperse cells in the 96-well plates.
Conclusions The CellRaft AIR System and CellRaft Array demonstrated superior performance compared to limiting dilution in the development of monoclonal CHO-K1 cell lines.
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