for Hybridoma Technology
Hybridoma technology includes cell fusion, the plating of master cell cultures, and antibody screening. The ultimate goal is to identify a single hybridoma cell producing a monoclonal antibody with a desired specificity or function. Achieving cell clonality is important from a process control as well as a regulatory perspective. Until recently, hybridoma-producing laboratories have pursued a labor-intensive and time-consuming serial path of hybridoma culturing and limiting dilution cloning to achieve clonality.
This Application Note addresses the need to verify hybridoma cell clonality in an automated fashion. The ImageXpress® Velos System offers sensitive, rapid, and robust screening of multi-well plates for the identification of wells containing a single cell as well as characterization of colonies grown from those cells.
Hybridoma cells were grown in clear polystyrene tissue culture plates. The hybridoma line, growth medium and the quantitation of human IgG were provided by Medarex (Milpitas, CA). The ImageXpress Velos System was configured with a 488 nm laser. The laser scatter signal was collected in channel 1 and fluorescence was filtered through a 510-540 nm band pass filter for channel 2. A 96-well Costar tissue culture plate was loaded into the plate nest of the ImageXpress Velos System for scanning and all instrument settings including the gains for the PMTs were automatically configured by the ImageXpress Velos System method (see Figure 1). Plates were typically scanned at a resolution of 10µ; scantime at setting is 120 sec.
Hybridoma Master Cultures
Hybridoma master cultures were established in 96-well clear polystyrene tissue culture plates after cell fusion on Day 0. The ability to use standard clear polystyrene tissue culture plates of 6, 12, 24, 48, or 96-well densities has been demonstrated previously. The 96-well plates were scanned on day 6, 8 and 11. A half-volume media replacement was done on day 7. As shown in Figure 2 the scatter image clearly shows the growth of the hybridoma colony versus number of days in culture. The lid remained on the plate during the scanning to maintain sterility
Single Cell Analysis in Tissue-Culture Plates
While it is possible to detect cells with laser scatter, a more robust approach is to stain them with (CMFDA, Molecular Probes, Eugene, OR) and measure fluorescence. Images of a single cell and a colony grown from that cell, both stained with CMFDA, are shown in Figure 3. A complication arises from laser induced fluorescence (LIF) from the clear polystyrene plates (Note: this is not an issue with black wall plates). These signals, seen in the right side of the wells in Figure 3, limit the ability to clearly define cells using an image threshold approach. However, the detection of single cells can be accomplished by using a fast Fourier transform (FFT) to remove the unwanted signal. The results of this process are shown in Figure 4, where the initial image shows a well with two CMFDA-stained cells, followed by a FFT filter process, and finally thresholding and measurement of single cells. Table I shows the number of cells counted per well on Day 0 from plating 0.5 cells/well into a 96-well plate using this technique. The resolution setting of 10µ provides a sufficient number of data points per cell for robust detection and allows a scantime of 120 sec.
Image after FFT Filter
Table I. Number of cells per well of a 96-well plate on Day 0
No Effect of CMFDA-labeling on Hybridoma Growth or Colony Formation
The repeated labeling of the hybridoma cells with CMFDA and scanning process had no effect on the cells ability to grow and form colonies, as shown by the representative colony in Figure 5. Moreover, additional studies demonstrated i) that antibody production was not affected by CMFDA-staining and ii) antibody detection could be linked to wells containing a single CMFDAlabeled cell (data not shown).
Compared with previously available methods, the use of the ImageXpress Velos System allows the fast analysis of large numbers of wells in multi-well plates for secreted products on the single-cell level. Critical to this is the ability to scan complete wells and perform multiparametric analysis of cells and colonies. Specifically we have demonstrated the capability to perform:
- Rapid cell counting of CMFDA-stained cells in standard tissue culture plates
- High throughput and rapid single cell detection
- Growth monitoring of colonies
- Multi-color fluorescence analysis of live clones (transfectomas)
The ability to rapidly identify wells containing a single cell has the potential of accelerating the antibody discovery process. The technology is broadly applicable for the detection of secreted products as well as the identification of transfected cells expressing the desired product. In addition, the platform provides the ability to normalize the amount of secreted product to the number of cells. These features are especially important for bioprocess development where the selection of high producing cell lines is an important step.