Aggregated Cells & Cells on Microcarriers

Nuclei counting was first described by Sanford et al. in 1951 and modified by van Wezel. Using this method, cells are permeabilized using hypotonic solutions and nuclei are stained and counted using a dye solution, e.g. crystal violet. This often leads to nuclei overestimation as nuclei cannot be distinguished accurately from debris.

To overcome this problem, the image cytometers by ChemoMetec A/S offer nuclei counting using fluorophores, enabling a clear distinction between nuclei and cellular debris and ensuring a fast and reliable method for nuclei determination.

Vero cells are anchorage dependent cells and can be cultivated on microcarriers for scaling up the production to achieve yields of several million cells per milliliter in large-scale fermenters. Monitoring microcarrier cultures is challenging and time-consuming, as the cells need to be detached in a multistep process from the microcarriers.

Untreated Vero cells appeared as a confluent cell layer on the microcarriers. Already after two minutes of incubation time, the cell layer was dissolved. Reagent A100 was not able to dissolve all aggregates, noticeable in non-lysed cell clumps between remaining aggregated microcarriers after one minute of incubation.

Results

HEK293 cells form cell aggregates of up to 3 mm in diameter. Using automated cell counters, many algorithms are not able to determine the correct number of cells due to the three-dimensional appearance, leading to imprecise results. The lysis using Reagent A100 led to a uniform distribution of nuclei and the algorithm was able to distinguish and count the single nuclei.

Image A shows a raw data scatter plot of DAPI area over intensity of stained cells in blue with the standard gating strategy. Multiple acquired images (one displayed in B) were used for automated cell count determination. Image C displays single nuclei in detail, and the image overlay for highlighting individual cells.

See full dataset in the PDF poster

The A100 & B assay was used to count Vero cells grown on Cytodex 1 microcarriers. Already after two minutes of incubation time with reagent A100, the maximum number of cell nuclei could be detected, and the total cell count determined.

Furthermore, we tested the same protocol for highly aggregated HEK293 cells. The A100 & B assay was able to dissolve the HEK293 aggregates, resulting in a single cell suspension. Stained cell nuclei formed a uniform distribution which doubled the amount of counted total cells in comparison to counting un-lysed cells.

For us at Hookipa, counting nuclei using the NucleoCounter^® NC-3000™ is a precise and fast way to determine total cell count and viability of highly aggregated HEK293 cells and Vero cells on microcarriers.

Authors

Philipp Andre and Andreas Aspöck.