How to count cells grown on Microcarriers more efficiently

Takes just two minutes with NucleoCounter® Cell Counters

Microcarriers offer a convenient method to grow adherent cells in bioreactors. Cells used in vaccine production, such as Vero cell lines and human mesenchymal stem cells (hMSCs) often rely on this method.1–4

Microcarriers allow for culturing a large number of cells in a smaller volume, reducing process complexity and labor requirements.

Microcarriers provide a large surface area for adherent cells to attach to while allowing them to proliferate in a bioreactor in free suspension (Figure 1).

Microcarrier cell culture allows for large-scale production and can easily be integrated into existing processes.5

microcarriers in bioreactor

Protocol—takes just two minutes

Before counting cells grown on microcarriers, cells are typically separated via trypsinization. The cells are then counted via the trypan blue exclusion method, using a hemocytometer. This process may take upwards of 40 minutes due to the number of additional manual steps—all of which may introduce human error (Figure 2)

Manual Vs NC-202 for microcarriers

When using the NucleoCounter® NC-202™, the process takes just two minutes. It works by fluorescently staining cell nuclei, in two steps (Table 1). Refer to our App Note for the full protocol.

Table 1. Counting Cells grown on microcarriers using the NC-202™

Step Action Result
1 Add lysis buffer, vortex, and count using the Via2-Cassette™ Total cell count
2 Count the cells, without lysing, using the Via2-Cassette™. Dead cell count

A faster & more accurate way to track microcarrier cell culture

Die Via2-Cassette™ mit all ihren Funktionen skizziert und erklärt.

The NC-202™ works with the disposable Via2-Cassette™ (Figure 3), where cell samples are loaded. Inside the cassette, the sample is instantly stained by the fluorescent dyes acridine orange (AO) and 4′,6-diamidino-2-phenylindole (DAPI), which are pre-loaded in the cassette. This technology combines cell sampling, staining and loading into a single workflow.

In the first step, cells are lysed using our Lysis 1 buffer, an acidic solution that rapidly releases nuclei from the microcarriers into suspension. When this sample is loaded into the NC-202™, the NC-View™ software provides total cell count.

In the second step, the intact microcarriers are sampled by the Via2-Cassette™ and inserted into the NC-202™ without any lysing buffer, just like any other cell sample. The DAPI inside the cassette stains dead cells, revealing the dead cell count. The software then provides viability for the entire sample.

Once the sample is loaded, the Via2-Cassette™ is placed into the NC-202™ and the user presses “run.” Just 30 seconds later, the NC-View™ software displays the cell count and viability

Automated. Precise. Fast.

Automated cell counting using the NC-202™ is an accurate and reliable method for measuring cell count and viability in microcarrier cultures.

The NucleoCounter® workflow removes several centrifugation, pipetting and incubation steps, minimizing the chance of human error. This results in better precision, reproducibility and final product, while saving time.

Learn more about the NucleoCounter® NC-202™

References

  1. Yang J, Guertin P, Jia G, Lv Z, Yang H, Ju D. Large-scale microcarrier culture of HEK293T cells and Vero cells in single-use bioreactors. AMB Express. 2019;9(1). doi:10.1186/s13568-019-0794-5
  2. Nienow AW, Rafiq QA, Coopman K, Hewitt CJ. A potentially scalable method for the harvesting of hMSCs from microcarriers. Biochem Eng J. 2014;85:79-88. doi:10.1016/j.bej.2014.02.005
  3. Kiesslich S, Kamen AA. Vero cell upstream bioprocess development for the production of viral vectors and vaccines. Biotechnol Adv. 2020;44. doi:10.1016/j.biotechadv.2020.107608
  4. Phillips BW, Horne R, Lay TS, Rust WL, Teck TT, Crook JM. Attachment and growth of human embryonic stem cells on microcarriers. J Biotechnol. 2008;138(1-2):24-32. doi:10.1016/J.JBIOTEC.2008.07.1997
  5. Lev R, Bar-Am O, Lati Y, Guardiola O, Minchiotti G, Seliktar D. Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier. Gels. 2023;9(3). doi:10.3390/gels9030243