Cell Counting & Viability in Microcarrier Cultures

Using NucleoCounter® Instruments for Adherent Cells

Scaling up the culture of stem cells, virus-producing cells, or other cells for drug production and therapeutic uses can be challenging. Microcarriers offer a convenient method for growing adherent cells in bioreactors. They allow for culturing a large number of cells in a smaller volume and help to reduce process complexity and labor intensity even in large-scale productions.

Microcarriers serve as a scaffold that adherent cells attach to, allowing them to proliferate while a bioreactor keeps the cell-microcarrier complex freely suspended in the media. Therefore, adherent cell lines are grown like suspension cells, simplify scaling and allow existing resources to be leveraged for process optimization and production.

Typical materials used for microcarriers are acrylamide, alginate, dextran, gelatin, glass and polystyrene, which are then coated with Matrigel or other types of base membrane polymers, or Matrigel-like coatings for xeno-free culture systems used in cell therapy applications.

NucleoCounter® Measures Cell Count & Viability in Microcarrier Cultures

Cell count and viability are crucial parameters for optimizing and monitoring large-scale bioproduction. Cells grown on microcarriers have traditionally been counted using a multistep process that involves trypsin digestion and trypan blue staining. This method is both time-consuming and inaccurate.

The NucleoCounter® detects cells by staining cell nuclei. The cells are made visible using the fluorescent dyes acridine orange (AO) to determine total cell numbers and 4′,6-diamidino-2-phenylindole (DAPI), which are highly specific to DNA, detecting cell nuclei accurately, even in the presence of cellular debris. Cell sampling, fluorescent staining and counting chamber loading are combined into a single workflow by the unique cassette technology, which is loaded into the NucleoCounter® NC-202™ or NC-3000™, and together with the instruments, calculate total cell count and viability.

Use the NucleoCounter® NC-202™ if you need cell data parameters on cell count, viability, cell concentration, DebrisIndex™, and cell aggregation. Use the NucleoCounter® NC-3000™ when you want to combine cell count data with one of our cell assays or co-stain for other proteins of interest using the FlexiCyte™ Assay.

A Faster & More Accurate Way to Measure Microcarrier Cultures

Automated cell counting using a NucleoCounter® is an accurate and reliable method for measuring cell count and viability in microcarrier cultures. The NucleoCounter® instrument protocol makes use of a lysis step immediately before the cell counting step, rapidly releasing nuclei from the microcarriers into suspension.

The operator simply loads the sample containing released nuclei into the disposable Via2-Cassette™, pre-loaded with fluorescent dyes for automated fluorescent staining. The image acquired by the NucleoCounter® is then automatically processed by its software, which handles data acquisition, image analysis and data presentation. The entire procedure takes less than five minutes and can be performed without the use of pipettes, dilutions and multi-step staining procedures.

A comparison of the traditional trypsin digestion method and the NucleoCounter® workflow shows that the NucleoCounter® removes several centrifugation, pipetting and incubation steps.

References

  1. AT Lam, AK Chen, J Li et al.: Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture. 2014; Stem Cell Res Ther, Sep 15;5(5)
  2. AT Lam, J Li, AK Chen et al.: Cationic surface charge combined with either vitronectin or laminin dictates the evolution of human embryonicstem cells/microcarrier aggregates and cell growth in agitated cultures. 2014; Cell Therapy, Jul 15;23(14):1688-703
  3. TR Heathman, A Stolzing, C Fabian et al.: Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate. 2016; Cancer Research, Apr;18(4)
  4. TR Heathman, VA Glyn, A Picken et al.: Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process. 2015; Biotechnol Bioeng, Aug;112(8)
  5. AT Lam, J Li, AK Chen et al.: Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures. 2015; Biores Open Access, Apr 1;4(1)
  6. AK Chen, X Chen, AB Choo et al.: Expansion of human embryonic stem cells on cellulose microcarriers. 2010; Curr Protoc Stem Cell Biol., Chapter 1
  7. PA Marinho, DT Vareschini, IC Gomes et al.: Xeno-free production of human embryonic stem cells in stirred microcarrier systems using a novelanimal/human-component-free medium. 2013; Tissue Eng Part C Methods., Feb;19(2)
  8. M Lecina, S Ting, A Choo et al.: Scalable platform for human embryonic stem cell differentiation to cardiomyocytes in suspended microcarrier cultures. 2010; Stem Cell Res Ther, Dec;16(6)

Newsletter
Contact us
Get a quote