Using NucleoCounter® Instruments to Monitor the Cell Count of Microcarrier Cultures

Introduction

NucleoCounting provides a reliable and accurate approach to determine the cell count and viability in microcarrier cultures. Two reagents are used in this method, and these reagents quickly release the nuclei from the microcarriers.

The nuclei released are placed in a disposable Via1-Cassette™, which, in turn, is placed inside the NucleoCounter™. This instrument automatically carries out fluorescent staining, image acquisition, and image analysis. It takes less than 5 minutes to complete the entire process and can be carried out without using pipettes.

Microcarriers: A scalable culture system for adherent cells

Figure 1. Microcarrier bioreactor. Microcarriers offer a convenient method for growing adherent cells in bioreactors.

It can be difficult to scale up the production of cells and viruses. When there is a need for high production volumes, cells culture flasks are not a viable option for industrial scale production. Microcarriers make it easy to grow adherent cells in bioreactors, and they act as a scaffold to which adherent cells can bind to.

This enables the adherent cells to proliferate easily, while the cell-microcarrier complex is allowed to suspend freely in the media by a bioreactor. Therefore, adherent cell lines are cultured like suspension cells, simplifying the scaling up process and enabling the current resources to be used for production and process optimization.

NucleoCounting

The NucleoCounting method fluorescently stains cell nuclei in order to detect the cells. The fluorescent dye DAPI, which is very specific to DNA, is used to label the cells and it allows the cell nuclei to be accurately detected even when cellular debris is present.

The unique Via1-Cassette™, which is inserted into a NucleoCounter NC-200™ or NC-3000™ instrument that measures the total cell count and viability, combines fluorescent staining, cell sampling, and counting chamber loading into a single workflow.

NucleoCounting saves time when counting cells in microcarrier cultures

During optimization and monitoring of large-scale bioproduction, cell count and viability are very important parameters. Traditionally, cells that are cultured on microcarriers are counted using a multistep method, where trypan blue staining and trypsin digestion are used. However, this method is not only inaccurate, but also takes a long time.

The NucleoCounter workflow demonstrates that the NucleoCounter™ eliminates several pipetting, incubation, and centrifugation steps when compared to the conventional trypsin digestion method. In less than 5 minutes, the entire cell counting process can be completed using the NucleoCounter™ instrument.

Literature:

  1. Lam AT, Chen AK, Li J, et al., (2014), Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a definedmicrocarrier spinner culture., Stem Cell Res Ther, Sep 15;5(5):11010.1186/scrt498
  2. Lam AT, Li J, Chen AK, et al., (2014), Cationic surface charge combined with either vitronectin or laminin dictates the evolution of human embryonicstem cells/microcarrier aggregates and cell growth in agitated cultures., Cell Therapy, Jul 15;23(14):1688-703, 10.1089/scd.2013.0645
  3. Heathman TR, Stolzing A, Fabian C, et al., (2016), Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrierculture using human platelet lysate, Cancer Research, Apr;18(4):523-3510.1016/j.jcyt.2016.01.007
  4. Heathman TR, Glyn VA, Picken A, et al., (2015), Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrierprocess., Biotechnol Bioeng, Aug;112(8):1696-70710.1002/bit.25582
  5. Lam AT, Li J, Chen AK, et al., (2015), Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-CoatedMicrocarriers Results in Efficient Growth in Agitated Cultures, Biores Open Access, Apr 1;4(1):242-5710.1089/biores.2015.0010
  6. Chen AK, Chen X, Choo AB, et al., (2010), Expansion of human embryonic stem cells on cellulose microcarriers., Curr Protoc Stem Cell Biol., Sep;Chapter 1:Unit 1C.1110.1002/9780470151808.sc01c11s14
  7. Marinho PA, Vareschini DT, Gomes IC, et al., (2013), Xeno-free production of human embryonic stem cells in stirred microcarrier systems using a novelanimal/human-component-free medium., Tissue Eng Part C Methods., Feb;19(2):146-55.10.1089/ten.TEC.2012.0141
  8. Lecina M, Ting S, Choo A, et al., (2010), Scalable platform for human embryonic stem cell differentiation to cardiomyocytes in suspended microcarriercultures., Stem Cell Res Ther, Dec;16(6):1609-19.10.1089/ten.TEC.2010.0104

About ChemoMetec A/SChemoMetec A/S

Founded in 1997, Chemometec specialises in the design, development and production of high quality instruments using patented technology for a wide range of applications in cell counting and evaluation.

Chemometec work closely with life science companies, research institutes, universities, hospitals, specialist clinics and a wide range of food and beverage manufacturers, matching our technical development expertise to customer needs - with quality results, reliability, cost-efficiency and ease-of-operation as our guiding principles.

Chemometec support a growing customer base worldwide with responsive technical services and the ready availability of a wide range of consumables.


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Last updated: Jun 15, 2017 at 5:39 AM

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