Immobilised Cells – Microcarriers

Measuring the cell density of immobilized animal cells grown on microcarriers can be very challenging. With microcarrier cultures, apart from the errors of sampling the heterogeneous suspension, the off-line method (usually based on nuclear counts) only provides a total cell count. Radio Frequency Impedance (RFI) provides a unique on-line method for estimating the live cell biomass in real time. 

Figure 1 shows the application of RFI to monitor CHO cells grown on microcarriers during long term culture runs at Genzyme Inc (USA) for both the growth and harvesting phases. The process has been scaled up and the RFI probes are used in cGMP production. 

Figure 1 – Using RFI probes at Genzyme (USA) to monitor CHO cells grown on microcarriers

Figure 2 demonstrates the use of capacitance to monitor growth of Vero cells at Xenova (UK) on  Cytodex 1 microcarriers, the drops in capacitance correlate with exchanges of media. 

Figure 2 – Vero cells grown on surface microcarriers and monitored with a RFI probe (Courtesy of Xenova, UK)

Figure 3 presents data from a 20-day cultivation of baby hamster kidney (BHK) cells attached  to macroporous microcarriers (courtesy of Novo, Denmark). Capacitance, conductivity, and the  off-line cell counts are shown as a function of time. Samples were taken daily, and the cell  counts were estimated by crystal violet staining followed by manual counting of released nuclei  in a hemocytometer. Vertical lines shown in both capacitance and conductivity indicate the daily  exchange of medium (the impeller is turned off to settle the cells). The off-line cell count on  the macroporous carriers was unable to provide any meaningful data on the progression of the  culture. By contrast, RFI provided valuable real-time information on the viable cell count. A small  daily drop in capacitance signals indicates that essential nutrients are being depleted before  the medium is exchanged.

Figure 3 – Biomass of BHK cell cultivation, cells were attached to macroporous microcarriers and on-line monitoring  commenced for 20 days in a 10 L fermentor (Data courtesy of Novo, Denmark) 

Summary of the benefits

  • Monitor immobilized cells in situ and in real time. 
  • No influence on measurement from microcarriers.  
  • Eliminates/reduces need for sampling 
  • Perform cell density measurements non-disruptively 
  • Obtain fingerprint of the process in real time 
  • Troubleshooting the process 
  • Control critical events during the process