Application Note - Real-Time Cell Detachment Monitoring in Microcarrier Culture by Incyte Sensors

Controlled cell detachment during  scaling up of microcarrier-based culture

Microcarrier-based platforms have revolutionized scalable cell culture for diverse applications, from vaccine production to cell therapy and cultured meat. These cultures progress through attachment, growth, and detachment phases, with viable cell density (VCD) monitoring via capacitance sensors already established as compliant with PAT (Process Analytical Technology) guidelines for enhanced process optimization and control. However, real-time monitoring of cell detachment, critical for timely bioreactor transfer and scale-up, remains challenging, as traditional methods rely on off-line sampling and microscopy, which disrupt workflow, reduce precision, and increase contamination risk, especially at larger scales.

In our study at ACIB Co., we applied in-line capacitance-based monitoring to track MA 104 cell detachment from Cytodex 1 microcarriers using Hamilton’s Incyte sensor. This integration provided real-time insights into detachment status, enabling immediate decision-making and improved process consistency. By supporting automated, real-time data collection, capacitance monitoring strengthens PAT’s role in scaling microcarrier cultures, extending its applications beyond vaccines to hMSC and cultured meat production. 

In-situ monitoring of cell detachment from  microcarrier by Incyte sensor

The real-time permittivity signal generated by the Incyte sensors was evaluated during the detachment of MA 104 cells from Cytodex 1 microcarriers, conducted in a 1-liter DASGIP bioreactor system (Eppendorf, DASGIP® Bioblock parallel system, 700 mL working volume) (Figure 1). Data acquisition occurred throughout the cell detachment process (maximum 1 hour), utilizing two different generations of Incyte Arc sensors (Incyte Unit DN12) and Incyte  Arc Expert (Figure 2) with different final concentration of Trypsin-EDTA solution as the dissociation reagent.

The Incyte Arc Expert incorporates an integrated preamplifier, representing a significant design enhancement. The capacitance measurements were performed at a frequency of 1 MHz for all experiments, a parameter optimized for mammalian cell applications. During the detachment process, off-line samples were systematically collected to assess cell density and viability using the Trypan Blue assay and calculating the detachment efficiency. Complete dissociation of cells from microcarriers was verified through microscopic examination.

Result

The results depicted in Figure 3 demonstrated the detachment efficiency (Figure 3a) and the evolution of the permittivity signal from Incyte sensor (Figure 3b) during cell detachment process for varying final concentration of the Trypsin.

Upon the addition of the enzyme solution, the permittivity signal exhibited a pronounced decline in the f irst 5 minutes. Based on the off-line measurements shown in Figure 3a, it can be concluded from Figure 3b that complete cell detachment from the microcarriers (MCs) led to a decrease in the permittivity signal to levels below 10 pF/cm. Higher enzyme concentrations (> 0.82 mg/mL) facilitated a more rapid and pronounced decrease in the permittivity signal compared to lower concentrations (0.64 mg/mL). Conversely, at very low enzyme concentrations (0.43 mg/mL), while the signal initially declined due to dilution of the solution, it failed to achieve the same minimal permittivity values and instead stabilized at a higher plateau.

The results from the on-line measurement of the permittivity signal during in-situ cell detachment demonstrate the potential of the Incyte sensor as a tool for monitoring enzyme activity and predicting the likelihood of complete cell detachment. A predictive model was established using permittivity signals recorded between minutes 4 and 7 of the detachment process. The model forecasts the permittivity  signal at 20 minutes, enabling the prediction of the f inal cell detachment outcome. Figure 4 illustrates the predictive accuracy (Root Mean Square Error (RMSE) = 4.6) and performance of the developed model.

To validate the consistency and reproducibility of the signal obtained with the Incyte sensors, experiments were conducted using the Incyte Arc sensor at enzyme concentrations of 0.86 g/L and 0.43 g/L. The results, compared in Figure 5, showed similar outcomes, with the primary difference being a steeper decline observed with the Incyte Arc sensor. This faster response indicates improved sensor technology, likely due to enhanced sensitivity. 

The experiments demonstrated

The implementation of real-time cell detachment monitoring using a capacitance sensor demonstrated its effectiveness in assessing detachment efficiency and predicting detachment status. This feature supports prompt decision-making for the next  steps in the process and improves overall control. Moreover, it reduces the reliance on offline sampling.

Furthermore, the proposed real-time monitoring method shows significant potential, especially for detaching stem cells from microcarriers, as microcarrier-based cell propagation became an established and widely accepted procedure for them. Due to the sensitivity of these cells, accurately predicting the optimal timing for enzyme solution inactivation can help prevent potential damage to essential surface markers that are vital for their differentiation.

Authors: Atefeh Ebrahimian, Harald Kühnel Austrian Center of Industrial Biotechnology (ACIB), FH campus Wien