Parameter Optimization and Capacitance-Based Monitoring of In Situ Cell Detachment in Microcarrier Cultures
Abstract
:1. Introduction
2. Materials and Methods
2.1. Static Culture in T-Flask
2.2. MC Cell Culture in Bioreactor
2.3. In Situ Cell Detachment in Bioreactor and Subculturing to the Subsequent Bioreactor
2.4. Selection of Critical Parameters for Experimental Design in In Situ Cell Detachment Studies
2.5. Analytical Techniques
2.5.1. Cell Count Determination
2.5.2. Trypsin Activity Assay
2.5.3. Apoptosis and Necrosis Analysis
2.5.4. Online Viable Cell Monitoring Using Capacitance Sensor
2.6. Experimental Design for Model Development of Cell Detachment from MCs
2.7. Model Development of In Situ Cell Detachment from MC
2.8. Statistical Analysis
3. Results and Discussion
3.1. Effect of Agitation Speed on Cell Detachment from MCs and Subsequent Cell Growth
Trypsin ratio (mL/gMC) | 50 | 90 | ||
Volume (V) (mL) * | 245 | 329 | ||
Agitation speed (N) (rpm) | 100 | 200 | 100 | 200 |
0.261 | 0.523 | 0.261 | 0.523 | |
4429 | 8859 | 4429 | 8859 | |
0.0088 | 0.0708 | 0.0066 | 0.052 | |
98.5 | 58.5 | 106.0 | 63.0 | |
Smallest possible eddy size λ min (μm) ** | 68.9–78.3 | 40.9–46.5 | 68.9–78.3 | 40.9–46.5 |
3.2. Effect of Incubation Time on Cell Detachment from MCs and Subsequent Cell Growth
3.3. Effect of Trypsin Volume on Cell Detachment from MCs and Subsequent Cell Growth
3.4. Effect of Washing Steps on Cell Detachment Efficiency and Subsequent Cell Growth
3.5. Monitoring Cell Growth on MCs Using Capacitance Sensor
3.6. Monitoring In Situ Cell Detachment by Capacitance Sensor
3.7. Decision-Making Tools for In Situ Cell Detachment
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Parameter | Lower Limit | Upper Limit | Other Parameters’ Setpoints |
---|---|---|---|
Agitation speed (rpm) | 100 * | 200 * | 90 mL/gMC, 30 min, 2X PBS wash |
Incubation time with enzyme (min) | 20 | 150 | 90 mL/gMC; 100 rpm, 2X PBS wash |
Trypsin–EDTA (0.25%) volume, mL/gMC | 50 | 90 | 20 min, 100 rpm, 2X PBS wash |
PBS washing steps (40% of initial volume) | 1 | 2 | 20 min, 100 rpm, 90 mL/gMC |
3c | 10c | 50c | 250 L | 1000 L | |
---|---|---|---|---|---|
Bioreactor working volume (L) | 1.25–3.75 | 3.3–10 | 18–40 L | 125–250 | 500–1000 |
Actual culture volume (L) | 2.5 | 10 | 40 | 190 | 900 |
MC concentration (g/L) * | 3.0 | 3.8 | 3.9 | 3.8 | 3.8 |
Vessel diameter (cm) | 14.7 | 20.4 | 33.7 | 59.7 | NA ** |
Blade width + off-bottom clearance (cm) | 7.0 | 9.0 | 16.5 | 30.0 | NA |
Minimum required volume to cover the impeller | 1.18 | 2.943 | 14.71 | 83.93 | NA |
20% remaining (L) *** | 0.7 | 2 | 8 | 50 | NA |
Minimum volume of enzyme solution (L) | 0.44 | 0.94 | 6.71 | 33.93 | NA |
Minimum volume of enzyme solution/gMC | 46.2 | 24.8 | 43.1 | 47.0 | NA |
Run No. | MC Conc. (g/L) | Culture Volume (mL) | Trypsin–EDTA Conc. (mg/mL) * | Trypsin–EDTA Volume (mL/gMC) | Final Trypsin Conc. (mg/mL) | Final Trypsin Activity (USP/mL) ** |
---|---|---|---|---|---|---|
1 *** | 3 | 700 | 2.5 | 90 | 1.65 | 1465.2 |
2 **** | 3 | 700 | 2.5 | 50 | 1.30 | 1150.9 |
3 | 3 | 700 | 1.25 | 90 | 0.86 | 767.2 |
4 | 3 | 700 | 1.25 | 90 | 0.82 | 732.6 |
5 | 3 | 700 | 1.25 | 50 | 0.64 | 575.4 |
6 | 3 | 700 | 0.830 | 50 | 0.43 | 383.6 |
Strain | Medium Condition | Washing Ratio (%iv *) | Washing Steps | Reference |
---|---|---|---|---|
Vero | SCM ** | 30 | 2–3 | [42] |
hMSC | SCM | 41.5 | 3 | [44] |
hMSC | SFM/SCM | NA | 2 | [37] |
Vero | SFM *** | - | 0 | [17,50] |
hMSC | SFM | 20 | 2 | [38] |
hMSC | nm **** | 100 | 1 | [51] |
Sensor | Final Enzyme Conc. (g/L) | Predicted Signal at 20 min | Predicted Status | Measured Signal at 20 min | Actual Status |
---|---|---|---|---|---|
Incyte Unit DN12 | 0.43 | 50.3 | No active enzyme, no detachment | 55.6 | No cell detachment |
Incyte Arc | 57.1 | No active enzyme, no detachment | 59.8 | No cell detachment | |
Incyte Unit DN12 | 0.86 | 0 | Fast and complete detachment | 6.54 | Complete cell detachment but slow |
Incyte Arc | 0 | Fast and complete detachment | 6.91 | Complete cell detachment but slow |
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Ebrahimian, A.; Schalk, M.; Dürkop, M.; Maurer, M.; Bliem, R.; Kühnel, H. Parameter Optimization and Capacitance-Based Monitoring of In Situ Cell Detachment in Microcarrier Cultures. Processes 2024, 12, 1887. https://doi.org/10.3390/pr12091887
Ebrahimian A, Schalk M, Dürkop M, Maurer M, Bliem R, Kühnel H. Parameter Optimization and Capacitance-Based Monitoring of In Situ Cell Detachment in Microcarrier Cultures. Processes. 2024; 12(9):1887. https://doi.org/10.3390/pr12091887
Chicago/Turabian StyleEbrahimian, Atefeh, Mona Schalk, Mark Dürkop, Michael Maurer, Rudolf Bliem, and Harald Kühnel. 2024. "Parameter Optimization and Capacitance-Based Monitoring of In Situ Cell Detachment in Microcarrier Cultures" Processes 12, no. 9: 1887. https://doi.org/10.3390/pr12091887