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CTS Rotea Counterflow Centrifugation System Sample Data

Performance and application data

 

The CTS Rotea System is well positioned to provide high purity and cell viability for cell processing of many different cell types.

T cell and leukopak processing: High recovery and viability

Cell samples washed and concentrated using the Rotea instrument exhibit excellent viability and recovery in small volumes. T cell quality and composition are maintained after Rotea instrument processing, and PBMC isolation can be completed in much less time than is possible with high density media protocols.

Figure 1. Viability and recovery during T cell washing and concentration. Gentle processing enables >90% cell recovery while maintaining cell viability through T cell washing and concentration. High reproducibility was demonstrated over 10 runs.

Bar graph showing 96% viability and 94% recovery of T cells washed and concentrated using the Rotea system

Figure 2. 200x T cell concentration. The CTS Rotea system was used to concentrate 500 million T cells in a starting volume of 1,000 mL down to 5 mL, achieving 97% cell recovery while maintaining viability of 94%.

Bar graph of input and viability vs output and viability of concentrated cells

Figure 3. High PBMC recovery during leukopak processing. The CTS Rotea system can achieve approximately 90% PBMC recovery from a Leukopak bag in combination with a red blood cell lysis buffer across multiple donors (n=7).

Box plot showing 88.9% PBMC recovery from a leukopak bag

The low volumes of efficiently washed and concentrated cells from the CTS Rotea system can be transferred directly to the CTS Xenon Electroporation System for transfection steps. Using these two highly compatible Cell Therapy systems saves times and reduces workflow complexity.
 

To learn more about automated PBMC isolation, T cell wash and concentration, and CAR T cell manufacturing using the CTS Rotea system and how the Rotea and Xenon systems can work together to support highly efficient and effective cell therapy workflows, use the link below to see all CTS Rotea system application notes.

Efficient times for T cell processing and leukopak PBMC isolation

Even at high input volumes, the Rotea system allows for rapid cell processing with high cell recovery. In less than four hours, users can effectively wash and concentrate large volumes of T cells or separate PBMCs from leukopak bags.

Input volume (L)
 Processing time (min)
 Cell recovery
T cell wash and concentrate (1 x 106 cells/mL)
0.5 11 >90%
1.0 18
3.0 38
5.0 58
10.0 114
20.0 226
Input volume (L)
 Processing time (min)
 Cell recovery
PBMC isolation
~0.25 (¼ leukopak bag) 25
 >90%
~0.5 (½ leukopak bag) 45
~1.0 (full leukopak bag) 85
5 116
10 230

Save time during leukopak PBMC isolation using the Rotea system vs Ficoll media

Flow cytometry data shows that PBMC cell separation in less than 30 minutes with the CTS Rotea instrument produces similar results to a two-hour Ficoll media separation.
 

Figure 4. PBMC composition from CTS Rotea system vs manual Ficoll polymer. A single-donor leukopak was split in two, and PBMCs were separated using the CTS Rotea System or a manual Ficoll media protocol. The PBMCs were assessed using successive gating to identify monocytes, B, NK, and T cells. Processing with the CTS Rotea System (A) took less than 30 minutes and produced results highly similar to those from the two-hour Ficoll separation (B).

Flow cytometry graphs showing results from Ficoll separation or the CTS Rotea system separation of PBMCs

Maintenance of T cell quality and composition

Figure 5. T cell quality and composition unaffected after washing and concentration. After PBMCs were isolated from healthy donors using the CTS Rotea System, the T cells were expanded in culture and assessed for CD4 and CD8 markers using flow cytometry. They were then washed and concentrated using the CTS Rotea System and assessed again. Comparison showed that CD4 and CD8 populations were consistent between the two analyses, demonstrating that the relative proportions were unaffected by Rotea system processing.

NK cell processing: high recovery, viability, and maintenance of cytotoxicity

The CTS Rotea system can also perform successful processing of NK cells at flow rates up to 80 mL/min, including automated washing and concentration of human NK cells expanded in Gibco CTS NK-Xpander Medium. In addition to high viability and recovery of NK cells, these cells also maintained their cytotoxicity following processing.

Figure 6. NK cell viability and recovery. Viability and recovery were averaged over four washing and concentration runs.

Figure 7. Maintenance of NK cell cytotoxicity after washing and concentration. NK cells washed and concentrated using the Rotea system maintained cytolytic function and were able to kill K562 target cells in a dose-dependent manner.

Graphed results of viability staining following processing with the Rotea system

Highly effective harvesting of MSCs from Cell Factory systems

MSCs have emerged as a promising cell type for cell therapy aimed at treating a variety of diseases. Closed and automated cell processing systems such as the Rotea system can support the manufacture of MSC-based cell therapies by promoting scalability, regulatory compliance, and quality control.
 

This includes the use of a custom StemPro SFM XF media for expansion of mesenchymal stem cells (MSCs) in monolayer and microcarrier suspension-based cultures and use of closed system passaging and harvesting of MSCs from multi-layered flasks using the Rotea system.

 

MSCs harvested from Cell Factory Systems using the Rotea system have been shown to maintain high cell viability and expression of known MSC markers.

Figure 8. MSC concentration. 10X concentration of MSCs from an input volume of 450 mL to an output volume of 45 mL was performed on the Rotea system.

Bar chart showing concentration of MSCs from 450 mL to 45 mL using the Rotea system.

Figure 9. MSC viability. MSC viability was maintained near 98% after processing with the Rotea system.

Bar chart showing high MSC viability before and after Rotea system processing

Figure 10. MSC total cell recovery. Following processing with the Rotea system, an MSC recovery rate of 97.3% was observed for cells in a 10-layer culture (Cell Factory Systems).

Bar chart showing a high rate of MSC recovery before and after Rotea processing of a 10-layer culture.

Figure 11. Retention of typical MSC markers. Flow cytometric analysis showed that all cells retained high expression of typical MSC markers CD73, CD90, and CD105 following processing with the Rotea system.

Bar chart showing high expression levels (>90%) for MSC markers CD73, CD90, and CD105. Negative MSC markers (CD34 and CD45) showed little to no expression.

In addition to the data presented here, researchers have designed a semi-automated process using counterflow centrifugation to passage and harvest MSCs from multi-layered flasks.

Reduced turbidity during lentiviral clarification

Gentle counterflow centrifugation using the Rotea system allows for reduction in turbidity levels during lentiviral clarification to levels comparable to manual centrifugation but in a closed, low shear environment. The Rotea system enables faster processing times between 25-75 mL/min for larger scale applications and furthermore, processing using the Rotea system results in minimal loss of infectious titer. 

Figure 12. Comparable turbidity clarification following lentiviral clarification. Lentiviral clarification with the Rotea system results in a significant reduction in turbidity; similar results were seen using standard manual centrifugation.

Bar graph showing a significant reduction in turbidity (NTU) following lentiviral clarification using the Rotea system.

Figure 13. Infectious titers following lentiviral clarification. CTS Rotea system runs maintain infectious titers above 2 x 108 after clarification.

Bar graph showing infectious titer levels (TU/mL) following lentiviral clarification using the Rotea system.

Rapid and effective dissociation of PSC spheroids

The CTS Rotea system provides an efficient and effective closed system for the dissociation of pluripotent stem cell (PSC) spheroids grown in suspension cultures. Utilizing this closed-system environment significantly decreases the risk of contamination compared to water bath methods.

Figure 14. A comparison of spheroid dissociation from a 3 L suspension culture vessel using the CTS Rotea system and a water bath. Spheroids grown for 5 days and dissociated using both the CTS Rotea system and a water bath exhibited similar performance in terms of (A) total cell yield and (B) cell viability. Final cell yields and cell viabilities were obtained from cell counts performed immediately after harvesting the single-cell output from the dissociated spheroids. Notably, the Rotea system can dissociate spheroids significantly faster than the water bath method. (C) All single cells harvested from the Rotea system were able to re-nucleate into spheroids when reseeded into new suspension culture vessels. (D) After 5 days of growth, the final cell yields of the reseeded spheroids were also shown to be similar, indicating that the Rotea system can gently dissociate spheroids into single cells capable of being utilized for downstream applications.

Effective residuals washout

Prior to use for cell therapy, cells may be thawed, selected, expanded, genetically altered, differentiated, and/or cryopreserved. Each of these steps may require a different medium or buffer that can contain substances harmful to cells. The CTS Rotea system can effectively remove residual substances with minimal cell loss.

Figure 15. Washing efficiency of the CTS Rotea system. Percentage of (A) glucose, (B) IL-2, or (C) HSA removed from T cell culture medium are shown for each wash volume. 

Scatter plots and tables showing different wash volumes used in the CTS Rotea to remove residual substances.

Explore a library of Rotea system resources

Visit the Rotea system resource page to download application notes, find publications, watch webinars, and more.

For Research Use or Manufacturing of Cell, Gene, or Tissue-Based Products.

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