Autologous CAR-T cell therapy manufacturing demands exceptional precision at every stage. As demand for these therapies grows, reproducibility and contamination control have become defining challenges for manufacturers looking to scale.
Manual workflows introduce variability at multiple points in the process, increasing contamination risk and undermining the product consistency that patient safety depends on. Even small deviations compounded across a manufacturing run can compromise final product quality.
This poster presents data from a fully integrated, modular automated workflow that combines counterflow centrifugation with automated formulation and multi-dose dispensing, demonstrating how closed-system processing can deliver reproducible, high-quality CAR-T cell outputs.
Download this poster to learn:
- How automated, closed-system workflows compare to manual processing across key quality metrics including cell recovery, viability, and phenotypic integrity
- The volumetric accuracy and output consistency achievable across clinically relevant fill volumes in both cryovial and cryobag formats
- How T cell proliferative capacity and phenotype are preserved through cryopreservation and post-thaw expansion under automated processing conditions
Introduction
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© 2026 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the
property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
For Research Use or Manufacturing of Cell, Gene, or Tissue- Based Products.
Acknowledgement: Illustrations in figures were created with BioRender.com
Autologous CAR-T manufacturing requires consistent cell processing and
precise, closed-system formulation and formulation fill–finish to ensure product
quality and reproducibility. However, manual workflows can introduce variability,
increase contamination risk, and negatively impact final product consistency
when compounded across the manufacturing process.
In this study, we evaluated a modular, automated workflow integrating the
Gibco CTS Rotea Counterflow Centrifugation System for cell concentration
and washing with the Gibco CTS Compleo Formulation and Fill System for
automated formulation and multi-dose dispensing.
The objective was to assess cell recovery, viability, and phenotypic integrity
following processing, as well as to evaluate volumetric accuracy and output
consistency across clinically relevant fill volumes.
Young J. Kim*, Ranganatha Somasagara*, Norbert Hernandez*, Pushpalatha Chaluvappa*, Narendra Sadhwani*, Derek Wampler*, Lisbeth Lopez*, Carl Dargitz*, Namritha Ravinder*
*Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008.
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Primary human activated T cells were processed using an integrated, closed
workflow combining the CTS Rotea Counterflow Centrifugation System and
CTS Compleo Formulation and Fill System. Cells were concentrated and
buffer-exchanged on the CTS Rotea system into PBS + 2% HSA (~20 mL), then
aseptically transferred to the CTS Compleo system via a sterile, closed
connection.
Cells were formulated with DMSO (1:1) to a final composition of 5% DMSO and
1% HSA, and dispensed into cryovials (4 mL) and cryobags (10 mL). Cryobags
were frozen using a controlled-rate freezer, thawed after 96 hours, and cultured
for short-term expansion prior to analysis.
Key endpoints included cell recovery, viability, phenotype, and proliferation
assessed across workflow stages (pre-processing, post-processing, and pre-
/post-thaw).
Target
volume
Volume Concentration
% Error % CV % Error % CV
4 mL 2.5 % 1.3 % 2.3 % 2.7 %
10 mL 1.1 % 1.5 % 1.7 % 2.3 %
Figure 1. Total cell recovery across integrated workflow. (Top) Cell recovery
remained high throughout processing, with minimal loss observed across the
Rotea–Compleo steps. A reduction in recovery was observed post-thaw, consistent
with expected losses during cryopreservation. (Bottom) Comparable recovery trends
were observed between automated (Rotea–Compleo) and manual workflows at both
post-processing and post-thaw stages. Values represent mean ± SD with individual
replicates shown (n = 3).
Figure 3. Cell viability across workflow compared to manual processing.
Viability remained >90% throughout processing, with comparable performance
between the automated Rotea–Compleo workflow and manual controls. Values
represent mean ±SD with individual replicates (n = 3).
Figure 4. Post-thaw T cell expansion and proliferation metrics. Percent divided,
proliferation index, and expansion index were comparable between the Rotea–
Compleo workflow and manual processing, indicating preserved proliferative
capacity post-thaw. These results demonstrate that automated processing does not
impair T cell expansion potential. Values represent mean ± SD with individual
replicates (n = 3).
Post-expansion
endpoint
Day 0: Processing
Pre-Rotea
Post-Rotea
Pre-Compleo
(in-process)
Post-Compleo
Category Observation (Rotea-Compleo vs. Manual)
T cell lineage (CD4 / CD8) Comparable lineage distribution
Central memory (CD27 / CCR7) Comparable phenotype distribution
Activation (CD25 / CD69) Similar activation post-thaw
Regulatory-associated (PD-1) No increase in exhaustion post-thaw
Cryopreservation Post-thaw
Day 3 Day 3-7 Day 7
Freezing Thawing Culture
Expansion
* Viability, cell count, and phenotype assessed at indicated timepoints.
Day 0
Method
Integrated modular T cell processing and
formulation-fill using automated system
Total cell recovery after integrated workflow
End-to-end workflow and analysis timepoints
Cell viability across workflows
Total cell recovery
Step recovery during processing
(% of input from previous step)
Cell viability across workflow
Day 0 Input
Day 0 Output
Day 4
Post-thaw
Day 7
Post-expansion
60
80
100
97%
94% 94%
91%
96%
93% 92% 93%
Viability (%)
Rotea-Compleo Manual
Post-thaw T cell expansion metrics
RC Manual
0
20
40
60
80
58%
56%
% Divided
RC Manual
0.0
0.5
1.0
1.5
2.0
1.53
1.58
Proliferation index
RC Manual
0
1
2
3
2.11 2.31
Expansion index
% Divided
Proliferation
index
Expansion index
• % Divided: Proportion of cells entering division
• Proliferation index: Average number of divisions
• Expansion index: Overall cell population expansion
T cell subset composition across workflow stages
0
50
100
150
Frequency of cells (%)
Pre-Processing
Post-Processing
T cell subset compositions
0
50
100
150
RC Manual
0
10
20
30
40
50
Frequency of cells (%)
RC Manual
0
10
20
RC Manual
0
50
100
150
RC Manual
0
10
20
30
40
50
Frequency of cells (%)
CD69 CD25 PD-1
Rotea-Compelo
Manual
0
50
100
150
Frequency of cells (%)
CD4 CD8 CD27+ / CCR7+ CD27- / CCR7-
Pre-Processing
Post-Processing
Post-processing
(Day 0)
Post-thaw
(Day 4)
Post-expansion
(Day 7)
Post-thaw
(Day 4)
Post-expansion
(Day 7)
*RC: Rotea-Compleo
*RC: Rotea-Compleo
T cell activation state
Figure 5. T cell subset composition across processing, cryopreservation, and
expansion. CD4/CD8 lineage distribution and memory phenotypes (CD27/CCR7)
remained consistent following processing, post-thaw, and after expansion.
Comparable profiles were observed between the automated Rotea–Compleo
workflow and manual controls, indicating preservation of T cell phenotype across
workflow stages. Frequencies represent mean values of individual replicates (n = 3).
*RC: Rotea-Compleo
Final output volume & concentration
Figure 7. Final output volume and concentration. Values are expressed as % of
target. Results were consistent across conditions, with values near 100% indicating
accurate delivery. Error and variability (%CV) are summarized in the table (mean ±
SD, n = 3).
4 mL 10 mL
0
5
10
10.1mL
4.1mL
Target Volume
Volume (mL)
Delivered volume vs target Concentration (% of Target)
% Error % CV
Run 1.8 % 0.85 %
Output line 1.8 % 1.49 %
Volume delivery repeatability
Volume across runs Volume across output lines
4 mL 10 mL
0
50
100
100% 99%
Target Volume
% of Target
1 2 3
0
50
100
101% 104% 101%
Run
Volume (% of Target)
W V U T
0
50
100
103% 102% 101% 101%
Output Line
Volume (% of Target)
Figure 8. Volume delivery repeatability across runs and output lines. Volume
expressed as % of target was consistent across repeated runs and output lines,
demonstrating low variability (%CV). Values represent mean ± SD with individual
replicates (n ≥ 3).
Trademarks / Licensing
Pre-Rotea
Post-Rotea
Pre-Compleo
Post-Compleo
Post-Thaw
0
50
100
77%
92%
100% 101%
100%
Recovery (%)
(normalized to input)
Figure 6. T cell activation and exhaustion marker expression following
cryopreservation and expansion. Activation markers (CD25, CD69) and the
exhaustion marker PD-1 showed comparable levels between the automated Rotea–
Compleo workflow and manual controls at both post-thaw (Day 4) and postexpansion
(Day 7). No increase in PD-1 expression was observed following
automated processing. Values represent mean of individual replicates (n = 3).
Cell recovery across workflow steps and post-thaw
RC Manual
0
50
1 00
90%
85%
Post-Thaw Recovery (%)
Rotea Compleo Manual
0
50
100
96%
91%
100%
Step Recovery (%)
Figure 2. Cell recovery across automated workflow compared to manual
processing. Step recovery remained high (>90%) throughout processing, with
comparable performance between the automated Rotea–Compleo workflow and
manual controls. Post-thaw recovery was similarly maintained, indicating minimal
additional loss introduced by automation. Values represent mean ± SD with
individual replicates shown (n = 3).
Post-thaw recovery
(% of input from previous step)
*RC: Rotea-Compleo
T cell activation state post-thaw & post-expansion
Cell recovery compared to manual processing
Post-processing Post-thaw
0
50
100
150
96%
82%
92%
77%
Recovery (%)
(normalized to input)
Rotea-Compleo
Manual
ns ns
