Atypical cell types, such as large or sticky cells, can clog cell sorters, show poor recovery, and ultimately undermine downstream results.
Researchers can address these challenges with gentle microchip-based cell sorting technology and specially designed cartridges for large, complex cells up to 50 µm in diameter.
This poster describes how microchip-based gentle sorting enables high-purity isolation of large glioblastoma cells while preserving their viability and function.
Download this poster to discover:
- A workflow to generate tumor-reactive T cells and autologous tumor cell lines
- Functional validation data confirming the effectiveness of microchip-based gentle sorting
- How this process can be applied across other tumor-infiltrating lymphocyte subsets of interest
Elvira Criado-Moronati1, Emre Balta1, Tümay Telatar1, Ana Kerin1, Donata Maciaczyk1, Johanna Engeln1, Marieke Domdey1, Arina Riabinska1, Denis Frison1,
Cristina Berganza Irusquieta1, Nadine Chelius1, Thomas Broggini2, Teresa Schmidt3, Martin Glas3,4,5, Ian Hardy1, Andrzej Dzionek1, Lorenz Jahn1
Poster Session II: 109
A workflow for generating tumor-reactive T cells from
GBM-derived cavitron ultrasonic aspirator (CUSA) fluids
Scan the QR code
to download the
poster (PDF)
Introduction
without headline
top margin
with headline
top
without headline
bottom
top
with headline
left margin right margin
Figure 1
Results
1 Composition of GBM-CUSA digests
without headline
top margin
with headline
top
without headline
bottom
top
with headline
left margin right margin
Tumor cells Endothelium Leukocytes
0
20
40
60
80
100
% of viable cells
T cells B cells NKs Myeloid
0
20
40
60
80
100
% of leukocytes
CUSA
104
105
106
107
108
T cell numbers
A Cell composition C TIL numbers
CD4+ CD8+
0
20
40
60
80
100
% of T cells
✱✱
B
T cell subsets
Phenotype of TILs
Tn Tscm Tcm Tem Teff
0
20
40
60
80
100
% of TIL subset
CD4
CD8
✱✱✱ ✱✱✱✱
Immune cell subsets
D
E
Figure 2
2 CD137 enrichment and TIL expansion
without headline
top margin
with headline
top
without headline
bottom
top
with headline
left margin right margin
Unselected CD137neg CD137pos
100
101
102
103
104
Fold expansion
Before
O/N culture
After
O/N culture
0 10 20 30 40
✱✱✱✱
% CD137pos (of T cells)
A
B C
CD137 upregulation TIL expansion
Tscm Tcm Tem Teff
0
20
40
60
80
100
% of CD4+ cells
Tscm Tcm Tem Teff
0
20
40
60
80
100
% of CD8+ cells
Unselected CD137neg CD137pos
D Phenotype of expanded TILs
Figure 3
3 Generation of eGFP+ tumor cell lines
without headline
top margin
with headline
top
without headline
bottom
top
with headline
left margin right margin
Before Sorting Unsorted Fraction Sorted Fraction
eGFP
SSC-A
eGFP
SSC-A
eGFP
SSC-A
Frequency of eGFP+ tumor
cells before sorting
Purity of eGFP+ tumor
cells after sorting
Viability of eGFP+ tumor
cells after sorting
eGFP positive (%)
eGFP positive (%)
Viability (%)
A B
C
Figure
4 Funcionality of expanded TILs
without headline
top margin
with headline
top
without headline
bottom
top
with headline
left margin right margin
0 12 24 36 48 60 72 84 96 108 120
0
1
2
3
4
Time (hours)
Green Integrated Intensity/mm2
Unselected
CD137neg
CD137pos
Tumor Only
Unselected
CD137neg
CD137pos
Unselected
CD137neg
CD137pos
0
2000
4000
6000
0
200
400
600
Perforin (pg/ml)
Granzyme B (pg/mL)
Cell Killing Assay Secretion of cytotoxic molecules
Top 10 clonotypes in CD137pos TRTs
GFP-expressing tumor cells (green) after 5 days co-culture
Tumor only Tumor +
Unselected TILs
Tumor +
CD137neg TILs
Tumor +
CD137pos TILs
Frequency
A C
B D
Figure
Summary
1Research and Development, Miltenyi Biotec, Bergisch Gladbach, Germany. 2Department of Neurosurgery, University Hospital Frankfurt, Frankfurt, Germany. 3Division of Clinical Neurooncology, Department of Neurology
and Center for Translational Neuro- and Behavioral Sciences (C-TNBS), University Hospital Essen, University Duisburg-Essen, Essen, Germany. 4Department of Neurology and Neurooncology, St. Marien Hospital,
Lünen, Germany. 5Department of Neurooncology, Center for Neurology, University Hospital Bonn, Germany.
Figures 1 and 3A were generated with BioRender.com
Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. For further regulatory notes, refer to miltenyibiotec.com/regulatory-notes.
MACS, gentleMACS, MACSQuant, the Miltenyi Biotec logo, TexMACS, Tyto and the Tyto logo are registered trademarks or trademarks of Miltenyi Biotec B.V. & Co. KG and/or its affiliates in various countries worldwide.
All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only.
Copyright © 2025 Miltenyi Biotec and/or its affiliates. All rights reserved.
Glioblastoma (GBM) is an aggressive brain cancer
with limited treatment options and a poor prognosis,
highlighting the need for more effective therapies.
Tumor-infiltrating lymphocyte (TIL) therapy has
shown promise and represent a viable strategy for
GBM treatment. To support the development of TILbased
therapies, we established a workflow to generate
tumor-reactive T cells and autologous tumor
cell lines using the fluid collected during surgical resection
with a cavitron ultrasonic aspirator (CUSA).
To enrich for tumor-reactive T cells, we magnetically
enriched CD137+ T cells following an overnight incubation
of the GBM CUSA dissociate (A, B). These
CD137+ TILs expanded rapidly over 14 days, with expansion
folds comparable to those of unselected and
CD137- TILs (C). Phenotypic analysis of the expanded
TILs revealed the presence of both Tcm and Tem subsets
(D).
• We developed a workflow for the generation of
tumor-reactive T cells from GBM-derived CUSA
fluids, using the activation marker CD137.
• This workflow also enabled the successful
generation and transduction of primary
autologous tumor cell lines, which were used for
the functional validation of TILs.
• Integration of the MACSQuant® Tyto® cell sorter,
along with newly developed cartridges,
facilitated the gentle enrichment of diverse cell
types, including GBM tumor cells. It can also be
utilized to sort tumor-reactive T cells.
• CD137 can be substituted with alternative
markers to support the analyis of other TIL
subsets of interest.
• This workflow provides a valuable tool for both
translational research and the clinical
development of TIL-based therapies.
Functional validation confirmed the tumor reactivity
of CD137+ T cells. Co-culture of expanded CD137+ T
cells with autologous eGFP+ tumor cells for five days
resulted in significant tumor control or elimination,
associated with the secretion of cytotoxic molecules
such as perforin and granzyme B (A-C). In contrast,
unselected or CD137- TILs did not mediate tumor
clearance. Additionally, expanded CD137+ TILs contained
dominant TCR clonotypes that were not detected
in unselected or CD137- subsets, as well as in
the initial tumor repertoire and the patient´s peripheral
blood, indicating selective enrichment and expansion
of potential tumor-reactive TCRs within the
CD137+ population (D).
We successfully established primary autologous GBM
tumor cell lines in vitro from fresh CUSA tumor dissociates.
To enable their use in fluorescence-based killing
assays, tumor cells were transduced to express
eGFP and subsequently sorted using the MACSQuant®
Tyto® cell sorter. This instrument uses a closed cartridge
system with a mechanical valve for steril, gentle
cell sorting (A). Sorting large GBM cells required a
newly developed cartridge, designed to support the
enrichment of large, fragile cell types while maintaining
cell viability and purity (B, C).
We first characterized the cellular composition of
CUSA fluid after tumor dissociation, identifying
GLAST+ tumor cells, CD31+ endothelial cells, and diverse
CD45+ immune cell subsets, including CD4+
and CD8+ T cells, CD20+ B cells, CD56+ NKs, and CD11b+
myeloid cells (A-C). On avarage, 1 x 107 T cells were
obtained from GBM-derived CUSA digests (D). T cells
were phenotypically classified into naive (Tn), stemcell
memory (Tscm), central and effector memory
(Tcm and Tem), and effector (Teff) subsets based on
CD45RA, CD62L, and CD95 expression (E).
