Application Note

Enhance Your TIL Expansion withExCellerate™ T Cell Media

Tumor infiltrating lymphocytes (TILs) are a heterogeneous
population of lymphocytes that have migrated into atumor. Largely comprised of T cells, TILs are activelyengaged within the tumor microenvironment. Theirfunctional impacts are wide ranging due to the immune
system complexities within tumors,1,2 and TILs can either
suppress or activate immune responses. In addition, TIL
subsets highly expressing CD3 and either CD4 or CD8 3-6
can exert anti-tumor and anti-proliferative effects in solidtumors, and their presence is associated with improved
clinical outcome.2,7 These encouraging findings havepropelled the development of TIL therapy for multiple
cancers in recent years.

TIL therapy is an adoptive cell therapy that utilizes TILs
from a patient’s own tumor. Biopsy-derived TILs areadapted to infiltrate and survive in the patient’s tumor,
an important capability for immune cell therapies. In TIL
therapy, these cells are expanded to therapeutic scale
ex vivo and delivered back into the patient.8 TIL therapy
has shown significant promise against a wide variety
of solid tumors including melanoma, head and neck,colorectal, and early stage lung cancers.5,6,9-12

ExCellerate Media for TIL andOrganoid Culture
ExCellerate™ Human T Cell Expansion Media is
optimized for the ex vivo culture of human T lymphocytes
in preclinical research. ExCellerate media is xeno-freewhich means that it is devoid of non-human animalderived products and therefore provides a more controlled
and stable culture environment for T cell expansion.
Organoid culture, pioneered by Hans Clevers and Toshi
Sato at the Hubrecht Institute in the Netherlands, is an invitro model system that is valuable for evaluating TILtumor cell interactions. The interactions of TILs withtumor organoids requires the generation of large numbersof TILs in a robust and reproducible process. Commonlyused methods of TIL expansion rely on the use of human

Learn more about ExCellerate

serum with conditioned media containing high-dose
IL-2 (Figure 4), a time-consuming process that results insignificant experimental variability.
In order to develop a more reproducible method forexpanding TIL cultures, Rosemary Millen, a previous
post-doctoral researcher in the Clevers lab, carried out amedia comparison to understand the impact ExCellerate TCell Media has on the cultures. This comparison showed a
15% - 30% increase in CD3+ T cells cultured in ExCellerate
media compared with the conditioned media (Figure1). These findings are consistent with studies on PBMCs
performed at Bio-Techne (Figures 2 and 3).

TILs Grown in ExCellerate Media Show Higher Proportion of Viable
CD3+ T Cells
Lymphocytes

Viable Cells

250K

CD3+ T Cells
250K

105

200K

200K
150K

100K

SSC

23.7%

DAPI

150K

SSC

TIL 1CM10499.5%

103050K

50K

-103

0250K

0250K

105

200K

200K
104

100K

99.8%

103SSC

150K

36.8%

DAPI

150K

SSC

TIL 1
ExCellerate

84.1%

100K

050K

50K

-103

0250K

96.7%

100K

0250K

105

200K

200K
104

100K

SSC

150K

53.8%

DAPI

SSC

TIL 2
CM

150K

96.6%

103050K

50K

-103

0250K

72.5%

100K

0250K

105

200K

200K

54.2%

100K

1099.7%

3

SSC

150K

DAPI

SSC

TIL 2
ExCellerate

104050K

050K

100K 150K 200K 250K

FSC

96.1%

100K
50K

-103

0150K

0050K

100K 150K

SSC

200K 250K-1030103104105

CD3ε-FITCFigure 1. Comparison of ExCellerate Human T Cell Expansion Media and conditioned media (CM). TILs were cultured from two separate patient biopsies (TIL1 andTIL2) for two weeks in CM or ExCellerate media in the presence of recombinant human IL-2 (6000 IU/mL). CD3+ T cell expansion is shown by flow cytometry andmicroscopy images. Data provided by Rosmary Millen.

AB
Total CD8+ Cell Number (104)

Total Cell Number (104)200150100500Day 0Supplier #1Day 5Supplier #280706050403020100Day 9Day 0ExCellerateTM T CellExpansion MediaMediaDay 5Media 2Day 9ExCellerateTM T CellExpansion MediaFigure 2. ExCellerate Human T Cell Expansion Media supports greater T cell expansion compared to other commercial T cell media. 2.5 x 106 PBMCs werecultured for 9 days using ExCellerate media or other commercially available media (Supplier #1 or #2), in combination with GMP-grade Recombinant Human IL-2,IL-7, and IL-15. The numbers of total T cells (A) and CD8+ T cells (B) were determined at Days 0, 5, and 9 of culture.

105TIL expansion

labCD8α1041030103100101102
CD3ε103104

Figure 3. Phenotypic analysis of human T cells expanded in ExCellerate
Human T Cell Expansion Media. Human PBMCs were cultured for 9 days inExCellerate media with Recombinant Human IL-2. CD8+ T cells were enrichedduring the culture as shown by flow cytometry with Mouse Anti-Human CD3
epsilon APC-conjugated Antibody and with Mouse Anti-Human CD8 alphaPE-conjugated Antibody.

+ high-dose IL-2
Figure 4. Expansion of TILs from fresh tissue specimens. Biopsy or surgicalresection from patients are divided into small pieces and placed into a cell
culture dish with conditioned media supplemented with recombinant human
IL-2.

Moving Forward
As you advance your TIL program towards clinicalstudies, it is important to develop TIL expansion andcoculture processes that are as robust as possible.
Adopting xeno-free and serum-free ExCellerate media
will eliminate the risk of contamination with mammalianpathogens, reduce manufacturing risk by reducing batch

failure, and eliminate variability due to trace animalcomponents. Better expansion and greater consistency inyour TIL cultures will accelerate your progress in ongoingstudies and enable your TIL therapy to reach the patients
who need it.

References
1. Romagnoli, G. et al. (2017) Morphological evaluationof tumor-infiltrating lymphocytes (TILs) to investigate
invasive breast cancer immunogenicity, reveal
lymphocytic networks and help relapse prediction: aretrospective study. Int. J. Mol. Sci. 18:1936.
2. Galon, J. et al. (2006) Type, density, and location ofimmune cells within human colorectal tumors predictclinical outcome. Science 313:1960.

7. Pinto, M. P. et al. (2018) Patient inflammatory status andCD4+/CD8+ intraepithelial tumor lymphocyte infiltration
are predictors of outcomes in high-grade serous ovariancancer. Gynecol. Oncol. 151:10.
8. Paijens, S. T. et al. (2021) Tumor-infiltrating lymphocytes in
the immunotherapy era. Cell. Mol. Immunol. 18:842.
9. Lee, S. and Margolin, K. (2012) Tumor-infiltrating
lymphocytes in melanoma. Curr. Oncol. Rep. 14:468.

3. Yu, A. et al. (2018) Presence of lymphocytic infiltratecytotoxic T lymphocyte CD3+, CD8+, and immunoscore
as prognostic marker in patients after radical cystectomy.
PLoS One 13:e0205746.

10. Idos, G. E. et al. (2020) The prognostic implications oftumor infiltrating lymphocytes in colorectal cancer: asystematic review and meta-analysis. Sci. Rep. 10:3360.

4. Tomšová, M., et al. (2008) Prognostic significance of CD3+
tumor-infiltrating lymphocytes in ovarian carcinoma.
Gynecol. Oncol. 108:415.

11. Millen, R. et al. (2020) CD8+ tumor-infiltrating
lymphocytes within the primary tumor of patients withsynchronous de novo metastatic colorectal carcinoma do
not track with survival. Clin. Transl. Immunol. 9:e1155.

5. Giatromanolaki, A. et al. (2021) Prognostic relevance ofthe relative presence of CD4, CD8 and CD20 expressing
tumor infiltrating lymphocytes in operable non-small celllung cancer patients. Anticancer Res. 41:3989.

12. Horne, Z. D. et al. (2011) Increased levels of tumorinfiltrating lymphocytes are associated with improved
recurrence-free survival in stage 1A non-small-cell lungcancer. J. Surg. Res. 171:1.

6. Borsetto, D. et al. (2021) Prognostic significance of CD4+and CD8+ tumor-infiltrating lymphocytes in head andneck squamous cell carcinoma: a meta-analysis. Cancers(Basel). 13:781.

Additional Resources forTIL Expansion• ExCellerate™ Cell Expansion Media• T Cell Manufacturing for Cell Therapy• Serum-Free and Animal-Free Cell Culture• T Cell-Based Therapies eBook• Transitioning to Animal-Free and GMP

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