Presented by Astarte Biologics (astartebio.com) at the Society for Immunotherapy of Cancer (SITC) 33rd Annual Meeting. This research explores a series of studies comparing the performance of several culture media.

1. Comparison of Culture Media for
In Vitro T Cell Expansion and Function
Ponni Anandakumar, Benjamin A. Tjoa, and P. Anne Lodge, Astarte Biologics, Bothell, WA
astartebio.com Getting on with discovery.
Background
Identification of a reliable culture media to support in vitro T cell studies has
become an important link in the chain of various Immuno-Oncology strategies.
While many labs have chosen one favorite media for their T cell culture needs, it
may be prudent to identify alternatives that can perform suitably, whether one
works in the development of cell-based assays to screen potential drug candidates
or generates and expands antigen-specific T cells. To address this issue, we have
conducted a series of studies comparing the performance of several culture
media.
Methods
A list of culture media (including several classic media + supplements as well as
several new media) was compared to several commercially available T cell media
in the generation of primary MLR (mixed-lymphocyte reaction), antigen-recall
assay (e.g., CMV, tetanus), antigen-specific T cell proliferation assay, as well as in
anti-CD3/CD28 driven T cell expansion culture.
Cells
PBMC were obtained from normal donors in our study protocol. The protocol
for collection of PBMC has been reviewed and approved by an accredited
independent review board (IRB) and participants have given their informed
consent for use of their cells in research. B lymphoblastoid cells used in some
experiments were derived from these PBMC as were antigen-specific T cells.
Recall Antigen Assay
PBMC are added to U-bottom 96 well plates at 250,000 cells per well in a 100 μL
volume. The antigens are prepared at 2x the desired final concentration and added
to the wells at 100 μL. Tetanus toxoid and CMV antigens are from our catalog
and used at 1 μg/mL, PHA was purchased from Sigma and used at 1 μg/mL and
the lipopolysaccharide (LPS) was also purchased from Sigma and used at a final
concentration of 100 ng/mL. Cultures of PBMC and antigen were incubated at
37˚C, 5% CO2
and after 4 days culture medium was collected for cytokine analysis.
Cytokine Analysis
Cytokines were analyzed using a U-Plex kit from Meso Scale Discovery. Interferon
gamma (IFNγ) and tumor necrosis factor alpha (TNFα) were routinely measured in
the recall antigen assay.
Antigen-Specific Proliferation Assay
Tetanus toxoid-specific T cells were used to evaluate the effect of media
formulations on antigen specific proliferation. Autologous B-LCL were inactivated
by incubation with 50 μg/mL of mitomycin C for 45 minutes at 37˚C. After this
incubation, mitomycin C was removed by 3 washes with PBS. The inactivated
cells were plated at 20,000 cells per well in a U-bottom 96 well plate. Peptide
antigen was added to indicated concentrations and tetanus toxoid-specific T cells
were added at 20,000 cells per well. The cultures were incubated for 4 days and
proliferation was measured by addition of CellTiter Glo™.
Mixed Lymphocyte Reaction
B-lymphoblastoid cells were used to stimulate PBMC in a mixed lymphocyte
reaction. The batch of PBMC and B-LCL were kept constant for this series of
experiments. Proliferation of the B-LCL was prevented by incubation of the
cells with 50 μg/mL of mitomycin C (Sigma) for 45 minutes at 37˚C. After this
incubation, mitomycin was removed by 3 washes with PBS and following the last
wash the cells were suspended in the desired medium. These APC were added at a
range of cell numbers per well in a 100 μL volume. PBMC were added at
20,000 cells per well in a volume of 100 μL of the appropriate medium. The culture
was incubated for 5 days and proliferation was measured by the addition of
CellTiter Glo™.
Stimulation with Anti-CD3 and Anti-CD28
PBMC were stimulated with ImmunoCult™, an anti-CD3, anti-CD28 conjugate from
Stem Cell Technology. This reagent was used at a range of concentrations in test
medium. PBMC were added at 100,000 cells per well in 96 well plates or cultured in
bulk for flow cytometric analysis.
Flow Cytometric Analysis
Fluorescent antibodies were purchased from BioLegend and used at the volumes
recommended by the manufacturer. Antibodies were incubated with cells for
15 minutes in the dark, washed once and data was acquired using a FACScan
cytometer. Further analysis was performed using FCS Express (DeNovo Software).
Results
Classical media supplemented with several defined components can support
primary in vitro responses as measured by cytokine production. Sustained T
cell proliferation demanded additional supplementation and revealed greater
differences between media. One representative data from these studies is included
in this abstract. This experiment demonstrates the effect of human AB serum
(HS) or fetal bovine serum (FBS) added to the culture medium X-VIVO™ 15 (Lonza,
Walkersville, MD). At low peptide concentrations (3 and 10 ng/mL), the presence of
HS and FBS inhibits T cell proliferation compared to X-VIVO™ 15 alone.
Conclusions
• Supplementation of media with human serum does not support optimal
cytokine production in the recall antigen assay.
• Minimal supplementation of DME/F12 is sufficient to support cytokine
production and proliferation in short-term (5-day) assays.
• Supplementation of culture medium with serum supports growth but
requires higher concentrations of antigen.
• Choice of culture medium influences not only growth rate but phenotype
and function of resulting T cells.
Figure 1
Each panel shows a different donor PBMC tested for antigen response and the IFNγ produced in DME/F12
supplemented with either fetal bovine serum or human AB serum at 10% or using X-VIVO™ 15.
Figure 2
Each panel depicts data from a different PBMC donor. Cells were tested for recall antigen response
using either DME/F12 supplemented with ITS+Premix and human serum albumin or X-VIVO™ 15.
Figure 3
An MLR conducted in the presence of three media formulations. X-VIVO™ 15 supplemented with
human serum and DME/F12 with ITS+Premix and human albumin supported greater cell growth at
low concentrations of stimulating B-LCL.
Figure 4
Tetanus toxoid-specific T cells were incubated with APC and the indicated concentrations of peptide antigen.
After 4 days incubation at 37˚C the proliferation was measured by addition of CellTiter Glo™.
Figure 6
Proliferation stimulated by anti-CD3+anti-CD28 using a second donor’s PBMC. ImmunoCult™
was added to culture medium at 6.25 μL per mL and a series of 1:2 dilutions as well as a control
without ImmunoCult™. after 4 days culture the proliferation was measured using CellTiter Glo™
Figure 7
Phenotypic analysis of T cells following stimulation with anti-CD3+anti-CD28. PBMC were stimulated using ImmunoCult™ in 3 different
serum-free medium formulations. IL-2 was added after the initial 24 hours and after 6 more days the cells were collected and analyzed for
expression of CD4, CD8, CD45RA, CD45RO, and CD62L. X-VIVO™ 15 in row 1, Medium A in row 2 and Medium B in row 3.
Figure 5
Proliferation stimulated by anti-CD3+anti-CD28. ImmunoCult™ was added to culture
medium at 6.25 μL per mL and a series of 1:2 dilutions as well as a control without
ImmunoCult™. After 4 days culture the proliferation was measured using CellTiter Glo™.