Lonza Walkersville, Inc.
bioscience.lonza.com
scientific.support@lonza.com
Scientific Support: 800-521-0390
Document # CT-OP005 05/24
Walkersville, MD 21793-0127 USA© 2024 Lonza Walkersville, Inc.

Instructions for Culturing Gamma Delta (γδ) T Cells Using
TheraPEAK® T-VIVO® Cell Culture Medium
I. Introduction
A second protocol describes an isolation processthat is tailored to deplete αβ T cells from PBMCs
and then uses anti-CD3/anti-CD28 co-stimulationto activate and expand γδ T cells (Vδ1 and Vδ2 T-cellsubsets) using the TheraPEAK® T-VIVO® Medium.

TheraPEAK® T-VIVO® Cell Culture Medium
TheraPEAK® T-VIVO® Cell Culture Medium isoptimized to support cell therapy applications
utilizing human T cells. It only uses recombinanthuman proteins and does not require serum orserum substitute. TheraPEAK® T-VIVO® CellCulture Medium is manufactured in compliancewith cGMP and all ingredients are chemicallydefined and non-animal origin (NAO). It does notcontain cytokines, antibiotics or phenol red.

II. Storage
TheraPEAK® T-VIVO® Cell Culture Medium should
be stored at 2 to 8 °C, protected from light.

Gamma delta (γδ) T cells
Gamma delta (γδ) T cells represent a relatively small
subset of T cells and are defined by the expressionof heterodimeric T-cell receptors (TCRs) composedof γ and δ chains, and can be further identified bydifferent Vδ (variable δ) chains. For example, Vδ1T cells are predominant in the peripheral tissues
and Vδ2 T cells constitute the majority of blood γδ
T cells.
γδ T cells have attractive properties for cancer
immunotherapy including their inherent ability toinfiltrate tissues and to directly recognize and killtransformed cells independent of HLA-antigenpresentation. Moreover, γδ T cells provide aplatform for allogeneic cell therapies as they do
not cause graft-versus-host disease (GVHD).
The first protocol describes a process that uses
zoledronic acid as the activating agent andperipheral blood mononuclear cells (PBMCs) as thestarting cell source for Vδ2 T cells expansion, using
TheraPEAK® T-VIVO® Medium without the additionof human serum. Vδ2 T cells can be expanded bymore than 1,000-fold in 14 days using this protocol.

III. Instructions for use
Media preparation
TheraPEAK® T-VIVO® Cell Culture Medium should
be supplemented with cytokines, such as IL-2, tosupport γδ T-cell expansion. The amount ofcytokines required may vary depending on the user
applications, however for this protocol, it issuggested to use 100 IU/mL of recombinant humanIL-2. The medium with cytokine may be stored at
2 to 8 °C for up to 7 days. When in use, minimize
exposure of medium to light.
General guideline for Vδ2 T-cells activated withzoledronic acid and cultured in T-flasks
For optimal gas exchange in static T-flasks, it isrecommended that the medium height be less than3mm.
1.

Page 1 of 3

Prepare fresh peripheral blood mononuclear
cells (PBMCs) or thaw frozen PBMCs in a 37 °C
water bath according to standard thawing
protocols.

2. Wash the cells with TheraPEAK® T-VIVO® CellCulture Medium.
3. Centrifuge the cells at 200 – 300 x g for
5–10 minutes and remove wash buffer.
4. Resuspend the cells in TheraPEAK® T-VIVO®
Cell Culture Medium with cytokine. Determine
viable cell concentration and cell viability using
standard cell counting protocols.
5. Plate 2.0 x 10e6 viable PBMCs in 1 mL
TheraPEAK® T-VIVO® Cell Culture Medium
with cytokine into one well of a 24-wellplate for small scale expansion. For larger
cell numbers, plate at 2.0 x 10e6 viable
PBMCs/mL of medium in the appropriate
vessel to allow for sufficient gas exchange.

2. Wash the cells with TheraPEAK® T-VIVO® Cell
Culture Medium.
3. Centrifuge the cells at 200 – 300 x g for5 – 10 minutes and remove wash buffer.
4. Resuspend the cells in TheraPEAK® T-VIVO®
Cell Culture Medium. Determine viable cellconcentration and cell viability using standard
cell counting protocols.
5. If start with cryopreserved PBMCs, allow
PBMCs recover in TheraPEAK® T-VIVO® Cell
Culture Medium overnight at 37 °C in ahumidified incubator with 5% CO2, before
isolating the γδ T cells.

6. Stimulate Vδ2 T cells by adding zoledronic acidto final concentration of 5 – 10 μM.
7. Incubate the culture vessel at 37 °C in ahumidified incubator with 5% CO2.
Every 2 – 3 days, change 50% of themedium.
8. When the cells become confluent, transfer theentire culture into a larger vessel and add fresh
medium to the appropriate volume.
9. Continue to expand the T-cell culture byadding fresh TheraPEAK® T-VIVO® Cell Culture
Medium with cytokine every 2 – 3 days and readjusting the cell density to 1.0 x 10e6 viable
cells/mL. Use larger T-flasks as needed.
10. Harvest cells when the desired cell number isachieved and proceed to downstream
applications.
General guideline for γδ T-cell expansion with
anti-CD3/anti-CD28 co-stimulation afterdepleting αβ T cells from PBMCs
Both γδ T cells and αβ T cells express CD3 andCD28, hence it is critical to deplete αβ T cells prior
to expansion using this protocol. αβ T cells may beremoved from PBMCs with a variety of commercial
cell separation products.
1.

Prepare fresh peripheral blood mononuclear
cells (PBMCs) or thaw frozen PBMCs in a 37 °C
water bath according to standard thawing
protocols.

6. Isolate γδ T cells from PBMCs.
A variety of commercial cell separation
products may be used to isolate γδ Tcells from PBMCs. Non-γδ T cells, i.e. αβT cells, NK cells, B cells, dendritic cells,
granulocytes, monocytes, stem cells
etc. may be depleted using a cocktailof antibodies. At minimum, αβ T cells
should be depleted using αβTCRspecific antibodies.
7. Determine viable cell concentration of theisolated γδ T cells using standard cell counting
protocols.
8. For research purposes, plate 0.5 – 1.0 x 10e6
viable γδ T cells in 1 mL TheraPEAK® T-VIVO®
Cell Culture Medium with cytokine into one
well of a 24-well plate. Alternatively, a 96-wellplate may be used, in which case, plate 5.0 –10.0 x 10e3 viable cells in one well of a 96-wellplate in 150 μL medium.
If only αβ T cells are depleted from PBMCs,
plate 1.0 – 2.0 x 10e6 viable cells in 1 mL
TheraPEAK® T-VIVO® Cell Culture Medium
with cytokine into one well of a 24-wellplate for small scale expansion.
9. Stimulate T cells for expansion using
commercial anti-CD3 and anti-CD28 T cell
activation products as recommended by thesuppliers. For example, use 10μL T Cell
TransAct™ (Miltenyi) for 1 mL medium.
10. Incubate the culture vessel at 37 °C in ahumidified incubator with 5% CO2.
11. Continue to expand the T-cell culture byadding fresh TheraPEAK® T-VIVO® Cell Culture
Medium with cytokine every 2 – 3 days.

Page 2 of 3

Count the cells if possible and re-adjust thecell density to 0.5 – 1.0 x 10e6 viable
cells/mL. Use larger T-flasks as needed.
12. Harvest cells when the desired cell number isachieved and proceed to downstreamapplications.

IV. General recommendations
Refer to the general TheraPEAK® T-VIVO® Cell
Culture Medium instruction for recommendations
on adopting processes that maintain good
dissolved oxygen levels during the cell expansion
processes, and optimizing processes that involve
Nucleofection® or electroporation.
Lentivirus transduction in γδ T cells
Lentivirus pseudotyped with the common VSV-G
envelope protein enters cells using the low-density
lipoprotein receptor (LDL-R) as the major entry
receptor, however, LDL-R expression is very low inresting T cells. Stimulation of T cells through theT-cell receptor upregulates the LDL-R expression
within 24-72 hours after stimulation, and permits
efficient VSV-G pseudotyped lentivirustransduction. Therefore, for optimal lentivirustransduction of γδ T cells, it is recommended toactivate the cells by crosslinking CD3 and CD28
followed by lentivirus transduction between 24 and72 hours after activation.

Product use statement

TheraPEAK® Products are not for human or animalin vivo or diagnostic use, including use as a diluent
or as an excipient. All TheraPEAK® Products areproduced according to applicable GMP standards
and follow the USP/EP guidance for cell and genetherapy raw materials. It is the end user's
responsibility to ensure full compliance withapplicable regulations based on their use of
Lonza's products in their specific process. All
trademarks belong to Lonza, and are registered inUSA, EU and/or CH or belong to third-party owners
and are used only for informational purposes. All
third-party copyrights have been reproduced withpermission from their owners. The information
contained herein is believed to be correct,
however, no warranty is made, either expressed orimplied. Any user must make his own
determination and satisfy himself that the products
supplied by Lonza Group Ltd or its affiliates and theinformation and recommendations given by Lonza
Group Ltd or its affiliates are (i) suitable forintended process or purpose, (ii) in compliancewith all applicable laws, including all environmental,
health and safety regulations, and (iii) will not
infringe any third party’s intellectual property
rights. The user bears the sole responsibility for
determining the existence of any third-party rights,
as well as obtaining any necessary licenses andapprovals.
For more details: http://www.lonza.com/legal.
©2024 Lonza, Inc. All rights reserved.

Page 3 of 3