The following protocol has been optimized for freezing PBMCs and T cells using PRIME-XV FreezIS DMSO-Free (Catalog # 91140)
Cryopreservation Medium. The procedure was performed using buy coat from six healthy donors as starting material, both with a manual
method and an automated method.
Cryopreservation Protocol for PBMCs and
T Cells Using PRIME-XV FreezIS DMSO-Free
Cryopreservation Medium
Isolation and freezing of PBMCs from buffy coat using
Ficoll-Paque reagent
1. Buffy Coats were diluted 4x in DPBS.
2. 16 mL of Ficoll-Paque reagent was pipetted into a Leucosep tube.
Tubes were centrifuged at 1000 xg for 30 sec.
3. Diluted cell suspension was transferred into prepared Leucosep tube
and filled up with PBS to a total volume of 50 mL/tube.
4. Tubes were centrifuged at 1000 xg for 10 minutes at Room Temperature
(RT) in a swinging-bucket rotor centrifuge without brake. Three layers
formed above the barrier of Leucosep Tube: a plasma layer, the
interphase containing PBMCs and a small layer of Ficoll-Paque.
The plasma layer was discarded.
5. The interphase above the barrier, which contains PBMCs, was
transferred to a new 50 mL conical tube.
6. The conical tube was filled with DPBS and centrifuged at 300 xg for
10 minutes at RT. Supernatant was carefully removed.
7. For removal of platelets, cellular pellet was resuspended in 50 mL of
DPBS and centrifuged at 200 xg for 15 minutes at RT. Supernatant was
carefully removed. Step 7 was repeated twice to ensure platelet removal.
8. Cell pellet was re-suspended in RPMI 1640. Cell counting was performed
using trypan blue and Countess II automated cell counter.
EXPERIMENTAL PROCEDURE: MANUAL METHOD
MATERIALS: REAGENTS
PRIME-XV T Cell CDM
(FUJIFILM Irvine Scientific; Cat. # 91154)
IL-2
(CellGenix; Cat. # 1420-050)
CD3
(Miltenyi Biotec; Cat. # 130-093-387)
CD-28
(BioLegend; Cat. # 302934)
PRIME-XV FreezIS DMSO-Free
(FUJIFILM Irvine Scientific; Cat. # 91140)
PRIME-XV FreezIS
Cryopreservation Medium
(FUJIFILM Irvine Scientific; Cat. # 91139)
FBS
(Gibco, Cat. # 10101-145)
DPBS 1x
(Lonza; Cat. # 17-512F)
WFI
(Galenica Senese, Cat. # AA0013004)
Cryoserv DMSO
(Mylan/Lagitre, Cat. # 67457-178-50)
Includes Human Albumin solution
(Kedrion; Cat. # AIC 021111024)
NaCl Solution 9 mg/mL
(Galenica Senese; Cat. # AA0492027)
Human Serum Albumin Antibody
(R&D System; Cat. # IC1455P)
CD3
(BD Biosciences Cat. # 51-9007098)
CD4
(BD Biosciences Cat. # 561843)
CD8
(BD Biosciences Cat. # 561947)
CD14
(BD Biosciences Cat. # 561712)
CD19
(Miltenyi Biotec; Cat. #130-110-351)
CD56
(BD Biosciences Cat. # 561903)
PD1
(BD Biosciences Cat. # 561272)
SHEATH Fluid
(Sony; Cat. # AE700713)
FACS beads
(Sony; Cat. # LE-B3001)
9. An aliquot of freshly isolated PBMCs (1×10
5
cells/marker) was analyzed
by FACS to assess CD3
+
, CD14
+
, CD56
+
, and CD19
+
population
percentages.
10. Cells were frozen at 2.5×10
6
cells/mL and 5×10
6
cells/mL in four
different freezing media:
• Freezing medium solution 1:
90% FBS + 10% DMSO
• Freezing medium solution 2:
PRIME-XV FreezIS Cryopreservation Medium
• Freezing medium solution 3:
90% Human Serum Albumin + 10% DMSO
• Freezing medium solution 4:
PRIME-XV FreezIS DMSO-Free Cryopreservation Medium
11. Cells were transferred to a freezing container, Nalgene Mr. Frosty, and
cells were incubated at -80°C for 48 hours.
12. After 48 hours, the vials were moved to a liquid nitrogen vapor tank.
Thawing, activation, and expansion of PBMCs
1. After four months, a vial of PBMCs for each of the aforementioned
conditions was thawed. Cells were resuspended using double volume
of PRIME-XV T Cell CDM (Cat. # 91154) expansion medium and softly
centrifuged at 300 xg for 10 minutes. Acceleration and brake were set
at the minimum value depending on the centrifuge model available.
After centrifugation, cells were resuspended in 1 mL PRIME-XV T Cell
CDM expansion medium supplemented with 300 IU/mL IL-2, 50 ng/mL
of anti-CD3 (clone OKT-3), and 125 ng/mL of anti-CD28 (clone CD28.2).
2. Cell number, viability was assessed using trypan blue and Countess II
automated cell counter.
3. An aliquot (1×10
5
cells/marker) of cells was analyzed by fluorescence-
activated single cell sorting (FACS) to determine CD3
+
, CD14
+
, CD19
+
,
and CD56
+
population percentages.
4. Cells were seeded in a G-Rex 24-well plate using 8 mL/well of
PRIME-XV T Cell CDM expansion medium supplemented with 300 IU/
mL IL-2, 50 ng/mL of anti-CD3 (clone OKT-3), and 125 ng/mL of anti-
CD28 (clone CD28.2). Seeding density was 1×10
6
cells/well.
5. After 72 hours, 6 mL of the medium was removed. Cell counting and
viability assessment was performed using trypan blue and Countess
II automated cell counter. Medium was replenished with 6 mL of fresh
PRIME-XV T Cell CDM expansion medium supplemented with
300 IU/mL IL-2.
6. Step 5 was repeated on day 7 and day 10.
7. On day 13, the culture was stopped. 6 mL of spent medium were
removed and the cells were resuspended in the remaining 2 mL. Cell
counting and viability assessment was carried out using trypan blue
and Countess II automated cell counter. Cells (1×10
5
cells/marker) were
analyzed by FACS to evaluate CD3
+
, CD4
+
, CD8
+
, and PD1
+
population
percentages.
8. The remaining cells were frozen at 2.5×10
6
cells/mL in the four different
freezing media (see section I., step 10).
Cryopreservation Protocol for PBMCs and T Cells Using
PRIME-XV FreezIS DMSO-Free Cryopreservation Medium
(CONTINUATION)
Protocol developed and owned by Anemocyte Srl.
Data provided courtesy of Anemocyte Srl.
9. Cells were transferred to a freezing container, Nalgene Mr. Frosty, and
cells were incubated at -80°C for 48 hours.
10. After 48 hours, cells were moved to a liquid nitrogen vapor tank.
11. After 1 month, 1 vial for each condition was thawed to perform cell
counting and viability assessment, as well as FACS analyses on surface
markers expression.
EXPERIMENTAL PROCEDURE: AUTOMATED METHOD
1. Buffy coats were diluted with 4 volumes of PBS.
2. Diluted buffy coats were washed to remove the platelet using Platelet
Free protocol and settings recommended by instrument supplier with the
kit CT 60.1.
3. The output bag obtained from PlateletFree protocol contained 220 mL
of buffy coats washed to reduce the number of platelets. This bag was
loaded as input for the separation protocol using Ficoll-Paque reagent
(NeatCell protocol). PBMC isolation was performed using CT 90.1 kit with
the setting recommended by instrument supplier.
4. A final bag containing 50 mL of PBMCs was resuspended in RPMI 1640
was automatically obtained. Cell number and viability were assessed
using trypan blue and Countess II automated cell counter.
5. An aliquot of cell suspension corresponding to 2.4×10
7
cells for each
donor was transferred to a 15 mL centrifuge tube and centrifuged at
300 xg for 5 minutes. The cell pellet was resuspended in PRIME-XV
T Cell CDM expansion medium supplemented with 300 IU/mL IL-2,
50 ng/mL of anti-CD3 (clone OKT-3), and 125 ng/mL of anti-CD28
(clone CD28.2). The remaining cells were frozen in different
DMSO-containing media and not used for this set of experiments.
6. Cells were seeded in a G-Rex 24-well plate. One G-Rex plate was
prepared for each donor using 8 mL/well of PRIME-XV T Cell CDM
expansion medium supplemented with 300 IU/mL IL-2, 50 ng/mL of
anti-CD3 (clone OKT-3) and 125 ng/mL of anti-CD28 (clone CD28.2).
Seeding density was set at 1×10
6
cells/well.
7. After 72 hours, 6 mL of medium was removed to perform cell counting
and viability assessment using trypan blue and Countess II automated
cell counter. Medium was replaced using 6 mL of fresh PRIME-XV T Cell
CDM expansion medium supplemented with 300 IU/mL IL-2.
8. On day 7, cells were analyzed by FACS to assess CD3
+
, CD4
+
,
and CD8
+
population percentages.
9. Expanded T cells were frozen at 5×10
6
cells/mL in four different
freezing media:
• Freezing medium solution 1:
90% FBS + 10% DMSO
• Freezing medium solution 2:
PRIME-XV FreezIS Cryopreservation Medium
• Freezing medium solution 3:
90% Human Serum Albumin + 10% DMSO
• Freezing medium solution 4:
PRIME-XV FreezIS DMSO-Free Cryopreservation Medium
10. Cells were transferred to a freezing container, Nalgene Mr. Frosty, and
incubated at -80°C for 48 hours.
11. After 48 hours, the cells were moved to a liquid nitrogen vapor tank.
12. After four months, a vial of expanded T cells for each of the
aforementioned conditions was thawed and analyzed by FACS to test the
percentage of cells positive for CD3, CD4, and CD8. Simultaneously, cell
number and viability after thawing was evaluated using trypan blue and
Countess II automated cell counter.
Frozen 5×10
6
/mL
Number of Viable Cells
Viability (%)
Time Days
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