Reference Pbmc

(c) 2009 Cellular Technology Limited- CONFIDENTIAL -

Cryopreserved human PBMC libraries with defined HLA-type and antigen / peptide-reactivity for accelerating and standardizing human immunological research.


Step 1 towards standardization: Functionally loss-free freezing of PBMC

In 2003 CTL scientists introduced protocols for functionally loss-free freezing of PBMC:

Kreher et al. : CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays. J. Immunol. Methods, 278:79-93, 2003

(IFN-γ, IL-2, IL-4 and IL-5 were measured)


1a. The role of temperature:Blood should be shipped, stored, and processed all times at room

temperature, not chilled. For freezing, the cells and the DMSO-containing cryomedium must be at room temperature (not ice-cold) when mixed. When thawing, bring cells rapidly to 37 oC.

Live

Apoptotic

Dead


1b. Following this protocol, we found that high and low frequency, high and low avidity T cells are preserved, loss free. No “resting” needed after thawing.

NIAID Contr. HHSN266200400098I, ELISPOT Qualification, Vaccinia

Ex vivo/ Ex cryo


• Since 2005, CTL offers a reference PBMC library, presently consisting of over 70 PBMC donors to chose from, each• High resolution HLA-typed (Class I and Class II)

• Immune-characterized for• 20 antigens, recognized by CD8 and CD4 cells

• IFN-γ, IL-2, IL-4, IL-5, IL-17, Granzyme B

• High and low frequency T cell responses

• High and low avidity T cell responses

• Essentially unlimited availability of identical cell material: between 500 and 1,000 aliquots of 10 million PBMC/vial from each donor available from each draw.

• Reference PBMC are essential for assay comparisons, qualification/ validation, and harmonization across institutions

Step 2 towards standardization: Reference PBMC


Example: response profile of donor 19

LP-19

Peptide Conc. (Log M)

-12 -11 -10 -9 -8 -7 -6 -5 -4

SP

U/4

00

,00

0 P

BM

Cs

0

100

200

300

400

500

600

700

CEF-3CEF-4CEF-5CEF-6CEF-7sigmoidal dose-response (variable slope)


• In 2005, CTL introduced serum free media for all steps of PBMC processing and testing • Freezing (CTL-Cryo™)

• Thawing (CTL-Deaggregate ™)

• Washing (CTL-Wash ™)

• Testing (CTL-Test ™)

Since, we and our customers have seen no serum that performs significantly better, but many sera that perform (much) worse.

Step 3 towards standardization: Serum Free Media


Wouldn't if be fantastic if you...

• Did not need to chase your colleagues with a needle anymore to obtain some peripheral blood mononuclear cells (PBMC) for your experiments?



• Did not need to bother with IRBs just to obtain some blood from healthy volunteers because we have taken care of this for you?



• Can do experiments that you have never done before because you can obtain as many PBMC as you need (up to 10 billion cells from the same donor at the same time of bleed)?



• Can immediately repeat experiments with PBMC of the same donor and of the same bleed, if the reviewer asks you to provide that one additional control?



• Can share the same PBMC with collaborators anywhere on the planet?



• Can prepare PBMC for an experiment more simply and more quickly than isolating such cells from blood by yourself?



• Have immediate access to a library of PBMC that are pre-characterized, with established HLA-types and antigen reactivities?



• Can design your experiments by selecting test subjects from a panel of characterized human donors?



• Can use such pre-characterized cells as positive and negative controls for any immune monitoring effort, and as reference samples to control the consistent performance of your assays?



• Have access to a large number of human donors that you can screen for an immune reactivity and/or genetic marker of your own individual interest?



• Do not have to worry about the human material you work with is HIV , Hepatitis B, etc infected?


CTL has made this dream come true by offering immune characterized cryopreserved PBMC libraries!


Identifying reference PBMC samplesIL-2 responses to CMV antigen in human PBMC donors

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50

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Sample

IL-

2 s

po

ts / 4

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0 c

ells

IL-4 responses to CMV antigen in human PBMC donors

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Sample

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ts / 4

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ells

Utilization of ePBMC for ELISPOT assay development, qualification and validation Single color human, murine and macaque IFN-γ assay, Vaccinia/MVA- specific NIAID ContrNo. HHSN266200400098I, highlights


Vaccinia WR virus titration

Vaccinia WR virus concentration

4m

oi2m

oi

1 m

oi

0.5

moi

0.25

moi

0.12

5 m

oi

0.06

2 m

oi

0.03

1 m

oi

0.01

5 m

oi

0.00

7 m

oi

0.00

3 m

oi

0.00

1 m

oi

No.

IF

N

spot

s/40

0,00

0 ce

lls

0

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100

150

200

250CS180 CS189 321050806P 318050806V 20060706A

a. Optimal antigen dose


b. Accuracy


c. Linearity (and range)

r=0.998


d. Precision –

Inter assay repeatability


e. Precision

Intermediate precision

(using low responder)


0

0.1

0.2

0.3

0.4

0.5

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1

0 6 12 18 24 30 36 42 48Duration of CMV stimulation (h)

% M

ax

imu

m S

po

t N

um

be

r

IFN-g IL-2IL-4IL-17

Kinetics of cytokine production after CMV stimulation


IL-2

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r =0.998

IL-4

050

100150200250300350400450500

r =0.998

IL-17

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r =0.956

TNTC

Number of PBMC per Well

SF

U p

er

We

ll

IFN-g

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r =0.937

Linearity of CMV induced cytokine spots

Reference Pbmc

Technology

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