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                                                             6/11/2007                                                                                                                                                                              (c) 2006 Cellular Technology Ltd.   1
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                    The Effects of Cell Processing on Cryopreserved
                    Human Peripheral Blood Mononuclear Cells

                                                 Hari Ramachandran and Paul Lehmannn (unpublished results)

                                                                                                                                                                              Presentation created by Ioana Moldovan

                                                                                                                                                                                                                                                   Cellular Technology Ltd.




                                                             6/11/2007                                                                                                                                                                                 (c) 2006 Cellular Technology Ltd.   2
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                A 40                                                                                                                                                                                                                               B 120
                  Cell Recovery (*10 )
               6




                                           30




                                                                                                                                                                                                                                                   Ce ll Viability (%)
                                                                                                                                                                                                                                                                         90


                                           20                                                                                                                                                                                                                            60                                                               Fig. 1 Comparison of the speed
                                                                                                                                                                                                                                                                                                                                          at which thawed PBMCs were
                                           10                                                                                                                                                                                                                            30
                                                                                                                                                                                                                                                                                                                                          diluted (fast vs slow) and of the
                                            0                                                                                                                                                                                                                                 0                                                           temperature of the thawing
                                                                                   Warm                                                                                             Cold                                                                                                            Warm               Cold               medium (warm vs cold). Cell
                C 40                                                                                                                                                                                                                               D 120                                                                                  recovery (panels A and C) and
                                                                                                                                                                                                                                                                                                                                          cell viability (panels B and D) are
                  Ce ll Recov ery (*10 )
               6




                                           30                                                                                                                                                                                                                            90
                                                                                                                                                                                                                                                   Ce ll Viability (%)


                                                                                                                                                                                                                                                                                                                                          shown in the 4 possible
                                           20                                                                                                                                                                                                                            60                                                               combinations: warm/slow (WS),
                                                                                                                                                                                                                                                                                                                                          warm/fast (WF), cold/slow (CS)
                                           10                                                                                                                                                                                                                            30                                                               and cold/fast (CF). Panels E and
                                            0                                                                                                                                                                                                                                   0
                                                                                                                                                                                                                                                                                                                                          F are summaries for cell recovery
                                                                                      Slow                                                                                           Fast                                                                                                           Slow               Fast               and cell viability for the four
                                           E 20                                                                                                                                                                                                                          F 100
                                                                                                                                                                                                                                                                                                                                          methods of media addition.
                                            Ce ll Recovery (*10 )




                                                                                                                                                                                                                                                                                               95
                                           6




                                                                    15
                                                                                                                                                                                                                                                                         Ce ll Viability (%)




                                                                                                                                                                                                                                                                                               90
                                                                    10
                                                                                                                                                                                                                                                                                               85

                                                                     5
                                                                                                                                                                                                                                                                                               80

                                                                     0                                                                                                                                                                                                                         75
                                                                                     WS                                              WF                                              CS                                             CF                                                              WS       WF        CS        CF
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A                       15                                                                                                                                                                                                                 B 120
                                                                                                                                                                                      p≤0.001
 Cell Recovery (*10 )




                                                                                                                                                                                                                                                                                                                                Fig. 2 Cell recovery (A) and
6




                                                                                                                                                                                                                                                                  90




                                                                                                                                                                                                                                            Cell Viability (%)
                        10                                                                                                                                                                                                                                                                                                      cell viability (B) by AOEB
                                                                                                                                                                                                                                                                  60                                                            staining and quantified under
                                5                                                                                                                                                                                                                                                                                               addition of warm or cold
                                                                                                                                                                                                                                                                  30
                                                                                                                                                                                                                                                                                                                                thawing medium. Cell
                                0                                                                                                                                                                                                                                           0
                                                                                                                                                                                                                                                                                                                                recovery (C) and cell viability
                                                                         Warm                                                                                                Cold                                                                                                          Warm               Col d             (D) differs according to the
                                                                                                                                                                                                                                                                                                                                staining technique (TB vs
                        C                       10                                                                                                                                                                        Warm D 120                                                                                    Warm    AOEB) for both warm and
                                                                                                                                                                                                                          Cold
                                                                                                                                                                                                                                 100
                                                                                                                                                                                                                                                                                                                        Cold    cold addition of thawing
                        Cells Recovery (*106)




                                                8
                                                                                                                                                                                                                                                                                                                                medium. TB = Trypan Blue;
                                                                                                                                                                                                                                                                 Cell Viability (%)




                                                                                                                                                                                                                                                                                      80
                                                6                                                                                                                                                                                                                                                                               AOEB = Acridine Orange and
                                                4
                                                                                                                                                                                                                                                                                      60
                                                                                                                                                                                                                                                                                                                                Ethidium Bromide
                                                                                                                                                                                                                                                                                      40
                                                2
                                                                                                                                                                                                                                                                                      20

                                                0                                                                                                                                                                                                                                      0
                                                                                                    TB                                                                                       AOEB                                                                                              TB                 AOEB




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A                        20                                                                                                                                                                                                                        B                          105
  Cell Recovery (*10 )
6




                                                                                                                                                                                                                                                   Cell Viability (%)
                         15                                                                                                                                                                                                                                                        95


                         10                                                                                                                                                                                                                                                        85                                       Fig 3. Effect of the number
                                                                                                                                                                                                                                                                                                    5                       of washes on cell recovery
                          5                                                                                                                                                                                                                                                        75
                                                                                                                                                                                                                                                                                                                            (panels A and C) and cell
                          0                                                                                                                                                                                                                                                        65                                       viability (panels B and D). All
                                                                                     1X              2X                                                                                                                                                                                    1X             2X                samples were stained with
                                                                                      Number of Washes                                                                                                                                                                                      Number of Washes
                                                                                                                                                                                                                                                                                                                            AO/EB and warm wash
C                        12                                                                                                                                                                                                                        D                               99
                                                                                                                                                                                                                                                                                                                            medium was used. Panels C
                         10                                                                                                                                                                                                                                                                                                 and D are summary figures
 Cell Recovery (*10 )
6




                                                                                                                                                                                                                                                              Cell Viability (%)




                                                                                                                                                                                                                                                                                   96
                          8                                                                                                                                                                                                                                                                                                 for all the individual donors,
                          6                                                                                                                                                                                                                                                        93                                       shown in panels A and B.
                          4
                                                                                                                                                                                                                                                                                   90
                          2

                          0                                                                                                                                                                                                                                                        87
                                                                                     1X              2X                                                                                                                                                                                    1X               2X
                                                                                      Number of Washes                                                                                                                                                                                       Number of Washes




                                                             6/11/2007                                                                                                                                                                                                                  (c) 2006 Cellular Technology Ltd.                          5
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A                         15                                                                                                                                                                                                                       B 110
Cell Recovery (*10 )




                          12
6




                                                                                                                                                                                                                                                                                                                        Fig. 4 Comparison of cell




                                                                                                                                                                                                                                                   Cell Viability (%)
                                                                                                                                                                                                                                                                         100
                          9                                                                                                                                                                                                                                                                                             recovery (A) and cell
                                                                                                                                                                                                                                                                          90
                          6                                                                                                                                                                                                                                                                                             viability (B) after PBMCs
                                                                                                                                                                                                                                                                          80                                            were thawed and allowed
                          3
                                                                                                                                                                                                                                                                                                                        to sit in DMSO for 0, 30
                          0                                                                                                                                                                                                                                               70                                            or 60 minutes. All
                                                                        0                                            30
                                                                                                                  Time (min)
                                                                                                                                                                                                             60                                                                      0           30
                                                                                                                                                                                                                                                                                              Time (min)
                                                                                                                                                                                                                                                                                                                   60
                                                                                                                                                                                                                                                                                                                        samples shown were
C                         10
                                                                                                                                                                                                                                                                                                                        stained with AO/EB and
                                                                                                                                                                                                                                                   D                     100                                            washed twice with warm
   Cell Recovery (*10 )




                             8                                                                                                                                                                                                                                                                                          medium. (C) Average
6




                                                                                                                                                                                                                                                    Cell Viability (%)




                             6
                                                                                                                                                                                                                                                                          96
                                                                                                                                                                                                                                                                                                                        number of cells
                                                                                                                                                                                                                                                                                                                        recovered from all
                                                                                                                                                                                                                                                                          92
                             4                                                                                                                                                                                                                                                                                          donors at each time
                             2                                                                                                                                                                                                                                            88                                            point. (D) Average
                                                                                                                                                                                                                                                                                                                        number of viable cells
                             0                                                                                                                                                                                                                                            84                                            obtained from all donors
                                                                                 0                                               30
                                                                                                                              Time (min)
                                                                                                                                                                                                                                   60                                                 0          30                60
                                                                                                                                                                                                                                                                                                                        at each timepoint.
                                                                                                                                                                                                                                                                                              Time (min)




                                                             6/11/2007                                                                                                                                                                                                         (c) 2006 Cellular Technology Ltd.                            6
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         A                              700                                                                                                                                                                          B                             300
                                        600                                                                                                                                                                                                        250
            Spots Produced by




                                                                                                                                                                                                                      Spots Produced by
                                        500
                                                                                                                                                                                                                                                   200                                         Fig. 5 Functionality in the IFNγ




                                                                                                                                                                                                                        CD4+ T Cells
              CD8+ T Cells




                                        400

                                        300
                                                                                                                                                                                                                                                   150                                         ELISpot assay of cells stimulated
                                        200                                                                                                                                                                                                        100                                         by the CEF peptide pool,
                                        100                                                                                                                                                                                                        50                                          designated as CD8+ T cells
                                                  0
                                                                                      Warm                                                                         Cold
                                                                                                                                                                                                                                                     0
                                                                                                                                                                                                                                                          Warm                   Cold
                                                                                                                                                                                                                                                                                               (panels A, C, E) and of cells
         C                               800                                                                                                                                                                         D                             30 0                                        stimulated by recall antigens,
                                         700                                                                                                                                                                                                       25 0                                        designated as CD4+ T cells
                                                                                                                                                                                                                       Spots Produced by
        Sp ots Produced by




                                                                                                                                                                                                                                                                                               (panels B, D, F). Cells were
                                         600
                                                                                                                                                                                                                          CD4+ T Cells




                                                                                                                                                                                                                                                   20 0
           CD8+ T Cells




                                         500
                                         400                                                                                                                                                                                                       15 0
                                                                                                                                                                                                                                                                                               treated with warm or cold
                                                                                                                                                                                                                                                                                               medium (panels A and B), washed
                                         300                                                                                                                                                                                                       10 0
                                         200
                                         100
                                                                                                                                                                                                                                                    50
                                                                                                                                                                                                                                                                                               once or twice (panels C and D),
                                           0
                                                                                                                                                                                                                                                                                               and allowed to sit in DMSO for 0,
                                                                                                                                                                                                                                                     0

                                                                                              1X                                                                        2X                                                                                    1X                     2X

                                                                                              Number of Washes
                                                                                                                                                                                                                                                               N umber of Wa she s
                                                                                                                                                                                                                                                                                               30 or 60 minutes. 0=0 minutes
         E                                900
                                          800
                                                                                                                                                                                                                     F                             300
                                                                                                                                                                                                                                                                                               sitting thawed in DMSO; 30=30
                                                                                                                                                                                                                                                   250
                                                                                                                                                                                                                                                                                               minutes in DMSO; 60=60 minutes
             Sp ots Produced by




                                                                                                                                                                                                                     Sp ots Produced by




                                          700
                CD8+ T Cells




                                                                                                                                                                                                                        CD4+ T Cells




                                          600
                                                                                                                                                                                                                                                                                               in DMSO.
                                                                                                                                                                                                                                                   200
                                          500
                                                                                                                                                                                                                                                   150
                                          400
                                          300                                                                                                                                                                                                      100
                                          200
                                                                                                                                                                                                                                                   50
                                          100
                                            0                                                                                                                                                                                                        0
                                                                                      0                                            30                                              60                                                                     0            30                 60




                                                             6/11/2007                                                                                                                                                                                               (c) 2006 Cellular Technology Ltd.                       7
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         A                                                                                                                                                                                                           B
                                                                                                                                                                                                                                                             99
                                                         58
                         Cell Reco very (*106 )




                                                                                                                                                                                                                                                             97




                                                                                                                                                                                                                              Cell Viability (%)
                                                         48                                                                                                                                                                                                  95

                                                                                                                                                                                                                                                             93
                                                         38
                                                                                                                                                                                                                                                             91
                                                         28
                                                                                                                                                                                                                                                             89
                                                                                                                                                                                                                                                                                                             Fig. 6 Experiments performed by
                                                                                                                                                                                                                                                             87
                                                                                                                                                                                                                                                                                                             using medium containing benzonase
                                                         18
                                                                          Without                                                                     With                                                                                                             Without              With
                                                                         Benzonase                                                                  Benzonase                                                                                                         Benzonase           Benzonase
                                                                                                                                                                                                                                                                                                             or without benzonase. Cell recovery
         C                                                50                                                                                                                                                         D
                                                                                                                                                                                                                                                             99                                              (A) and cell viability (B) for
                                                                                                                                                                                                                                                                                                             individual donors are shown. The
                                Cell Reco very (*106 )




                                                                                                                                                                                                                                       Cell Vi ability (%)



                                                          40
                                                                                                                                                                                                                                                             96
                                                                                                                                                                                                                                                                                                             average cell recovery (C) and
                                                          30                                                                                                                                                                                                 93
                                                                                                                                                                                                                                                                                                             average cell viability (D) of all tested
                                                                                                                                                                                                                                                                                                             donors is represented. Panel E shows
                                                          20                                                                                                                                                                                                 90                                              the number of IFNγ spots produced
                                                                                                                                                                                                                                                                                                             by cells stimulated with recall
                                                                            With out                                                                    With                                                                                                           With o ut             With
                                                                           Benzonase                                                                  Benzonase                                                                                                       Ben zo n ase         Ben zo n ase

         E                                               250                                                                                                                                                         F                                       140                                             antigens, designated as CD4+ cells.
                                                         200
                                                                                                                                                                                                                                                             120
                                                                                                                                                                                                                                                                                                             Panel F shows the percentage
             Spots p ro duced b y




                                                                                                                                                                                                                         % Resp o nse b y




                                                                                                                                                                                                                                                             100
                                                                                                                                                                                                                                                                                                             response of all the donors tested for
                                                                                                                                                                                                                          CD 4+ T Cells
               CD4+ T Cells




                                                         150                                                                                                                                                                                                  80

                                                         100
                                                                                                                                                                                                                                                              60                                             recall antigens.
                                                                                                                                                                                                                                                              40
                                                          50
                                                                                                                                                                                                                                                              20

                                                           0                                                                                                                                                                                                      0
                                                                           With out                                                                   With                                                                                                              With o ut            With
                                                                          Benzon ase                                                                Benzonase                                                                                                          Ben zo nase         Ben zon ase




                                                               6/11/2007                                                                                                                                                                                                             (c) 2006 Cellular Technology Ltd.                         8
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                           Summary
                           • Cryopreserved PBMC give the best recovery and viability when
                             thawed quickly, by adding slowly warm thawing medium.

                           • Staining PBMC with acridin orange/ethidium bromide gives the
                             most accurate count on the UV microscope, but trypan blue
                             staining also gives comparable results.

                           • Two washes yield both good cell recovery and good cell
                             viability.

                           • PBMC should sit the least amount of time thawed in DMSO
                             before further processing. However, leaving them in DMSO for
                             up to 30 minutes doesn’t affect drastically the cell recovery
                             and cell viability.

                                                             6/11/2007                                                                                                                                                                             (c) 2006 Cellular Technology Ltd.   9
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                           Summary (cont’d)

                           • The functionality of thawed PBMC in the ELISpot assay was
                             best when cells were thawed quickly, by adding slowly warm
                             medium.

                           • The addition of benzonase to prevent cell clumping assures
                             that 100% of the thawed cells are recovered and viable to
                             produce cytokine upon antigenic stimulation.




                                                             6/11/2007                                                                                                                                                                             (c) 2006 Cellular Technology Ltd.   10

								
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