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Transcript of 1 Large Scale culture. 2 APPLICATIONS Basic Biology Cancer Biology Genomics Drug Discovery Cell...
1
Large Scale culture
2
APPLICATIONSAPPLICATIONS
Basic BiologyBasic BiologyCancer BiologyCancer BiologyGenomicsGenomicsDrug DiscoveryDrug DiscoveryCell TherapyCell Therapy
CHARACTERIZATIONCHARACTERIZATION
CD Surface AntigensCD Surface AntigensChemokine ReceptorsChemokine ReceptorsCytokinesCytokinesMitogensMitogensImmunological FeaturesImmunological Features
SOURCESOURCE
Bone MarrowBone MarrowAdipose TissueAdipose TissueUmbilical CordUmbilical CordAmniotic FluidAmniotic FluidSkeletal MuscleSkeletal Muscle& Others& Others
ESC/iPSCESC/iPSC
Multi-lineage Adult Stem Multi-lineage Adult Stem Cells Cells (i.e. (i.e. MAPC, MPLC, MIAMI, etc.)MAPC, MPLC, MIAMI, etc.)
MESODERM MESODERM DIFFERENTIATIONDIFFERENTIATION
EXPANSIONEXPANSIONISOLATIONISOLATION
ChondrocytChondrocyte e
(cartilage)(cartilage)
OsteocytOsteocyte (bone)e (bone)
Adipocyte Adipocyte (fat)(fat)
Myoblasts Myoblasts (muscle)(muscle)
TRANSDIFFERENTIATIONTRANSDIFFERENTIATION
NeuralNeuralHepaticHepaticEndothelialEndothelial
TenocytTenocyte e
(tendon(tendon))
Mesenchymal Stem Cells
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Human MSC Therapeutic ApplicationsHuman MSCs have become of interest for clinical application due to:
• Capacity for homing and engraftment
• Wide-range differentiation potential
• Immunosuppressive attributes
Potential MSC Therapies:
• Graft versus Host Disease
• Crohn’s Disease
• Bone Defects/ Genetic Disease
• HSC Transplantation
• Cardiac repair
• NIH Clinical Trials search for mesenchymal stem (stromal) cells = >90 studies (www.clinicaltrials.gov)
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Overview of MultiStem® Production Process
Lot Release & Product Characterization Testing
SterilityPotency
Identity and ViabilityStable Cytogenetics
Absence of tumorigenic potential in vivo
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MultiStem – Adherent Adult Stem Cell Platform-Athersys
IND Protocol Title Key elements
Phase I, Multicenter, Dose-Escalation Trial Evaluating Maximum-Tolerated Dose of Single and Repeated Administration of Allogeneic MultiStem® in Patients with Acute Leukemia, Chronic Myeloid Leukemia, or Myelodysplasia
IV-delivered productPhase I: open label – SAD, MADAdjunctive to BM/HSC transplant
Phase I, Multicenter, Dose-Escalation Trial Evaluating the Safety of Allogeneic AMI MultiStem® in Patients with Acute Myocardial Infarction
Catheter-delivered productPhase I: open label (w/ registry)
Phase I, Multicenter, Dose-Escalation Trial Evaluating the Safety of Allogeneic MultiStem® in Patients with Ischemic Stroke
IV-delivered productPhase I: blinded, placebo controlled
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The processing of human MSCs for therapeutic application should be a standardized GMP process, including defined:
(a) Starting material: donor, tissue origin, harvesting, enrichment method, etc.
(b) Tissue culture processing methods: culture medium, culture conditions, etc.
(c) Devices for cell culture: closed (or nearly closed) cell culture system
(d) Quality control:
> input material (donor) analysis
> active components or impurities
> expanded cell karyotype
Human MSC Therapeutic Applications
Sensebé and Bourin Transplantation. 2009 May 15;87(9 Suppl):S49-53. Review.
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Chondrogenesis
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Act
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ton
Sig
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Che
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Eph
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tor
Sig
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ER
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AP
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GM
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Inte
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Sig
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p38
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PD
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Sig
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PP
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PT
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Sig
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Sig
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Wnt
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Osteogenesis
0
0.5
1
1.5
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2.5
3
3.5
Dea
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Sig
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Sig
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Leuk
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Sig
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Adipogenesis
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Act
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Sig
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nce
Sig
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1/S
Che
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EG
F S
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Sig
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ER
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AP
KS
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Hun
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Dis
ease
Sig
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IGF
-1S
igna
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Insu
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Sig
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Inte
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Sig
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JAK
/Sta
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Leuk
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Sig
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Neu
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PD
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Sig
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A07
A14
* Analysis of active pathways in hMSCs suggests that PDGF, TGFβ and FGF are important signaling pathways for hMSC proliferation and differentiation
7 pathways 13 pathways
15 pathways
Day 14Day 7
3.31
2.82
8.28
2.29
3.46
3.12
0.71
5.38
8.03
9.76
3.4620
4.0910
6.924Tyrphostin (IC50 20 M)
10.772SU 5402 (IC50 10-20 M)
2.8820
6.3510
7.212SB431542 hydrate (IC50 10 M)
1.0140
5.0520
10.38-No inhibitor
Fold increase in viable cell numberConc (M)Inhibitor
Day 14Day 7
3.31
2.82
8.28
2.29
3.46
3.12
0.71
5.38
8.03
9.76
3.4620
4.0910
6.924Tyrphostin (IC50 20 M)
10.772SU 5402 (IC50 10-20 M)
2.8820
6.3510
7.212SB431542 hydrate (IC50 10 M)
1.0140
5.0520
10.38-No inhibitor
Fold increase in viable cell numberConc (M)Inhibitor
Ng et al. Blood. 2008 Jul 15;112(2):295-307 2008
Active Signaling Pathways in Human MSCs
(PDGF inhibitor)
(TGFβR inhibitor)
(FGFR inhibitor)
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StemPro® MSC SFM Kit (Cat# A10332-01)
Consists of the following:
1. StemPro® MSC SFM Basal Medium (1X)(500ml)
2. StemPro® MSC SFM Supplement (6.67X)(75ml)
Note: Additional components required:
• CELLstart (XenoFree Substrate) (Cat# A1014201)
• GlutaMAX (Cat# 35050) or L-Glutamine (Cat# 25030)
• TrypLE Express (Cat# 12604013) (Animal Origin Free)
StemPro MSC SFM
9
Total Cell Expansion: STEMPRO MSC SFM
0.0E+00
1.0E+06
2.0E+06
3.0E+06
4.0E+06
5.0E+06
6.0E+06
7.0E+06
8.0E+06
9.0E+06
0 1 3 5 7 10
Passage
Net
Tot
al C
ell N
umbe
r P
er F
lask
DMEM + 10% MSC-Qualified FBS
STEMPRO MSC SFM
Adipocyte (Oil Red O)
Chondrocyte
(Alcian Blue)
Osteoblast (Alkaline Phosphase)
0
200
400
600
800
1000
1200
1400
1600
1800
SCM D+PBT D D+P D+B D+T D+PB D+PT D+BT
Growth Factor Supplementation
Rel
ativ
e F
lou
resc
ence
Un
its
(RF
U)
StemPro MSC SFMDMEM + 10% FBS
P = PDGF-BB
B = bFGF
T = TGFβ1
Exp
ansi
on
Dif
fere
nti
atio
n
input cells = passage 5 human Bone Marrow MSCs (4-donor pool)
StemPro MSC SFM
Beadchip Gene Array Analysis
Chondrocyte (Toluidine Blue)
Chase et al. 2009 Submitted.
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Ste
mP
ro M
SC
SF
MM
EM
alp
ha
+ 1
0%
FB
S
H&E staining; Data published in Agata et al. Biochem Biophys Res Commun 2009;382(2):353-8
Beta Test Data – Dr. Hideaki Kagami – The University of Tokyo
in vivo ectopic bone assay
Experimental Observations:
1) Enhanced primary culture efficacy (more cells faster)
2) Lower alkaline phosphatase activity in undifferentiated cells
3) Greater responsiveness to osteogenic induction
4) Confirmed in vivo ectopic bone formation
Primary Culture
StemPro MSC SFM
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• A cGMP serum-free medium specially formulated for the growth and expansion of human mesenchymal stem cells (MSCs).
• Contains xenogenic components not amenable to most clinical applications
• Need for a second generation product: StemPro MSC SFM XenoFree
PERFORMANCE - BETTER CELLS - CONVENIENCEPERFORMANCE - BETTER CELLS - CONVENIENCE
StemPro MSC SFM
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StemPro MSC SFM XenoFree Kit (Cat# A11409SA)(Custom cGMP)
Consists of the following:
1. StemPro® MSC SFM Basal Medium (Cat# A10334-01) (500ml)(1X)
2. StemPro® MSC SFM Supplement (Cat# 09-0012SA) (5ml)(100X)
Note: Additional components required:
• CELLstart (XenoFree Substrate) (Cat# A1014201)
• GlutaMAX (Cat# 35050) or L-Glutamine (Cat# 25030)
• TrypLE Express (Cat# 12604013) or TrypLE Select (Cat# 12563011) (Animal-Origin Free)
Complete cGMP XenoFree workflow: (1) Medium; (2) Substrate; (3) Harvest enzyme
For order inquiries please contact: Sandy Kuligowski ([email protected]) or Lucas Chase ([email protected])
StemPro MSC SFM XenoFree
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StemPro MSC SFM XenoFree
Pas
sa
ge
1
DMEM + 10% MSC-Qualified FBS
Pas
sa
ge
9
Input cells = passage 5 human Bone Marrow MSCs (4-donor pool)
Adipocyte (Oil Red O)
Chondrocyte (Alcian Blue)
Osteoblast (Alkaline Phosphase)
Passage 5 Multi-lineage Mesoderm Differentiation - StemPro Differentiation Regents
Exp
ansi
on
an
d D
iffe
ren
tiat
ion
StemPro MSC SFM XenoFree: BM-MSC Expansion
StemPro
MSC SFM XenoFree: Doubling Time(Human BM-MSC)
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9
Passage
Do
ub
ling
Tim
e (H
ou
rs)
DMEM + 10% MSC-Qualified FBS StemPro MSC SFM XenoFree
StemPro
MSC SFM XenoFree: Net Population Doublings(Human BM-MSC)
0
2
4
6
8
10
12
14
16
18
20
Set-up 1 2 3 4 5 6 7 8 9
Passage
Net
Po
pu
lati
on
Do
ub
ling
s
DMEM + 10% MSC-Qualified FBS StemPro MSC SFM XenoFree
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MarkerPassage 5
(% Positive)Passage 9
(% Positive)CD73+/NEG- 99.3 99.9CD90+/NEG- 96.4 100.0CD105+/NEG- 96.5 99.9CD34+ 0.1 0.6
MarkerPassage 5
(% Positive)Passage 9
(% Positive)CD73+/NEG- 99.7 100.0CD90+/NEG- 97.9 100.0CD105+/NEG- 98.1 100.0CD34+ 0.2 2.3
StemPro MSC SFM XenoFree
DMEM + 10% MSC-Qualified FBS
NOTE: NEG = multiplex analysis of CD14, CD19, CD45 and HLA-DR.
Negative markers-rPE
CD
105-
Ale
xa F
luo
r® 7
00
Negative markers-rPE
CD
73-P
erC
P
CD90-FITC
Ne
ga
tive
mar
kers
-rP
E
CD90-FITC
CD
34-Q
do
t ®
800
Passage 5Passage 5 Passage 9Passage 9
Cells analyzed at Passage 5 and 9 = 46, XY= NORMAL KARYOTYPECells analyzed at Passage 5 and 9 = 46, XY= NORMAL KARYOTYPE
Mu
ltip
lex
Flo
w C
yto
me
try
Jolene Bradford – Life Technolgies Jolene Bradford – Life Technolgies
Kar
yoty
pe
An
alys
is
Minimal Defining Criteria*:(1)Adherence to plastic under standard culture conditions (10% FBS-containing medium)
(2) Characteristic Surface marker expression
Positive (≥95%) Negative (≤2%)
CD73 CD11b or CD14
CD90 CD34
CD105 CD45
CD79a or CD19
HLA-DR
(3) In vitro tri-lineage mesodermal differentiation (osteoblasts, chondrocytes, adipocytes)
*Dominici et al. 2006
StemPro MSC SFM XenoFree: BM-MSC Characterization
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Multi-Parameter Human MSC Characterization
FxCycle™ Violet stainDNA content
Click-iT® EdU Alexa Fluor® 647 azideCell proliferation
Qdot® 800CD34
HLA-DR
CD45
CD19R-PE
CD14
Alexa Fluor® 700CD105
FITCCD90
PerCPCD73
FluorophoreMarker
CD90-FITCCD
10
5-A
lexa
Flu
or®
70
0
99.6%
0.4%
CD90-FITC
Neg
ativ
e m
ark
ers
-rP
E
100%
CD90-FITCC
D3
4-Q
do
t® 8
00
100%
CD90-FITC
CD
73
-Pe
rCP 99.5%
0.5%
Flow cytometry combiningClick-iT® EdU proliferation analysisWITH
Antibody-based immunophenotypingSimultaneous testing for percentage proliferation with phenotype characterization
Click-iT EdU®
Bright fieldHoechstClick-iT® EdU
HumanMSCs
Clic
k-iT
® E
dU
A
lexa
Flu
or®
64
7 a
zid
e
FxCycle™ Violet DNA
21.5%
FxCycle™ Violet DNA content
Proliferating Human MSCs detected with Click-iT® EdU reagents
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StemPro MSC SFM XenoFree: BM-MSC Characterization
R2 = 0.910
DMEM + 10% FBS - P3
SP
MS
C S
FM
XF
- P
3
R2 = 0.957
DMEM + 10% FBS – P3
DM
EM
+ 1
0% F
BS
– P
9
R2 = 0.949
SP MSC SFM XF - P3S
P M
SC
SF
M X
F –
P9
NOTES:
• Input cells = P5 MSC (4-donor pool)
• P3 = 13 days in culture
• P9 = 42 days in culture
• Beadchip = HumanWG-6 v3.0
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StemPro MSC SFM XenoFree OSTEOBLAST
Oil Red O (Day 14)
Alcian Blue (Day 14)
Alkaline Phosphase (Day 14)
Alizarin Red S (Day 21)
OSTEOBLASTCHONDROCYTEADIPOCYTE
Exp
ansi
on
(P
3)
Dif
fere
nti
atio
n (
P3)
StemPro MSC SFM XenoFree: BM-MSC Primary Isolation
MarkerPassage 3
(% Positive)
CD73+/NEG- 99.8CD90+/NEG- 99.9CD105+/NEG- 99.7CD34+ 0.4
StemPro MSC SFM XenoFree - Primary Isolation
NOTE: NEG = multiplex analysis of CD14, CD19, CD45 and HLA-DR.
StemPro MSC SFM XenoFree: Primary Expansion(Human BM-MSC)
0.0E+00
2.0E+06
4.0E+06
6.0E+06
8.0E+06
1.0E+07
1.2E+07
0 1 2 3Passage
Ne
t C
ell
Nu
mb
er
Pe
r F
las
k
StemPro MSC SFM XenoFree
Human Serum Human Serum RemovalRemoval
Passage 3 Multi-lineage Mesoderm Differentiation - StemPro Differentiation Regents
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Human ADSC – SP MSC SFM XF
Shayne Boucher – Life Technologies
StemPro MSC SFM XenoFree: ADSC Expansion
NOTES:
• Input cells = passage 4 StemPro Human ADSCs
• Differentitaion = Passage 3, Day 14 – StemPro Differentiation Reagents
Adipocyte (Oil Red O) Chondrocyte (Alcian Blue)Osteoblast (Alk Phos)
StemPro
MSC SFM XenoFree: Doubling Time(Human ADSC)
0
20
40
60
80
100
120
140
1 2 3 4 5
Passage
Do
ub
ling
Tim
e (
Ho
urs
)
Control Medium StemPro MSC SFM XenoFree
StemPro
MSC SFM XenoFree: Net Population Doublings (Human ADSC)
0
1
2
3
4
5
6
7
8
9
10
Set-up 1 2 3 4 5
Passage
Ne
t P
op
ula
tio
n D
ou
blin
gs
Control Medium StemPro MSC SFM XenoFree
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StemPro MSC SFM XenoFree: Comparisons
NOTE: input cells = passage 5 Human BM-MSCs (4-donor pool)
Exp
an
sio
n
(Pas
sa
ge
3)
Net Population Doubling: Competitor Audit
0
2
4
6
8
10
12
14
16
18
Set-up 1 2 3 4 5 6 7
Passage
Net
Po
pu
lati
on
Do
ub
ling
s
10% MSC-Qualified FBS StemPro MSC SFM XenoFree Competitor X
P3 Competitor X
CD90-FITC
CD
105-
Ale
xaF
luo
r® 7
00
MarkerPassage 3
(% Positive)CD73+/NEG- 98.8CD90+/NEG- 100.0CD105+/NEG- 100.0CD34+ 0.3
MarkerPassage 3
(% Positive)CD73+/NEG- 98.4CD90+/NEG- 77.3CD105+/NEG- 100.0CD34+ 0.4
StemPro MSC SFM XenoFree
Competitor X
NOTE: NEG = multiplex analysis of CD14, CD19, CD45 and HLA-DR.
StemPro MSC SFM XenoFree Competitor X
Ch
on
dro
cy
te
(Alc
ian
Blu
e)
(Pas
sa
ge
3)
P3 StemPro MSC SFM XenoFree
CD90-FITC
CD
105-
Ale
xaF
luo
r® 7
00
Flow cytometry data generated by Jolene Bradford – Life Technologies
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Expand Qualify StoreIsolate
Growth factors & substrates
ELISA
Cell selectionbeads
Cell imaging
Gene expression
Media, supplements& single use
processing systems
Cryopreservationmedia
Flow cytometry
Monitor
Dissociation enzymes
Processingdevices
Cell therapy work flow
HLA typing
Immuneresponse
monitoring
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Data generated by Christian Prante, Wolfgang Prohaska and Knut Kleesiek - Ruhr-Universität BochumData generated by Christian Prante, Wolfgang Prohaska and Knut Kleesiek - Ruhr-Universität Bochum
The detailed MSC isolation and expansion workflow was employed to provide an autologous MSC product from human bone marrow aspirate under completely closed conditions using xeno-free cell culture reagents.
ClosedClosed System Xeno-Free MSC Isolation and Expansion
1. 2. 3. 4. 5.
MSC expansion using the
CLINIcell cassetteand StemPro
MSC SFM XenoFree Medium
BM aspirate
Mononuclear cell isolation using
the Sepax system MSC harvest and wash
using TrypLE Select and theCytoMate Cell
Washer
AutologousMSC
product
22Can achieve manufacturing runs equivalent to >200,000L of liquid media
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Closed system considerations
Media bag ONLY, not a bioreactor!
Pre-filled, stocked and ready when you need them (reduced lead time)
Closed system, ideal for aseptic processes/applications
Small Volume ”Pillow”-Universal Bags” [5L, 10L & 20L]
Large Volume Bags” [100L ,200L, 500L & 1000L]
Easily customized to integrate with
automated systems and process applications (connection methods, in-line filtration, flow rates, tubing lengths, re-circulation loops, etc.)
Conveniently designed for bench top to larger-scale applications
Ideal for ‘daisy-chaining’ set-up
New ‘9101’ PE Film
Old PE Film
(Stedim & Crestbury)
New ‘9101’ PE Film
EVA Film
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Bioreactor – Single use bioreactors
Prefilled Wave O-Series Cellbag
• Ready to ship - media formulations include Sf-900II and Freestyle 293• Convenient - Cellbag chambers are filled and ready to use• Customizable - you choose the GIBCO medium formulation and volume
Size Working Volume
Cellbag10L/O 0.5L – 5LCellbag20L/O 1L – 10L
Connection Methods
• Quick Connect (MPC)• SCD Tubing• Threaded Luers• UNIVERSAL BAGS!!!!!
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Processing using Dynal® ClinExVivo™ MPC
Primary magnet
Cell depletion bag
Secondary magnet
Sample collection
bag
Sample bag
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DYNAL® Magnetic Beads
Combining magnetic particles and single use cell expansion technologies
Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy.
Hollyman D. et al. J Immunother. 2009 Feb-Mar;32(2):169-80.
Phase I Clinical Trial underway at Memorial Sloan Kettering
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• Undifferentiated hESCs are efficiently depleted from the sampleUndifferentiated hESCs are efficiently depleted from the sample• Higher purity of differentiated cells Higher purity of differentiated cells
• Differentiated cells remain unaffected after purificationDifferentiated cells remain unaffected after purification
Dynabeads® SSEA-4
Dynabeads® SSEA-4 are designed to remove undifferentiated human embryonic stem cells from a culture, providing you with highly pure and differentiated stem cells for your translational applications
Differentiated Neural Stem Cells before and after depletion of SSEA-4+ cells
SS
EA
-4 A
PC
0.2 %14 %
SS
EA
-4 A
PC
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Von Kossa (Calcium Deposit)
Alkaline Phosphatase
Alcian Blue (Proteoglycan)
Oil Red O (Lipid Vessible)
Osteogenesis
Osteogenesis
Chondrogenesis
Adipogenesis
Day 12 Expanded MSCs
IgG
1
BM
MN
C
IgG1 CD14 CD14/45
IgG
1
CD
90C
D90
CD
105
CD
105
Da
y 12
MS
C
Day 12 expanded mononuclear cells reveals robust selection of CD90 and CD105-positive cells in the absence of hematopoeitic antigen expression (CD14 and CD45), indicative of human MSC expansion. Expanded cells reveal retained multipotency as shown by positive staining results for osteogenesis, chondrogenesis and adipogenesis.
Total Cell Expansion: 3x105 BM MNCs 2x107 MSCs
ClosedClosed System Xeno-Free MSC Isolation and Expansion
Data generated by Christian Prante, Wolfgang Prohaska and Knut Kleesiek - Ruhr-Universität BochumData generated by Christian Prante, Wolfgang Prohaska and Knut Kleesiek - Ruhr-Universität Bochum
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Human MSC Culture Systems- Testing
Lot release- pathogen, sterility, identityfunctional quality, viability, consistency
Efficacy tests- Karyotype, epigenome, contaminating cells, HLA type, etc
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SummarySummary• Therapeutic application of human MSCs will require standardized
GMP-compliant procedures and reagents
• We have developed two generations of cGMP-manufactured serum-free MSC expansion media, including StemPro MSC SFM XenoFree
• Expansion of cells in StemPro MSC SFM XenoFree provides retained MSC phenotype and multipotency with a stable karyotype
• XenoFree medium applied to a closed isolation/expansion protocol reveals a promising clinical culture system
• Continual collaboration will be critical to explore both in vitro and in vivo applications
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AcknowledgementsRuhr-Universität Bochum (Germany)Ruhr-Universität Bochum (Germany) Christian PranteWolfgang ProhaskaKnut Kleesiek
A*STAR (Singapore)Vivek TanavdeFelicia NgSusie KohKonduru S. R. Sastry
The University of TokyoHideaki KagamiHideki Agata
Case Western Reserve UniversityLuis Solchaga
Life Technologies:Uma LakshmipathyShayne BoucherMohan VemuriSandy KuligowskiDoug DannerSaki PhommachanhJean DonovanMaureen CookBob KendersonAlaine MaxwellKate WagnerMary Lynn TilkinsAndy CampbellJolene BradfordScott Clarke
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