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Educational Web Seminar
“Scaling up Cell Culture: Application of Closed Cell Culture Systems in Clinical Research”
Thursday, May 8, 201411:00 AM 11:00 AM -- 12:15 PM ET12:15 PM ET
Adrian Gee, MI Biol, PhDProfessor, Departments of Medicine and Pediatrics
Center for Cell and Gene TherapyBaylor College of Medicine
Juan Vera, MDProfessor, Department of MedicineCenter for Cell and Gene Therapy
Baylor College of MedicineBaylor College of Medicine
Darin Sumstad, CLS (ASCP)Lead Medical Technologist
Fairview Clinical Cell Therapy LaboratoryUniversity of Minnesota
Today’s web seminar presentation slides are available publicly at www.pactgroup.net
CE Credit and certificates of attendance provided upon request
The Accreditation Council for Continuing Medical Education (ACCME) is the governing body thataccredits AABB to provide continuing medical education credits for physicians. In accordance with theACCME Standards for Commercial Support, AABB implemented mechanisms, prior to the planning andimplementation of this CME/CEU activity, to identify and resolve conflicts of interest for all individualsin a position to control content of this CME/CEU activity.
Faculty Disclosure Nature of Relationship Manufacturer/ProviderAdrian Gee, MI Biol, PhD None Speaker N/A
Juan Vera, MD Yes Scientific Advisor Wilson Wolf ManufacturingJuan Vera, MD Yes g
Darin Sumstad, CLS (ASCP) None Speaker N/A
Debbie Wood None Planning Committee PACT Staff N/A
David Styers None Planning Committee PACT Staff N/A
Karin Quinnan None Planning Committee PACT Staff N/A
Laarni Ibenana None Planning Committee PACT Staff N/A
Holly Baughman None Planning Committee PACT Staff N/A
Sharon Moffett None AABB Staff N/A
Jared Case None AABB Staff N/A
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In this web seminar, speakers will discuss best practices for producing sufficient numbers of cells required for clinical use. Significant advantages of closed cell culture systems over the commonly used traditional open systems will be highlightedcommonly used traditional open systems will be highlighted. Speakers will share their clinical research results generated
through the application of these culture techniques.
Inclusion of companies in this web seminar does not indicate endorsement by either the speakers or PACT nor is it meant to implyspeakers or PACT, nor is it meant to imply that their products or services are superior
to those of other companies.
Mesenchymal Stromal CellsLarge-scale Culture
Adrian GeeCenter for Cell & Gene Therapy
Baylor College of MedicineHouston, Texas
Hanley et al: Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System
Cytotherapy in Press
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Human Stem Cell
Adult Stem Cell
Pancreatic
Adult Somatic Stem Cells
Gut
Eye
Mesenchymal
Muscle
Tendon
LigamentMarrow Stroma
Pancreas Islets
RetinaLimbal Stem cells
Neuronal
Epidermal
Hematopoietic
Hepatic
Cartilage
Fat
FatCord Blood
MarrowApheresis
Cord Blood
Bone Marrow
Peripheral Blood
Clinical Trials using MSCwww.ClinicalTrials.gov: 352 studies on Mesenchymal stem cells
• Osteoarthritis• Severe Brain Injury (Adipose)• Ischemic Stroke (BM)
• Ischemic cardiomyopathy (BM)
• Multiple sclerosis (Adipose, BM, Cord blood)
• Systemic sclerosis• Liver cirrhosis• Lateral epicondylitis (Adipose)
• Crohn’s disease (BM, Adipose)
• Pulmonary fibrosis (BM)
• Parkinson’s disease (BM)
• Acute respiratory distress (Adipose)• Myocardial infarction• Liver failure• Cleft lip and palate• Cartilage defects (BM)Lateral epicondylitis (Adipose)
• Hereditary ataxia• Rheumatoid arthritis (Cord Blood)• Osteoarthritis (BM)
• Ulcerative colitis (Cord blood)• Type 1 diabetes (Cord Blood)• Type 2 diabetes• Spinal Cord Injury (BM)
• Critical limb ischemia in diabetes (Adipose)• Tibial & Femoral fractures• Cerebral artery infarcts (BM)
Cartilage defects (BM)
• Retinitis pigmentosa (BM)
• Degenerative disc disease• Cerebellar ataxia (Adipose)• Ulcerative colitis (Adipose)• Lupus nephritis• Mental retardation• Muscular dystrophy (Cord Blood)• Amyotrophic lateral sclerosis • Chronic wound healing• Emphysema
Other uses for MSCs
Adult Somatic Stem Cells
Muscle
Tendon
Ligament
Cartilage
Fat
“Other”
Mesenchymal
P ti f G HD i h t l i l
Immunosuppressive Effect
Combat GvHDCondition Recipients for Transplant
• Prevention of GvHD in hematological malignancies
• Prevention of subclinical rejection in organ transplant
• Treatment of chronic GvHD
• Promotion of engraftment in unrelated BMT
• Treatment of steroid refractory GvHD
• Poor graft function
• Co‐infusion with cord blood stem cells
• Induction of renal transplant tolerance
• Co‐infusion in mismatched mini‐transplants
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Aim
• Generation of allogeneic MSC for treatment of stroke
• FDA wanted us to use a method in clinical trials – Ed Horwitz, CHOP,
• Used platelet lysate instead of serum
• Wanted to try to close up system
• Looked at methods that would do that
Traditional Culture Method
• Automated bioreactor
• Closed system
• Whole marrow as starting material
Terumo Quantum Bioreactor
material
• Cells must meet all criteria for MSC
• Cleared for IND manufacturing
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Terumo Quantum Bioreactor
• Stand‐alone device• Automated , hollow‐fiber bioreactor system
Hanley et al: Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System Cytotherapy in Press
Quantum Manufacturing Procedure
Wash out non‐
adherent cells @ 24‐
48hr
10‐14 days 7‐10 days
Flow rate starts @ 0.1ml/min.
Monitor lactate & glucose If >lactate 4mM then double feeding rate, until reaches 0.4ml/min and lactate is at
4mM
Flow rate starts @ 0.1ml/min.
Monitor lactate & glucose If >lactate 4.5mM then
double feeding rate, until reaches 1.6ml/min and
lactate is at 5mM
Hanley et al: Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System Cytotherapy in Press
0200400600800
10001200
0 10 20 30 40
MSC Line A
0200400600800
10001200
0 10 20 30 40
MSC Line B
Quantum
Flasks
Cells x 106
Expansion of MSCs in the Quantum and Flasks
0 10 20 30 40
‐300
200
700
1200
0 10 20 30 40
MSC Line C
0 10 20 30 40
0
500
1000
1500
0 10 20 30 40
MSC Line D
DaysDays
Cells x 106
Hanley et al: Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System Cytotherapy in Press
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Cell Doubling Times
FlasksQuantum
MSC A MSC B MSC C MSC D Mean
Hanley et al: Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System Cytotherapy in Press
MSC Phenotype & Viability
Flasks (n=3) Quantum (n=4)
Hanley et al: Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System Cytotherapy in Press
CFU‐F Recovery
Hanley et al: Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System Cytotherapy in Press
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MSC Differentiation Potential
Cells tested for ability to differentiate into:‐
• Adipogenic cells
O t i ll
From: Research Center for Molecular Medicine, Debrecen, Hungaryhttp://rcmm.dote.hu/research‐groups/oxidative‐sress‐and‐adp‐ribosylation/
• Osteogenic cells
• Chondrogenic cells
All three cell types seen
T cells suppression by MSC
Proliferation 100
75
50
FlasksQuantum
1:0 1:1 1:05 1:0.1 1:0.05 Unstim
% CD4 T Cell 50
25
0
Hanley et al: Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System Cytotherapy in Press
T‐175 Flask BioreactorPer Flask/Expansion Set
Seeding Cells 2 3
Exchanging Media 2 1
Cell Dissociation 6 3
T l 10 7
Open events using Flasks versus the Bioreactor
Total 10 7
Per Donor (340 Flasks)
340 Flasks 3,400 7
Number of Donors / Expansion Sets
16 Donors* 19 Expansion Sets
(1 donor)
Total 54,400 133
Hanley et al: Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System Cytotherapy in Press
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Considerations
• Growth assessment is indirect – lactate production
• Disposable must be pre‐coated with fibronectin
• Can be accomplished in 4 hours
• Cost of disposablep
• Offset by cost of labor
• Cost of bioreactor
• Offset by multiple applications (retrovirus manufacturing)
Regulatory Considerations
• Cleared by FDA for use in stroke Phase 1 trial
• Selling point – closed system
• Comparability of cells from flasks and bi tbioreactor
• Issues regarding allogeneic “cell bank”
• Number of patients to be treated
• Degree of testing required
Collaborators
• Patrick Hanley – Children’s National Medical Center, Washington D.C.
• Zhuyong Mei – CAGT
• April Durett – CAGT
• Graca Cabreira‐Harrison Texas Heart InstituteGraca Cabreira Harrison – Texas Heart Institute
• Mariola Klis, Wei Li, Yali Zhou – CAGT
• Peiman Hematti & Debra Bloom – University of Wisconsin
• Sean Savitz et al. ‐ University of Texas Health Science Center
• Brent Rice – Terumo BCT
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Optimizing culture of suspension cells using the G‐Rex
• Prolonged culture period • Extensive manipulation ‐risk of contamination
Limitations of conventional suspension cell culture methods
risk of contamination• Labor intensive• Require highly trained personnel
• Excessive use of reagents
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Limited volume of media and gas exchange
Conventional Cultureware
Wilson Wolf ManufacturingGas Permeable Devices (G‐Rex)
• Gas permeable membrane allows exchange of CO2 and O2
• Supports cell growth with large volumes of media
• Reduces feeding frequency and manipulation
• No rocking or stirringVera JF et al. JIT. 2010
G‐Rex 100 (100cm2)
SA: 100 cm2 Vol: 500 ml
SA: 100 cm2 Vol: 2000 ml
G‐Rex 100
G‐Rex 100L
SA: 10 cm2 Vol: 40 ml
G‐Rex 10
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G‐Rex vs Conventional cultureware
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15
sion
0
3
6
9
G‐Rex Plate
Fold expan
Questions poll #1 Have you heard about the G‐Rex?
Have you used the G‐Rex?
•Yes •No
•Yes•No
What cells have you expanded in the G‐Rex •T cells•Regulatory T cells•NK cells•Cell lines•Murine cells
Questions poll #2
What applications do you use your cells for?
•Preclinical/Basic research •Clinical•Both
How many cells do you require for your application?
•5x10e8 •5x10e8 to 1x10e10•1x10e10
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1st – What is the optimal seeding density?
2nd – What is the optimal volume of media to use?
3rd – How can cell expansion be monitored?
1st – What is the optimal seeding density?
2nd – What is the optimal volume of media to use
3rd – How can cell expansion be monitored?
Low seeding density results in greater fold expansion
75
95
115
on
‐5
15
35
55
1.00E+06 5.00E+05 2.50E+05 1.25E+05 6.50E+04
Cells/cm2 (x 106)
Fold Expansio
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Low cell density = Higher cell expansion
rs
Max cell density 10E+07 cells/cm2
or t
Optimal seeding density
Time
Cell numbe
Op
timal
tim
e fo
cultu
re h
arve
st
1st – What is the optimal seeding density = 1.2E+05 cells/cm2
2nd – What is the optimal volume of media to use?
3rd – How can cell expansion be monitored?
10ml of media/cm2 resulted in the maximum cell expansion
2 (x 106)
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12
15
Volume of media/cm2
Cells/cm
0
3
6
0.5 2 5 10 15 20
14
Addition of media at the culture onset resulted in short culture period
10
12
14
16 10mL/cm2 (2.5mL*4)
10mL/cm2 (5mL*2)
10mL/cm2
(x 106)
0
2
4
6
8
0 3 6 9 13 16 20 24
Cells/cm
2
Time in culture
1st – What is the optimal seeding density = 1.2E+05 cells/cm2
2nd – What is the optimal volume of media = 10ml/cm2
3rd – How can cell expansion be monitored?
Inverse correlation between cell number and glucose concentration
10
12
14
200
250
300GlucoseCells
g/dl] C
e
0
2
4
6
8
0
50
100
150
Day 0 Day 3 Day 6 Day 9
Glucose [m
g ells/cm2
Time in culture
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Glucose assessment can be used to predict cell output
er (x 10
6)
80
100
120
140 Hemocytometer
Flow
Formula
Cell numbe
Days in culture
0
20
40
60
80
0 4 8 12
1st – What is the optimal seeding density = 1.2E+05 cells/cm2
2nd – What is the optimal volume of media = 10ml/cm2
3rd – How to monitor cell expansion = Glucose consumption
Are this observations reproducible?
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Day 0
•Celgene
Multi center PACT study
Combination of optimal culture conditions using the G‐Rex
•25E+06 cells•1 L of media
G‐Rex 100M
•Celgene •City of Hope •CAGT
Combination of optimal culture conditions using the G‐Rex
Day 0 Day 12
•25E+06 cells•1 L of media
G‐Rex 100M G‐Rex 100M
•1.4E+09 cells
Multi center study of optimal G‐Rex culture conditions
100
1000
10000CelGeneBaylorCity of Hope
er (x 106)
1
10
100
Day 0 Day 12
Cell numbe
Days in cultureBajgain et al. Mol Therapy – Methods and Development, 2014 In press
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The culture conditions in the G‐Rex are linear scalable
x 106)
1000
10000 G‐Rex 5
G‐Rex 100
G‐Rex 500
Cell number (x
0.1
1
10
100
Day 0 Day 10
Bajgain et al. Mol Therapy – Methods and Development, 2014 In press
What about the cell harvest?
GatheRex device
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GatheRex device
Questions poll #3
How would you like to see the G‐Rex develop?
• Closed system devices• Larger capacity• Multiwell platformp
Conclusions •G‐Rex provides a simple yet highly efficient platform for the expansion of suspension cells
Optimal G‐Rex conditions:•Seeding density: 1.25E+05 cells/cm2
•Media volume: 10mL of media/cm2
•Simple culture assessment: Glucose•Over 100 fold expansion in 10 days of culture•No feeding/manipulation required•Robust and easily scalable•Validated by multi‐center study•GatheRex/semi‐automatic harvest process
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AcknowledgementCenter for Cell and Gene Therapy
Pradip BajgainLara Brenner Roopa MucharlaUsanarat AnurtahapanNatasha LaptevaAdrian P. Gee
Wilson Wolf Manufacturing
John WilsonDan Welch
Celgene Cellular Therapeutics
Bitao LiangXi h LAdrian P. Gee
Helen HeslopAnn LeenCliona RooneyMalcolm Brenner
University of Wisconsin ‐Madison
John CentanniKyle Ripple
Xiaohua Lu
City of Hope
Christine HallDavid HsuLarry Couture
Scaling Up Cell Culture: Application of Closed Cell Culture Systems in Clinical ResearchPACT Webinar - May 8, 2014
D i S t d CLS T h i l L dDarin Sumstad, CLS-Technical Lead
MSC’s Glioblastoma UCB T-Regulatory Cells
Disclaimer This discussion will be based on the
implementation of the GE WAVE bioreactor system in our production processes. The speaker does NOT endorse the specific purchase of this orendorse the specific purchase of this or any other equipment discussed.
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Objectives Discuss WAVE platform Outline techniques utilized for WAVE
implementation into existing academic cGMP production lab
Discuss supporting equipment used to Discuss supporting equipment used to make this process easier.
Discuss “lessons learned” and solutions to production bottlenecks.
Questions?
History Why evaluate a WAVE BioReactor?
Current processing workflow will allow for expansions in excess of 20 billion cells - the ‘old fashioned’ way○ High sterility risk
E t l l b i t○ Extremely labor intense○ PI’s always want more…..
Incoming external projects already established on platform
History
vs
Provided by
Space saving, processing time, and a decrease in contamination risk make bioreactors an attractive alternative to traditional culture flasks.
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History Evaluate Current Production Processes
Traditional T-Flasks, Cell Factories, Culture Bags○ Open System
Internal processes developed to ‘close’ as much as possible (Rigging sets etc )possible (Rigging sets, etc.)
○ Equipment Standard – CO2 incubator, PVC tube sealer, Sterile
connecting device (SCD)
○ Media Requirements Low/Medium – Max required ~ 50L
Getting Started
Provided by
Getting Started
Provided by
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Getting Started New processing workflows would be
required to accommodate our evolution into large scale bioreactor processing Equipment management
Media preparation management
Fluid transfer management
Training
Getting Started
Materials Compatibility Can already stocked items be used for the
culture?○ Review supplied production procedure and/or
provide researcher with a spreadsheet template to complete.
○ Compare with available materials and re-distribute for acceptability approval
Not all materials will be available in-house
Materials CompatibilityMaterials Compatibility
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Materials CompatibilityMaterials Compatibility
Media Management Base Media Configurations
Traditionally offered in bottles – not ideal for closed system processing
Aseptically transferring from hard to soft wall vessel is challengingvessel is challenging
Media Management Base Media Configurations
Most media manufacturer’s will ‘custom fill’ into bags, but this requires a substantial initial investment of media (300 - 400L) to be economically viable○ If custom fill is an option for institution, media
preparation / storage of large volumes of media may be difficultdifficult
○ High risk – Premature trial closure – large monetary value tied up in
media – that will eventually expire! Storage – Manufacturer may not be willing to store
- Current capacity to hold on-site- Additional capital equipment investment- Institutional liability – power loss, equipment failure, etc.
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Equipment Equipment Requirements
Bioreactor○ Multiple required – coverage required for
downtime
Tube Sealer / SCD larger tubing not Tube Sealer / SCD – larger tubing not compatible with current models
Accessory equipment○ Uninterrupted power supply(UPS), real-time
equipment monitoring, CO2 supply access
EquipmentLarge volume cell harvest
• Additional equipment is required for the efficient processing of final cultured product.
• Traditional centrifuge not optimal
• CellSaver 5+, Cobe 2991, LOVO etcLOVO, etc.
• Validation required
EquipmentCobe 2991 Cell Saver LOVO
Max Process Volume Unlimited* Unlimited* 5L*
Observed NC Recoveries > 70% > 80% > 95%
Process Rate 125 ml/min** 250 ml/min 150 ml/min
Min Final Product Volume (mL) 50 70 50
*Theoretical / > 10L processed on LOVO with similar results
**3 min centrifugation time
Optimization work required - dependent on cell types / load concentration
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Equipment BioProfile 400*
pH, pO2, pCO2, Na+, K+, Gluc, Lac, NH4+,
Glu, Gln, Osm
*Information obtained from Nova Biomedical
Equipment Vi-Cell XR*
Automation of the standard trypan blue assay
% Viability
Total cell concentration
Total viable cell concentration
Mean cell size
*Information obtained from Beckman Coulter
Final Result - Optimized Procedure
Initial cell culture protocol
Optimized cell culture protocol
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Lessons Learned Historical bottleneck = generating cells
shift production bottleneck to final formulation and media management (including waste)
Immediate need for media companies to Immediate need for media companies to supply base media in bags as a catalog item (2L / 5L / etc.)
Budget for supporting equipment
Special Thanks!Dr. Dave McKennaDiane KadidloLisa VanOrsowSheryl AdamsNancy BostromNancy ColeyAnh Do
Cell Therapy Clinical Laboratory / MCT Support Staff / PACT / GE
Anh DoLeyla HassanJulie LatourLien LeStacy LinnMichelle LucioKristen ReynaMolly RicciCindy Stanaway
Past and Present Collaborators
“Scaling Up Cell Culture: “Scaling Up Cell Culture: Application of Closed Cell Culture Application of Closed Cell Culture
Systems in Clinical Research”Systems in Clinical Research”
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Speaker Contact EmailSpeaker Contact Email
Adrian [email protected]
Juan VeraJuan [email protected]
Darin [email protected]
WebWeb Seminar Presentation SlideSeminar Presentation Slidess
Today’s web seminar presentation slides and presentation slides from previous web seminars are available publicly atweb seminars are available publicly at
www.pactgroup.net www.pactgroup.net
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CE CreditCE CreditPhysiciansThis activity has been planned and implemented in accordance with the Essential Areas and Policies ofthe Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship ofAABB and PACT. AABB is accredited by the ACCME to provide continuing medical education forphysicians (Provider number 0000381). AABB designates this educational activity for a maximum of 1hour of AMA PRA Category 1 credit™ toward the AMA Physicians Recognition Award. Each physicianshould claim those credits that he/she actually spent in the activity.
California Clinical Laboratory PersonnelAABB is an approved, accrediting agency for continuing education for California-licensed clinicallaboratory personnel. This event has been approved for a maximum of 1.0 contact hours. AABB’saccrediting agency number is 0011. California clinical laboratory personnel must provide a personalsignature and other required information on the attendance log.
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General AttendeesAdministrators, nurses (other than California-licensed nurses), clinical laboratory personnel (otherthan California- and Florida-licensed personnel), and other health-care professionals may receive acertificate of attendance.
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CE CreditCE Credit
Sign and fax roster to 240-306-2527
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Complete the online surveyhttps://www.surveymonkey.com/s/PACT_Webinar_Closed_Cell_Culture_Systems
Note: Please complete within 48 hrs of the web seminar
(Survey link above is embedded in the reminder email sent 05/07/14)
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