advanced human immune system mouse models · i-o: immune check point inh., adcc; macrophage...
TRANSCRIPT
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Slide #1
+33 456 44 81 [email protected]
Biopark, Le Forum 1 & 2 260, av. Marie Curie, FR-74160 Archamps, France
Advanced Human Immune System Mouse ModelsTMIn vivo pharmacology services
TODAY: INFLAMMATIONSébastien Tabruyn, PHDCSO
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Slide #2
Advanced Human Immune System MouseTM
NOD-Prkdcem26Cd52 ll2rgemCd22
NCG/NOG/NSG
Life-long reconstitution of full HUMAN immune system
Immuno-deficientMOUSE
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Slide #3
Highest Engraftment Rate and Quality Control
1) chemoablation 2) hCD34+ engraftment
Time(Week)O 14
4-week old animal
Customer Study
Monthly production:450 hu-NCG
QC hCD45+ ≥ 25%Average in 2019: >50%Ø 8/10 hu-mice (total
2450) pass
Quality control:complete flow cytometry panel of
human immune cells
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Slide #4
All Identified Human Immune Cell MarkersImmune populations Flow cytometry markersT cells CD3, CD4, CD8
Central and effector memory T cells CD45RO, CD45RA, CD62L, CCR7
Activated / Exhausted T cells CD69, CD25, TIM3, Lag3, PD-1, 41BB, Ox40, Granzyme B, IFNG, Ki67
Regulatory T cells CD25, FOXP3, CD127
Gamma-Delta T cells Vd2 TCR
Tetramer specific T cells Mart-1, NYESO-1, PRAME, WT-1
NK cells CD56, CD16, NKp46
Myeloid cells CD11b, CD33
Monocytes / macrophages CD14, CD16, CD163, CD86
Myeloid dendritic cells CD11c, CD141, CD209, CD86, HLADR
Plasmacytoid dendritic cells CD123, CD303, CD304, CD86, HLADR
Neutrophils, eosinophils, basophils, mastcells
SSC, CD15, CD16, CD24, CD123, HLADR
B cells CD19, CD20, CD22, IgD, IgM, IgG
Plasma cells, Plasmablasts CD27, CD38, CD138
Tumor cells CD146, PDL1, PDL2
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Slide #5
Transient expression for 48-72 hours of selected human cytokines by liver cells:
• Physiological level of expression of the chosen human cytokines• No over-stimulation (not constitutive nor deleterious)• Stable differentiation: up to 3 months downstream effect• Adaptable
For enhanced differentiation/boosting of:
Myeloid/Dendritic Cells: hGM-CSF + hIL3 + hIL4 + FLT3LNK Cells: hIL15
• via hydrodynamic DNA deliveryPlasmid DNA in 2 ml of saline for 10 sec. i.v.
Transient Human Cytokine Boost
-7 3 141
10
100
1000
10000
100000
FLT3L
Days after hydrodynamic boost
pg/m
L
-7 3 1410
100
1000
IL3
Days after hydrodynamic boost
pg/m
L
-7 3 141
10
100
IL4
Days after hydrodynamic boost
pg/m
L
-7 3 140
20
40
60
GMCSF
Days after hydrodynamic boost
pg/m
L
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Slide #6
NOG-EXL: NK cells (1.5%), Monocytes (6.5%), Granulocytes (5%) B-NCG: NK cells (2%), Monocytes (6%), Granulocytes (0.5%)
Level of human cells in peripheral blood (D7 after boost) hu-NCG
T cells
B cells
NK cells
Monocytes
Granulocy
tes
Dendriti
c cell
s0
2
4
6
820406080
% o
f hum
an c
ell
GM-CSF IL3 IL4 FLT3L hu-NCG
T cells
B cells
NK cells
Monocytes
Granulocy
tes
Dendriti
c cell
s0
2
4
6
820406080
% o
f hum
an c
ell
hu-NCG
T cells
B cells
NK cells
Monocytes
Granulocy
tes
Dendriti
c cell
s0
2
4
6
820406080
% o
f hum
an c
ell
IL15 hu-NCG
Myeloid lineage, DC and NK cells Boosting
Non BoostednoBoost: hGM-CSF + hIL3 + hIL4 + FLT3L hIL15none
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Slide #7
Myeloid and DC Boost in hu-NCG
-7 7 -7 70
20000
40000
60000
Days after hydrodynamic boost
Cel
ls/m
L
Vehicle
GM-CSF IL3 IL4 FLT3L
x 6
• Peripheral Blood
-7 7 -7 70
20000
40000
60000
Days after hydrodynamic boost
Cel
ls/m
L
Vehicle
GM-CSF IL3 IL4 FLT3L
x 6
-7 7 -7 70
5000
10000
15000
20000
25000
Days after hydrodynamic boost
Cel
ls/m
L
Vehicle
GM-CSF IL3 IL4 FLT3L
x 4
-7 7 -7 70
50000
100000
150000
Days after hydrodynamic boost
Cel
ls/m
L
Vehicle
GM-CSF IL3 IL4 FLT3L
x 13
-7 7 -7 70
1000
2000
3000
Days after hydrodynamic boost
Cel
ls/m
L
Vehicle
GM-CSF IL3 IL4 FLT3L
x 18
-7 7 -7 70
20000
40000
60000
80000
Days after hydrodynamic boost
Cel
ls/m
L
Vehicle
GM-CSF IL3 IL4 FLT3L
x 51
-7 7 -7 70
1000
2000
3000
4000
Days after hydrodynamic boost
Cel
ls/m
L
Vehicle
GM-CSF IL3 IL4 FLT3L
x 3
Myeloid cells(CD11b+)
Monocytes(CD11b+CD14+)
Conventional Dendritic cells(CD11b+Lin-HLADR+CD11c+)
Dendritic cells type 1(CD11b+Lin-HLADR+CD11c+CD141+)
Dendritic cells type 2(CD11b+Lin-HLADR+CD11c+CD1c+)
Plasmacytoid dendritic cells(CD11b+Lin-HLADR+CD11c+CD123+CD303+)
7
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Slide #8 TCS – 2019 – Confidential
NK cells
Monocyte
s
Granulo
cyte
s
Dendrit
ic ce
lls0
1
2
3
4
% o
f hum
an c
ell
Myeloid boosthu-NCG
NK cells
Monocyte
s
Granulo
cyte
s
Dendrit
ic ce
lls0
1
2
3
4
% o
f hum
an c
ell
hu-NCG
Tissues were harvested on Day 14 after boost (n=5)
NK cells
Monocyte
s
Granulo
cyte
s
Dendrit
ic ce
lls0
5
10
15
20
% o
f hum
an c
ell
hu-NCG
NK cells
Monocyte
s
Granulo
cyte
s
Dendrit
ic ce
lls0
5
10
15
20
% o
f hum
an c
ell
Myeloid Boosthu-NCG
Bone marrow Spleen Liver
x2.5x2.5
x2.5
NK cells
Monocyte
s
Granulo
cyte
s
Dendrit
ic ce
lls0
5
10
15
20
25
% o
f hum
an c
ell
hu-NCG
NK cells
Monocyte
s
Granulo
cyte
s
Dendrit
ic ce
lls0
5
10
15
20
25
% o
f hum
an c
ell
Myeloid Boost hu-NCG
x2.5
hCD45 = 2.4 x 105 cells/femur hCD45 = 6.7 x 105 cells/femur hCD45 = 9.9 x 106 cells/g hCD45 = 1.5 x 107 cells/g hCD45 = 4.5 x 106 cells/g hCD45 = 7.3 x 106 cells/g
Myeloid and DC Boost in hu-NCG• Bone Marrow, Spleen, Liver
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Slide #9 TCS - Confidential
Major advantages of hu-NCG
4. Human cytokine boosts available for increasingthe number of myeloid – NK – dendritic cells
Hydrodynamic injections of plasmid DNA encoding humanM-CSF, GM-CSF, IL3-4-15, FLT3L
2. Lifelong stability (> 18 months) of the immune system
No irradiation – No AnemiaHumanisation following chemoablation
1. No GvHDhigh purity of hCD34+ with < 2% hCD8+
3. Trusted source of human cord bloodEtablissement Français du Sang: EFS BesanconEthic/legal: informed/signed consent by the parents, anonymization, only for R&D purposes
5. Disease models compatibility:I-O: immune check point inh., ADCC; macrophage polarisation, oncolyic bacteria and viruses, vaccination, CRS (cytokine release syndrome)Infectious: HIV, Dengue, hCMVInflammation: IBD chronic and acute, RA, AsthmaImmunization: 100% human IgG
6. Ample historical data12’750 hu-mice produced last 5 years2020: >450 hu-NCG produced per month
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Slide #10
Cancer Immuno-Oncology• Solid tumors • Liquid tumors • PDX• Cytokines Storm (CRS)
Vaccines & ImmunoSafety
• 100% human IgG • Prophylaxis
Infectious Diseases
• HIV • Dengue
• hCMV
InflammationAutoimmune Diseases
• IBD• Asthma
• GVHD (PBMC)
Advanced Human Immune System Mouse ModelsTM
NEW Platform: hu-liver
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Slide #11
Inflammatory Bowel Disease (IBD)
TCS - Confidential
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Slide #12
Current IBD Models Have Limitations
• Chemically induced : DSS, TNBS, oxazolone,…
• Gene-manipulated : IL-2-/-, IL-2R-/-, and IL-10-/-
• Bacteria-infected models
• These models on immunocompetent mice : C57BL/6, BALB/c,…
Slide #12
NOT APPLICABLE for drug targeting human-specific cells/cytokines/targets
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Slide #13
Human Leukocyte Infiltration in hu-IBD Colitis
Human leukocytes infiltration at the site of inflammation (ALU staining) and human TNF-α secretion (mAb staining)
TCS - Confidential
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Slide #14
hCD4 Infiltration in hu-IBD Colon
TCS - Confidential
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Slide #15
Acute hu-IBD Colitis Protocol
Endpoints or Disease Progression Scores Clinical + Histology + Endoscopy
TCS - Confidential
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Slide #16
Acute hu-IBD Colitis : Peripheral blood
Human Immune cells in Peripheral blood
VehicleProduct-1Product-2
n=10
D0
Vehicle
Nicotine
PMI001
Vehicle
Nicotine
PMI001
0
2×105
4×105
6×105
hCD45/ml
*
D12
A
D7
D0
Vehicle
Nicotine
PMI001
Vehicle
Nicotine
PMI001
0
2×103
4×103
6×103
8×103
Treg/ml
B
D12D7
TCS - Confidential
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Slide #17
Clinical hu-IBD Colitis Scales and Scores
TCS - Confidential
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Slide #18
Scoring hu-IBD Colitis Symptoms with Humira
Body WeightStool consistency
Colon length(D13-Sacrifice)
Bleeding
TCS - Confidential
0 2 4 6 8 10 12 140
1
2
3
4
5
time (days)
body
wei
ght l
oss
scor
e humiraisotype control
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Slide #19
Diminution of hu-IBD Colitis Symptoms with anti-huTNFa
Therapeutic human mAb reduces inflammation in hu-IBD model
TCS - Confidential
0 2 4 6 8 10 12 14012345678910
time (days)
glob
al c
linic
al s
core
humiraisotype control
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Slide #20
Reduction of T-cell Infiltration by Cyclosporin-A in hu-IBD
TCS - Confidential
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Slide #21
3 % DSS
1 2 3 4 5 6 7 8 9 10 11 120
1
2
3
4
5
time (days)
Bod
y W
eigh
t Los
s S
core
A
1 2 3 4 5 6 7 8 9 10 11 120
1
2
3
4
5
time (days)
Dia
rrhe
a S
core
3 % DSS
B
****
***
***
1 2 3 4 5 6 7 8 9 10 11 120
1
2
3
time (days)
Rec
tal B
leed
ing
Sco
re
3 % DSS
C
*1 2 3 4 5 6 7 8 9 10 11 12
0
2
4
6
8
10
12
time (days)
Glo
bal C
linic
al S
core
3 % DSS
****
*
D
VehicleProduct-1Product-2
n=10
Acute hu-IBD Colitis – Case Study with Nicotin Derivatives
Clinical Scores
TCS - Confidential
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Slide #22
Acute hu-IBD Colitis – Colon Shortening
VehicleProduct-1Product-2
n=10
Vehicle Nicotine PMI00140
60
80
100
120
Col
on L
engt
h (m
m)
A
Vehicle Nicotine PMI00140
60
80
100
120
Col
on L
engt
h (m
m)
*
BD7 D12
TCS - Confidential
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Slide #23
Histopathology hu-IBD Colitis Scales and Scores
TCS - Confidential
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Slide #24
Inflammation and Ulceration in DSS induced hu-IBD
TCS - Confidential
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Slide #25
Acute hu-IBD Colitis – Case Study
Histological Score (D7) – Distal Part
VehicleProduct-1Product-2
n=10
Vehicle Nicotine PMI0010.0
0.5
1.0
1.5
Infla
mm
atio
n (S
core
)
A
*
Vehicle Nicotine PMI0010.0
0.5
1.0
1.5
Tiss
ue d
amag
e (S
core
)
B
**
Vehicle Nicotine PMI0010.0
0.5
1.0
1.5
2.0
Mon
onuc
lear
infil
tratio
n (S
core
)
C
*
TCS - Confidential
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Slide #26
hu-IBD Colitis MEICS (Murine Endoscopic Index of Colitis Severity) Scale
Advantages:
• Detailed description of colonic mucosa • Mucosal lesion follow-up over time • Early and accurate appearance of colitis compared to the clinical score alone. • Real time treatment efficacy on evolution of the lesions after drug treatment• Good correlation with histological analysis
days after induction
0
5
10
15
0
3
6
9
0 2 4 6
051015
0369
02
46
clin
ical
sco
reen
dosc
opic
sco
re
051015
0369
02
46
clin
ical
sco
reen
dosc
opic
sco
re
Acute DSS model
Martin-Rodriguez et al., in preparation
TCS - Confidential
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Slide #27 TCS - Confidential
Thickening of the colon
MEICS 0 1 2 3
Vascular Pattern
Fibrin visible
Granularity of the
mucosa surface
Stool consistence
/3
/3
/3
/3
/3
Murine EndoscopicIndex of Colitis Severity
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Slide #28
Acute hu-IBD Colitis – Case StudyEndoscopy scoring
TCS - Confidential
0 3 6 9 120
2
4
6
8
10
Day
Global Endoscopy Score
*
0 3 6 9 120
1
2
3
Day
Colon thickening
**
*
0 3 6 9 120
1
2
3
Day
Vascular pattern
*
0 3 6 9 120
1
2
3
Day
Visible Fibrin
0 3 6 9 120
1
2
3
Day
Granulosity of mucosal surface
*
0 3 6 9 120
1
2
3
Day
Stool consistency
*
Ab-1Vehicle
n=10
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Slide #29
Chronic hu-IBD Colitis Protocol
Endpoints or Disease Progression Scores Clinical + Histology + Endoscopy
TCS - Confidential
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Slide #30
Diminution of Chronic hu-IBD Colitis with CyclosporinA
0 10 20 30 400
2
4
6
8
10
time (days)
glob
al c
linic
al s
core
Cyclosporin ACtrl
3%DSS 3%DSS 3%DSS
0 10 20 30 400
1
2
3
4
time (days)di
arrh
ea s
core
3%DSS 3%DSS 3%DSS
0 10 20 30 400
1
2
3
4
5
6
time (days)
body
wei
ght s
core
3%DSS 3%DSS 3%DSS
Body WeightStool consistency
Global Clinical Score
Reduction of symptoms by administration of cyclosporin-A
TCS - Confidential
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Slide #31
Microbiome/MicrobiotaMicrobiome manipulations:
• fecal microbiota Transfer (FMT)• oral administration of in vitro expanded bacteria (pups/adults)• antibiotic treatments
• IBD (conventional and hu-mouse)• GVHD (engraftment of PBMC)• Immuno-oncology (Check-point inh. efficacy in hu-mouse)• Immuno-modulation (via flow cytometry)
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Slide #32
General Guidelines for IBD models
• Study size: at least 10 hu-IBD mice per group• Gender: female animals are recommended• Drug validation: each batch of DSS is calibrated• Animal randomization by: humanization rate, CD34+ donor• Positive controls and route of administration: CSA, anti-human TNFa
TCS - Confidential
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Slide #33
Cancer Immuno-Oncology• Solid tumors • Liquid tumors • PDX• Cytokines Storm (CRS)
Vaccines & ImmunoSafety
• 100% human IgG • Prophylaxis
Infectious Diseases
• HIV • Dengue
• hCMV
InflammationAutoimmune Diseases
• IBD• Asthma
• GVHD (PBMC)
Advanced Human Immune System Mouse ModelsTM
NEW Platform: hu-liver
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Slide #34
Involvement of Immune Cells in Asthma
1. Allergen captured by DC 2. DC present antigen to T cells to induce Th23. Th2 promote IgE production by B cells4. IgE bind to mast cells and basophils à local allergic
response5. Recruitment of eosinophils6. Induction of airway hyper-responsiveness, mucus
production, tissue damage
TCS - Confidential
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Slide #35
Presence of human DC, Mast Cells, Eosinophils, Basophils, Neutrophilsin Lungs
TCS - Confidential
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Slide #36
Asthma Induction Model in hu-mouse Models
Boosted IL-3 / GM-CSF hu-Mouse
TCS - Confidential
Study Design 1
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Slide #37
OVA challenge in Asthmatic hu-mouse Models
hCD45 – Total human leukocytes
PBSOVA
0
500
1000
1500
hCD45 in BALF
hCD
45 /
milli
on c
ells
BALF Lungs
TCS - Confidential
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Slide #38
OVA challenge increases neutrophils, Mast Cells and Basophils in lungs of hu-mouse
Neutrophils
PBSOVA
1000
10000
100000
mast cells/100 mg lung
Cel
ls /
100
mg
lung
Mast cells
PBSOVA
10
100
1000
basophils/100 mg lung
Cells
/ 10
0 m
g lun
g
Basophils
PBSOVA
10
100
1000
10000
eosinophils/100 mg lung
Cel
ls /
100
mg
lung
Eosinophils
TCS - Confidential
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Slide #39 TCS - Confidential
0 16 70
IMMUNIZATION PHASEOVA/alumPBS
77
Boost IL-3, IL-4, GM-CSF, FLT3-L
93
CHALLENGE PHASEOVA acuteOVA chronicPBS
7 23 30 37 44 51 65days
Asthma Induction Model in hu-mouse ModelsStudy Design 2
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Slide #40
IgE Production
TCS - Confidential
D14 D28 D42 D56 D70 D9310
100
1000
total IgE in plasma
tota
l IgE
, ng/
mL
PBS OVA acute OVA chronic
immunizations challenges
***
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Slide #41
H&E Staining
TCS - Confidential
bronchiole
blood vessel
alveolar duct
alveoli
terminal bronchiole
respiratory epithelium(ciliated cells, gobletcells…)
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Slide #42
Respiratory epithelium thickening
TCS - Confidential
PBS OVA chronic OVA acute
• Cellular Density• Globlet Cells – Mucus Production
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Slide #43
Patrick NEFPhD. CEO
Romain GRETDirector Business
Development
Sébastien TABRUYNPhD. CSO
Kiave-Yune HO WANG YIN, PhD. Director Innovation
Catherine VERHAEGHE, PhD.Director Operations
Hendrik CLAEYSGlobal Sales Manager
Slide #43
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Slide #44
Expertise• Full reconstitution of the human immune
system/functions in hu-mouse models • Protocol designs, tailor-made• Flow cytometry, histology, small animal
imaging, cell biology state-of-the-art• Data collection, analysis, reports
Application• Preclinical candidate selection• Efficacy models for cancer (I-O, PDX), infectious
(HIV, Dengue), inflammation (IBD), Vaccines (100% human IgG), immuno-Safety/Toxicity
• Combination strategy
About us• Bio Safety Level -2 and -3 animal and lab. certified
facilities (740 m2) + offices (200 m2) • High quality and flexibility• Complete Fee for Services• Attractive Pricing
European CRO located in France Near Geneva (Switzerland) 42 FTE, 11 PhD
Who We Are & What We Do
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Slide #45
Laboratory Analytical Platforms• Flow cytometry
• immuno-histochemistry (IHC)
• in vivo imaging for small animals
• TP1020 Automatic Tissue Processor• Microtome: HistoCore Arcadia H• Leica Aperio Versa (8 slides)• paraffin inclusion; cryosection; H&E staining; Histopathology; Immunohistology:• Chromogenic and Fluorescence; Digitalization and image Quantification
• Vilber Newton 7.0 (Bioluminescence – up to 5 animals)• Luciferase and autobioluminescence tumor growth monitoring
• Attune NxT Flow Cytometer (n=2)• Complete tissue and blood sample analysis• 14 channels each for complex human immune cell panel detection• Cell Sorter: BD FACSMelody TM Cell sorter
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Slide #46
One-Stop-Shop Fee-for-ServicesStudyDesign
Protocol Execution
Data Analysis Deliverables
§ Tailor-made protocols (budget, timeline, experimental plan)
§ Quotes§ SOP driven§ Designated Project
Officer§ Clear Role and
Responsibility
§ Raw data transfer§ Final Reporting with
expert analysis & discussion
§ Feedback
§ BSL 2 & 3 animal facility
§ State-of-the-art flow cytometry, histology, imaging, cellular biology
§ Direct access to your Project Officer
§ Frequent interim reporting + draft reports
§ Raw data collection§ Data analysis
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Slide #47
Our Next Webinar: 2020 SeriesOur Next Webinar 2020 SeriesJune 25th 2020: Infectious diseases: full human system mouse models for HIV, Dengue, CMV and Corona virus.
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Slide #48
NOW: Q&A Session
Type your question
Thank you for your attention