bioprinting exosome microenvironments -...
TRANSCRIPT
Bioprinting Exosome Microenvironments
Saigopalakrishna S. Yerneni1, Theresa L. Whiteside2, Lee E. Weiss1, 3, Phil G. Campbell1, 4
1Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA
2University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA
3The Robotics Institute, Carnegie Mellon University, Pittsburgh, PA
4Engineering Research Accelerator, College of Engineering, Carnegie Mellon University, Pittsburgh, PA
Nucleus
Multivesicularbodies
Nucleus
Autocrine
Membrane fusion
Receptor-ligand
InternalizationCell 1 Cell 2
JuxtacrineParacrineTelecrine
Exosome & it’s cargoRNA, DNA, Proteins, Lipids
What Are Exosomes? • 30-150 nm cell-secreted extracellular vesicles• Involved in cell-to-cell communication• Potential for clinical implications:
• Biomarkers• Drug delivery vehicles
Extracellular Matrix
Macrophages
Fibroblasts
Immune Cells
Growth Factors
Red Blood Cells
Exosomes
Cell Microenvironments Consists of ‘Solid-Phase’ and ‘Liquid-Phase’ Exosomes
Research to-date has primarily used ‘liquid-phase’ models to study exosomes physiology➢ Goal 1: Use bioprinting to engineer ‘solid-phase’
exosome-based microenvironments as test-beds for invitro and in vivo studies.
Delivery of exogenous growth factors with exosomes is yet to be explored➢ Goal 2: Demonstrate the feasibility of loading native
exosomes with a paradigm growth factor (BMP2) and usebioprinted solid-phase BMP2-exosome constructs tocontrol cell fate in vitro and in vivo.
Project Goals
Carnegie Mellon University 3
Inkjet-Based Biopatterning Technology
Carnegie Mellon University 4
Concentration of deposited exosomes is modulated by overprinting strategy (#OPs = no. overprints)
60 pL droplets
ExosomesPBS w/10% glycerol (v/v)
In vitro solid-phase testing
10 20
3040
#OPs
10 μg/ml exosome solution
Implantation in murine muscle pocket model
Uniform deposition on 4.5 mm dia.
accellular dermalmatrix (ADM)
discs (200 µm thick)
Cell culture
Picture of 60 pL droplet formationcaptured using ImageXpert® system
From: M0, M1, M2, THP1 cells
10 30 50 70 90 110 130 150 170 190 210 230 250 270 290 310 330
Time (µs)
Bioprinter
Collagen-coated coverslip
In vivosolid-phase testing
Validation of Printed Solid-Phase Patterning
Carnegie Mellon University 5
1mm
1mm
1mm 1mm
Overprint modulation of exosome microenvironment patterns
243 pgprotein/pattern
486 pgprotein/pattern
972 pgprotein/pattern
972 pgprotein/pattern
1944 pgprotein/pattern
2.43 protein/pattern
500µm
10 OP 20 OP 30 OP
30 OP 40 OP 50 OP1mm
Rel
ativ
e Fl
uore
scen
ce (A
. U.)
PKH26-labeled murine macrophage-derived exosome
microenvironments on collagen type-I coated coverslips
Exosome binding retention to various ECM substrates
2mm
HO
Binding retention of exosomes to
DermaMatrix™
0
20
40
60
80
100
120
24 Hours 48 Hours 72 Hours
% R
etai
ned
(Mea
n FL
uore
scen
ce
Inte
nsity
, A. U
.)
Pattern persistence on collagen type-I coated
coverslips
Solid-Phase Exosomes
are A
vailable for Cell U
ptake
Nuclei F-actin
Exosomes
Inhibitors: 10µg/ml heparin + 10µg/m
lβMC
D
16000000
14000000
12000000
10000000
8000000
6000000
4000000
2000000
0
30min
60min
3hours6hours
Relative Mean FuorescenceIntensity (A.U.)
Time
PCI-13-inhibitors
+inhibitors
0
2000000
10000000
8000000
6000000
4000000
12000000
14000000
3hours6hours
Relative Mean FluorescenceIntensity (A.U.)
Time
SCC-90-inhibitors
+inhibitors
60m
in
3hours
6hours
PC
I-13cells
-inhibitors+
inhibitors
SC
C-90
cells
-inhibitors+
inhibitors5
30m
in
30min
60min
Carnegie M
ellon University
6
Acid w
ash (2 m
in)
Cells
Image on
pattern
THP
1 exosomes
Effects of Solid-Phase M1/M2 Murine Macrophage-Derived Exosomes on C2C12 Myogenesis
NucleiActin
NucleiActin
NucleiActin
M0M1 M2100 ng/ml LPS
100 ng/ml IL10
M1 Exosomes = 1.90x1010 particles/mlM2 Exosomes = 1.57x1010 particles/ml
qNano Analysis
M1/M2 Exosome Combinatorial Array
10 OP M110 OP M2
20 OP M110 OP M2
20 OP M120 OP M2
F-actin MF-20
F-actin MF-20 F-actin MF-20 Nuclei
1.25mm
1.75mm
1mm
10 OP M1200 OP M2
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
1 2 3 4
Rel
ativ
e Fl
uore
scen
ce In
tens
ity (
A.U
.)
Overprints F-actin MF-20
10 M1/10 M2 20 M1/10 M2 10 M1/20 M2 20 M1/20 M2
Carnegie Mellon University 7
BMP2 Loading and Characterization
Cellular internalization of 125I-BMP2-loaded M0 exosomes
Internalization of PKH26labeled M0 exosaaaaaaomes
TEM of exosomes
100nm
125I-BMP2 retention in exosomes
Carnegie Mellon University 8
DLS Analysis
Minutes
Internalization of BMP2-exosomes
BMP2-exosomesNon-activated J774A.1 (M0)
exosome isolationSonication with BMP2
Purification by size-exclusion chromatography
OPs
Liquid-phase ALP expression
BMP2-exosomes (sonicated)
100 ng/ml BMP2
100 µm
Native exosomes (10 ug/ml)
Sonicated exosomes (10 ug/ml) BMP2 (100 ng/ml)
BMP2-exosomes (10 ug/ml)
500µm
10 OPs 20 OPs
30 OPs40 OPs 30 OPs
20 OPs10 OPs
40 OPs
0
10000
20000
30000
40000
50000
60000
10 20 30 40
Mea
n In
tens
ity (A
.U.)
05000
10000150002000025000300003500040000
1 2 3 4 5
Mea
n In
tens
ity (A
.U.)
Treatments
Solid-phase ALP expression
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BMP2-exosomes Induce C2C12 Osteoblastogenesis In Vitro
Bioprinted BMP2-exosomes Promote Heterotopic Ossification Formation In Vivo
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HO
HO
0
1
2
3
4
5
6
BMP2-Exosomes
Bone
Vol
ume
(µm
3 )
Exosomes without BMP2
BMP2-ExosomesExosomes without
BMP2
2mm 2mm
Heterotopic Bone Volume
Exos
omes
w
ithou
t BM
P2BM
P2-E
xoso
mes
5X 10X 20X
HO
• Optimizing of BMP2-loading in exosomes
• Elucidation of BMP2-exosome signaling
• Applications in musculoskeletal research
• Applications in transplantation biology
• Studying cancer biology
Carnegie Mellon University 11
30 OP
Future Directions
Acknowledgements• Dr. Lee E. Weiss• Dr. Phil G. Campbell• Dr. Theresa L. Whiteside• Philip and Marsha Dowd• All the members of Weiss,
Campbell and Whiteside labs
• Funding Source: Pittsburgh Infrastructure Technology Alliance (PITA)
Carnegie Mellon University 12
Thank You!