bioprinting exosome microenvironments -...

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


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


1mm

1mm

1mm 1mm

Overprint modulation of exosome microenvironment patterns

243 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


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


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


BMP2-exosomes Induce C2C12 Osteoblastogenesis In Vitro

Bioprinted BMP2-exosomes Promote Heterotopic Ossification Formation In Vivo


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


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)


Thank You!

bioprinting exosome microenvironments -...

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