Application of cell-seeded microcarriers strategy: organs and tissue on chip

BOTTOM-UP/SCAFFOLD-based/CELL-SEEDED MICROCARRIERS
Application of cell-seeded microcarriers strategy: organs and tissue on chip
Microcarriers posses a high specific surface
• Commercially available
microcarriers

Microcarrier-based tissue engineering

Table 1 Commercial microcarriers and their physicochemical parameters.

Name Size (mm) Density Material Refs
(g/mL)
Cytodex-1 60e87 1.03 Dextran matrix with positively charged diethylaminoethyl [25,38
groups throughout the matrix
Cytodex-2 60e87 1.04 Dextran matrix with N,N,N-trimethyl-2-hydroxyaminopropyl groups [25]
Cytodex-3 60e87 1.04 Dextran beads coated with denatured porcine-skin collagen [16,39
bound to surface
Cytopore 1 200e280 1.03 Cellulose [17,27
CultiSpher G 130e380 1.04 Cross-linked porcine gelatin [43,48
CultiSpher S 130e380 1.04 Cross-linked porcine gelatin [62]
Hillex 150e210 1.1 Dextran matrix with treated surface [25]
Glass coated 90e150 1.05 Glass [6]

• Surface of a
medium T-Flask is • Number of
suspension polymerization, by which Pis‚kin et al. [23] ob- such as trypsin or require mechanically scraping the cel
75cm2 tained a polydimethylsiloxane-OH microcarrier, which has a • 250.000 microcarriers
However, these measures can have adverse effects, such
microcarriers need
diameter of approximately 200 mm. Gabler et al. [24] pre- the proteolysis of the cell membrane and degeneration
have a total volume
to obtain a surface
pared a poly(lactide-co-glycolide) (PLGA) microcarrier by cell activities. Cell membrane proteins are degenerated
emulsification, while controlling the polymer concentration
of less than 0.2 cm
trypsin treatment and mechanical scraping3[32]. To remo
• Surface of a of 75cm is almost 2
and stirring speed to produce microspheres in the size this obstacle, a cell cultivation method using a liquid/liqu
range of 40e330 mm. Cartilage cells in the microcarrier
(0.2 ml)
interface system was proposed [33]; anchorage-depende
microcarrier (100 250.000
culture had a 100% survival at 3e5 days, but had degraded
by 3 months. Cytodex-1 microcarriers are a suitable sub-
animal cells can adhere, spread, and grow at the interfa
between the culture medium and a hydrophobic liqu
micron diameter)is strate for the propagation of articular bovine chondrocytes.
When cultured on a Cytodex-1 microcarrier, chondrocytes
Initially, researchers showed that the cells could grow at t
interface. This culture system developed from a simple h
0.000314 cm2 partly revert to their differentiated phenotype [25]. The drophobic liquid into using synthetic liquid pe
Application of cell-seeded microcarriers strategy: organs and tissue on chip

100 x Cell / microcarrier In normal cell expansion

on micorcarrieres we
have cell expansion only

In micro tissue fabrication

ECM amount

strategies, we can modulte

culture conditions in order to
promote the synthes of ECM
components
 approach for the optimization of micr
S: Zwietering Njs constant* based cell culture processes be devel
Application of cell-seeded microcarriers strategy: organs
n: kinematic viscosity (m2/s) takingandinto tissue
account onthe
chip
unique proper
g: gravitational constant (m2/s) of the platform of interest. The final p
rs: solid density (kg/mMicrocarriers
3
) need to be should suspendedbe built upon knowledge deve
rl: liquid density (kg/m3) around the basic performance charac
of the microcarrier chosen in the cont
• X: solidstoloading
In order ensure ((kg solids/kg
successful liquid) the
cell culture, × 100)
microcarriers must be kept in suspension
d of the stirred tank bioreactor implem
p: particle the
throughout diameter
duration (m)of the process.
D: impeller diameter (m) While the Zwietering correlation has b
used to help predict agitation require
*The Zwietering constant is a function of impeller and
• Depending upon the stirring velocity (N) we can obtain tank
geometry. Empirical values can be found in literature.
in three suspending
traditional regimes:
stainless steel bioreacto
it cannot be applied directly to the va
majority of single-use systems becau
A. B. C.
• empirical Zwietering constants2 do no
A On-bottom suspension: some
for the unique geometries of these ve
particles remain unsuspended at
The work presented herein describes
the bottom
approach for of the vessel.
defining agitation requir
• ofBaOff bottom suspension:
microcarrier-based cellnoculture pr
particles
using remaining
the Mobius ®
50onLthe bottom Bi
Single-Use
and collagen-coated
of the vessel for more microcarriers
than 1 to 2 as
model
secondsystem. The principles and app
• discussed
C Uniform are applicable to any micro
Suspension
stirred-tank bioreactor system.

• B and C are preferred but there are other aspects that can be taken in to account
 While the Zwietering correlation has been
Application of cell-seeded microcarriers
used to help strategy:
predict organs and tissue on
agitation chip
requirement
impeller and tank
n literature.
in traditional
Microcarriers needstainless
to be suspended steel bioreactors,
it cannot be applied directly to the vast
majority
The of single-use
minimum agitation rate Njs (rate of systems because
“just suspended”)
C.
empirical Zwietering constants2 do not exi
for the 0.1 g( ρ Sgeometries
unique − ρ L ) 0.45 0.13
N js = Sv [ ] nbof dthese 0.2
b D vessels
−0.85

The work presented ρL herein describes an

approach for defining agitation requiremen
• Njs = minimum agitation rate [REV/MIN]
of a microcarrier-based cell culture proces
• S = Zwietering Constant*
•using =the Mobius 502/s] L Single-Use Bioreac
®
n kinematic viscosity [m
•and
g collagen-coated microcarriers
= gravitational constant [m/s 2]
as a
• rs = solid density (Kg m3)
model
• rL
system. The principles and approac
= liquid density (Kg m3)
•discussed
nb = beads are applicable
mass fraction [%] to any microcarri
•stirred-tank
db bioreactor system.
= beads diameter
• D = impeller diameter

• * S depends by the impeller type (tabled value)

 While the Zwietering correlation has been
Application of cell-seeded microcarriers
used to help strategy:
predict organs and tissue on
agitation chip
requirement
impeller and tank
n literature.
in traditional
Microcarriers needstainless
to be suspended steel bioreactors,
it cannot be applied directly to the vast
majority
The of single-use
minimum agitation systems because
rate Njs (just suspended)
C.
empirical Zwietering constants2 do not exi
for the 0.1 g( ρ Sgeometries
unique − ρ L ) 0.45 0.13
N js = Sv [ ] nbof dthese0.2
b D vessels
−0.85

The work presented ρL herein describes an

approach for defining agitation requiremen
Problem related the agitation rate (Njs)
of a microcarrier-based cell culture proces
•using theofMobius
Lower value Njs ®
50 L Single-Use Bioreac
and • Non homogenous suspension
collagen-coated microcarriers as a
• Settling of cell and microcarriers
model
• Masssystem.
transfer non The
efficientprinciples and approac
discussed arenon
• Oxygen supply applicable
efficient to any microcarri
stirred-tank bioreactor system.
• High value of Njs
• Shear stress can damage microcarriers
• Hinder cell attachment
• Shear stress damage the cells
Application of cell-seeded microcarriers strategy: organs and tissue on chip

The shear stress (t) in the spinner flask

• For mo%on on flat surface the shear • In the spinner flask it is difficult to obtain
stress can be easily evaluated exact solution on microcarriers surface
• Simple geometry • Complex geometry
• Laminar Flow • Turbulent flow

Reynold
number
Laminar r fl nR dR 2 Turbulent
<1 Re =
h fl
> 1000
Shear at Wall
Shear 2 p nR dR
6 Fh fl t ISF≈=
Shear Force
t w ==h fl × 2
y=0 DR - dR
bh
F (flow rate), h viscosity, h height, nR (rotation rate), h viscosity,
b width dR impeller diameter, DR vessel diameter
Application of cell-seeded microcarriers strategy: organs and tissue on chip

The shear stress (t) in the spinner flask

• Shear forces on microcarriers in a turbulent flow. • The worst case is: eddies
Microcarrier-eddy interac8ons: (a) eddies much larger size comparable with
than beads; (b) mul8ple eddies the same size as beads, microcarriers size or
(c) eddies size same as inter-bead spacing. smaller
• The smallest eddy size
is (Kolmogorov ś
length ) lkol
1/ 4
æn3
ö
lKol = çç ÷÷
èe ø

P
e=
r fl VL

n cinematic viscosity, e energy dissipation rate, P power input, rfl, density, VL volume
ure bath of quiescent depth 36.4 mm, the
Application of cell-seeded microcarriers strategy: organs and tissue on chip
vector map to one side of the impeller shaft
Figure 4. [The The impeller (t) in the spinner
shear stress Reynolds flask
number
• Detailed inves+ga+on on the agita+on regimes within spinner flask bioreactor can be
1250, based on D=35 mm.]
obtained by coupling Par+cle Image Velocimetry (PIV) experiments and CFD analyses
Im
m
Particle Image Velocimetry
(PIV) experiment

Im

Par5cles Map Velocity Map

Ninth International Conference on CFD in the Minerals and Process Industries

CSIRO, Melbourne, Australia
10-12 December 2012

FLUID FLOW AND STRESSES ON MICROCARRIERS IN SPINNER FLASK

BIOREACTORS

1* 2 2 3 3
Petar LIOVIC , Ilija D. ŠUTALO , Robert STEWART , Veronica GLATTAUER and Laurence MEAGHER
magnet
Application of cell-seeded microcarriers strategy: organs and tissue on chip

The shear stress (t) in the spinner flask

Particle Image Velocimetry CFD

(PIV) experiment Simulation
[m]

Impeller magnet

Figure 5: Results of grid-dependence study using the

[m] 20 mm/s sliding-mesh CFD model of the Corning stirred-flask and
Figure 4: Experimental ensemble-averaged velocity vector the k-ω turbulence model, for the flow field in the plane of
the PIV light sheet: (left column) streamlines; (middle

Figure 5: Results of
map at laser light sheet location shown in Figure 3,
generated by the µPIV system for the case of 60 rpm column) u-velocity component; (right column) w-velocity
impeller speed. The reference vector magnitude (bottom of component; (top row) low-resolution mesh; (middle row)
medium-resolution mesh; (bottom row) high-resolution
 the high-stress regions
Application of cell-seeded microcarriers strategy: organs and tissue on chip
problems,
CONCLUSION however, the
The shear stress (t) in the spinner flask proportion of t
significant
the The paper presents
high-stress regions inaF
problems, however, therealistic
evidence of model
interaction ofofa the
significantHigh shear
proportion stress (tpopulation
of the microcarrier ) with
the high-stress regions instirred-flask.
problems, however, the evidence of interactionThe
of a C
CONCLUSION
Figure 8 is of some concern.
significant proportion of the microcarrier population with
improvement
the high-stress regions in Figure 8 is of some concern. throug
CONCLUSION
The paper presents a CFD
The paper presents a turbulence
CFD model that is shown modelling,
to be a
realistic model of therealistic modelwithin aof the hy
CONCLUSION
hydrodynamics Corning
The paper presents
stirred-flask. The CFD
realistic model through
a
of the
surface
CFD model
stirred-flask. modelling.
that
models feature
hydrodynamics
is shown
withinThe
room be
to
a Corning
fora Th
CFD
improvement targeted improvements to
stirred-flask.modelling,
turbulence CFD identifying
The improvement
stirrer models feature possibiliti
motion modelling through
room
and freefor
improvement through targeted improvements to
surface modelling.
identifying High
turbulence modelling, regions
The work
turbulence
turbulence
possibilitiesstirrer
shows CFD
for motion of
modelling
microcarrier stress
to be useful in
modelling, level
to stir
and free
exposure
) surface ofmodelling. The that
workcould
shows CFDcell to damage
be useful in
)
regions
identifying
stem
stress levels stem
surface
possibilities Or
cell differentiation
cell
modelling.
cause
for microcarrier
issues. As such, CFD
differentiat
exposure The
and
shows to wo
regions offor
promise
stem cell
stress
Low identifying
uselevels
as
differentiation
promise
that
risk-mitigation
Kolmogorovissues. As
possibilities
could cause cell
such,
for
tool
CFD
prioruse
damage and
to
shows
a
commissioning protocols used in for stem-cell bioreactors.
promise for use regions as commissioning of stress
risk-mitigation tool priorlevels to th
protoco
length
commissioning protocols used in for stem-cell bioreactors.
ACKNOWLEDGEMENTS stem cell differentiation
This work was supported(75 by µm) =
the Biomedical Materials and
ACKNOWLEDGEMENTS promise for use as r
This work was
Engineering
comparable
supportedACKNOWLEDGEME
(CMSE).
with Materials
Devices Theme of CSIRO Materials Science and
commissioning
by the Biomedical
protocols and
u
ure 8: Distribution acting on microcarrier particle
Engineering
microbeads
(CMSE).
size was support
Devices Theme of CSIRO Materials Science and
This work
faces by the fluid flow during operation of the Corning REFERENCES
ure 8: Distribution
red-flask at 60 rpm,acting on microcarrier
as predicted particlesolid
by the immersed ALLEGRUCCI,cell damage
Devices
and YOUNG, Theme
ACKNOWLEDGEMENT
C., L.E., (2007), of
aces by the fluid flow during operation of the Corning REFERENCES
Application of cell-seeded microcarriers strategy: organs and tissue on chip

The shear stress (t) in the spinner flask

• Effect on cell growth

Higher is the Agitation Rate (higher e) 1/ 4

æn 3 ö
• higher is the average shear and lKol = çç ÷÷
• lower is the smallest turbulent eddy èe ø
Application of cell-seeded microcarriers strategy: organs and tissue on chip
Starting a cell expansion phase: input parameters

• No Ini$al cell number Cell Bead Ratio

• Nb Ini$al microbeads number 150

• V Volume of the s$rrer

• N Rota$on speed

50 Optimal
• no cell concentra$on
range
100

No / V
• nb microbeads concentra$on

Nb / V
10
10
• x cell microbeads ra$o
0 Time -days 10
No/Nb
Application of cell-seeded microcarriers strategy: organs and tissue on chip
Starting a microcarriers cultivation : typical values
• V = 100 ml • nb regulates the sSrring rate
• N = 30 - 50 rpm • no regulates the final cell number and
• no = 25.000 cell / ml oxygen/nutrient demand
• nb = 0,5 mg / ml; or • V affects agitaSon rate and the amount of
= 2500 beads /ml available oxygen/nutrient
(1mg of beads contains almost • N regulate shear stress on cells
5000 microcarriers) • x affects the probability of cell – beads
• x = 10 collisions

Lab scale working volume • At the half of the process we have about 2 106
V = [200 – 500 ml] cells in the volume: what about oxygen
consumption?
• Consumption rate: Y molO2 cell-1 h-1
• Initial oxygen concentration: X molO2
• Consumption time: X / (Y*2 106 )
• Useful to optimize
• rotation rate
• medium exchange intervals
ells Culture
ing samplesRed
with Neutral werereagent.
also examined microscopically
This involved incubation
um Application of cell-seeded microcarriers strategy: organs and tissue on chip
toof determine
a culturethe proportion
sample (0.3 mL)of unoccupied
with an equal beads. Occu-of
volume
nse- pancy of the
Neutral Red Dynamic
reagent of
Cultispher-G cell-bead
beads
(0.1% wasattachment
Neutral confirmed
Red in D-PBS) by stain-
for
s • Comparison
ine ing20with Neutral
minof at
two kindsRed
20°C. Beads
of reagent. This
occupied
microcarriers: byinvolved
Cytodex cells incubation
appeared
vs. Cultisphere red
mor of under
• Comparison the
a culture
of twomicroscope.
sample (0.3 mL)
kinds of stirring: with an
Continuous vs. equal volume of
Intermittent
- Neutral Red reagent (0.1% Neutral Red in D-PBS) for
• The rate of cell attachment can be evaluated by measuring the disappearance of
e 20inmin at 20°C. Beads occupied by cells appeared red
cells the culture media
r Analysis
under of Attachment
the microscope. Kinetics
• Spilling a small volume from the
spinner and count
The rate of disappearance cellscells
of free concentraAon
was followed by
ne) • Cell count is made on single cells
ght an exponential• decay curve: (more than 3) are not
Cells aggregates
Analysis of Attachment Kinetics
ed taken C,
in to=account
Co . e-kr
hed The rate of disappearance of free cells was followed by
)
tat- anwhere C, is the
exponential decay curve:
cell-free concentration •at Cttime C,, is in the
= cell t,concentration
the original cell concentration, and k is the rate spinnerconstant.
dx-1 = Co . e-kr
This equation canC,be
• Co = initial cell concentration
expressed logarithmically as
dres • t = Time
• k = rate of cell disappearance
rk-
- where C, is the cell-free concentration
-In (C,/Co) = k * t at time t, C,, is
or rate of attachment [t-1]
1ere the original cell concentration, and k is the rate constant.
s Thus,
This a first-order
equation can be rate would logarithmically
expressed be represented asby a
Application of cell-seeded microcarriers strategy: organs and tissue on chip
Dynamic of cell-bead attachment
• Minimizing the cell aggregate forma0on and maximizing the cell / beads seeding
• Con0nuous vs. intermi:ent:
[Con0nuous = 40 rpm; Intermi:ent = 40 rpm for 5min@40rpm + 30 min@rest]
• Problem: cell aggregation leads to an uneven cell/bead distribution; high fraction of
unoccupied beads.
• Cell concentration decreases
faster under intermittent stirring
(with symbols)

• Percentage of cell forming

aggregates is lower under
intermittent stirring at each
culture time (black symbols)

• Intermittent stirring can be

used to reduce the formation of
0 20 40 60 80 100 120 140 cells aggregates
Time ( min )
Application of cell-seeded microcarriers strategy: organs and tissue on chip

Fabbrication of functional connective Fabbrica1on of func1onal comlex

tissues structures
 Macro-
Application of cell-seeded microcarriers strategy: organs and tissue on chipTissues

Dermis µT Airway µT Cervix µT

Human Skin

Human Airway

GUTµT Breast Cancer µT Cardiac µT

Human GUT

Human Cervix
Pancreas
Liver µT
Cancer µT

µ-Tissues
Applica'on of cell-seeded microcarriers strategy: organs and 'ssue on chip
Is 3D enough?
Pathologic events (UV, Tumor
Progressing,..) affect cell, ECM
and their interplay

Need Stroma-Responsive
3D tissues:
• Organization
• Composition
• Signaling
• Transport Properties
• Stifness

Increased Reliability

High Predictive Tissue

Pathology è ECM Remodeling

Applica'on of cell-seeded microcarriers strategy: organs and 'ssue on chip
Epidermis Stemness

Full thickness HSE

CTRL

UVA

Protectant

Collagen
Dermis Remodeling
HA

CTRL

UVA Wrinkle!
Responsive Dermis

Protectant
Application of cell-seeded microcarriers strategy: organs and tissue on chip
• Tissue Repair: spatiotemporal recapitulation of cellular processes in wound healing– scar formation
Week 1 Week 2 Week 3

ECM fiber orientation in the neo-formed tissue

neoformed Bssues evolved toward a configuraBon

resembling a scar Bssue featured by:
lower cell density
over expression of FN
preferential orientation of ECM fibrils
Lombardi et al. Adv. Mat Health. 2017
Application of cell-seeded microcarriers strategy: organs and tissue on chip

Melanoma model
Cell Nuclei S100+ Cells

Healthy
Human Skin
model

Radial
Melanoma
Stroma+
Ker/A375

VerCcal
Melanoma
Stroma+
Ker/Malme
3M
Application of cell-seeded microcarriers strategy: organs and tissue on chip
EPITHELIAL DIFFERENTIATION CERVICAL STROMA
OUR MODEL NATIVE TISSUE OUR MODEL NATIVE TISSUE

ABSORPTION PROPERTIES
OUR MODEL NATIVE TISSUE Human Cervix
Studies dealing with:
Equivalent
ü Epithelial differentiation-dependent HPV gene
regulation
ü Stromal remodeling in cervical ripening
ü Absorption studies
ü Abnormal cervix in infertility (mucus accumulation
after hormonal stimulus, etc.)
ü cervical cancer development studies
Application of cell-seeded microcarriers strategy: organs and tissue on chip
3D Stromal environment affects intestinal epithelium morphogenesis
Human GUT model Engineered Native
lysozyme

Muc2
Actin

physiologically relevant stromal

environment of the 3D-CSSE drives
the CaCo-2 cell differentiation
towards the four different type of
intestinal epithelial cells
(absorptive, mucus-secretory,
enteroendocrine and Paneth)
phenotype and promote, in
contrast to the 3D-CGE, the
production of the basement
3D-CGE=collagen gel equivalent De Gregorio et al. Biotech. and Bioeng.2017 membrane.
3D-CSSE=cell-synthe9zed stromal equivalent
 Gut–on-chip
Application of cell-seeded microcarriers strategy: organs and tissue on chip

increases ECM remodeling

inducing faster epithelial cell
differentiation

Villin
Mucus
Laminin 5
Full thickness
Application airway
of cell-seeded in vitro
microcarriers model
strategy: organs and tissue on chip

A novel Full Thickness Cystic Fibrosis model on a microfluidic

chip to study pathogenic mechanisms and evaluate therapeutic
strategies. Granted by FFC2017

Mucus
Cilia
CFTR α TUB cftr signal is expressed into
DAPI DAPI the apical side of the
epithelium in the normal
FT_NA model; in diseased cftr protein
can’t be found on the apical
cell membrane

CFTR α TUB
DAPI DAPI
the precence of cilia is
demostred by alpha
FT_CF

tubulin marker
Full thickness
Applica'on airway
of cell-seeded in vitro
microcarriers model
strategy: on'ssue
organs and chipon chip
Bronchis epithelium Lung connective tissue

Sigare7e smoke

On line detection

Cell!Prolifera-on!Rate! NON-Exposed
Collagen Exposed
Exposure!
-me!

Possible use of Lung-on-Chip :

ü environmental pollution on airway
tissues
Collagen!Assembly! ü Cystic Fibrosis
ü Asma
ü Lung cancer
ü Infections
Cell

!day!
 Liver–on-chip
Application of cell-seeded microcarriers strategy: organs and tissue on chip
Ethanol cytotoxicity assessment Hep-G2 microtissue

PGP
28mm
Dose-dependent decrease of cell viability and metabolic activity

Oxida6ve stress analysis via ROS detec6on assay

Corrado et al. Under revision Adv. Health. Mat.

 GUT-LIVER
Application AXIS:
of cell-seeded FIRST-PASS
microcarriers METABOLISM
strategy: organs and tissueON CHIP
on chip
GUT Equivalent Hepatic (HepG2)
microtissues
Live/dead

Gut injury Hepatic injury

Epithelial barrier integrity evaluation
Ros and Fat overexpression
Control ETOH 400mM

Control ETOH treated

Oil red

Claudin 1

ROS

The intestine-liver device is responsive to ethanol treatment which confirms the possibility of its application to drug
toxicity analysis or to evaluate the bioavailability of nutraceutical compounds.
GUT-MICROBIOTA
Applica'on on amicrocarriers
of cell-seeded chip strategy: organs and 'ssue on chip
Alterations in composition, diversity and metabolites derived from the gut microbiota are
associated with diseases affecting different organs of the human body.

Different factors can alter the composiFon of the gut microbiota. The gut microbiota
converts these inputs into metabolites, which can signal to different organs and Fssues
Applica'onmicroenvironment
Tumor complexity
of cell-seeded microcarriers strategy: organs and 'ssue on chip

Tumor microenvironment is the

results of the crosstalk among
cancer and stroma cells
interacting with their ECM
3D human pancreatic tumor in vitro
Applica'on of cell-seeded microcarriers strategy: organs and 'ssue on chip

Human tumor biopsy in vitro Histological and IHC staining

Imparato et al. International Materials Reviews 2015

3D humanof pancreatic
Applica'on tumor in vitro
cell-seeded microcarriers strategy: organs and 'ssue on chip
H&E Masson SHG
20x 20x 40x
pancreas
Normal

200µm 200µm
Pancreatic
cancer

200µm
200µm
3D human tumor
micro:ssue

200µm 200µm
Brancato V. Imparato et al. Acta Biomateria, 2016
Bioengineered tumoral microtissues recapitulate desmoplastic
Application of cell-seeded microcarriers strategy: organs and tissue on chip
reaction of pancreatic cancer

in vitro in vivo

Human tumor biopsy in vitro

Confocal and Multiphoton Imaging

Heatmap of the
‘‘extracellular
matrix” gene set Versican
highlighted the
up-regulated
expression of a
set of genes
involved in the Periostin
desmoplasia

Collagen

Brancato et al. Acta Biomat 2017

 Replicating
Application complexmicrocarriers
of cell-seeded dynamics of breast
strategy: cancer
organs andmicroenvironment
tissue on chip

Multi-Step Fabrication Process

Tumor-µTissue Tumor-Stroma Tumor-Vascular network

Interac7on Interac7on

75µm 100µm

Mazio C.. Imparato G. et al. Acta Biomaterialia, 2018

 Replicating
Application complexmicrocarriers
of cell-seeded dynamics of breast
strategy: cancer
organs andmicroenvironment
tissue on chip

• Tumor • Tumor
Microenvironment Heterogeneity
Remodeling
Single file
Protusive strand
Round Cluster
Solid Strand

T-µTP

N-µTP

T-µTP
HA - DAPI N-µTP

CLS – MCF7

T-µTP

N-µTP

Mazio C.. Imparato G. et al. Acta Biomaterialia, 2018

 Tumor
Application of cell-seeded microcarriers on chip
strategy: organs and tissue on chip

Edmond W. K. Young Integrative Biology 2013

 Tumor
Application of cell-seeded microcarriers on chip
strategy: organs and tissue on chip

Tumor-stroma
interaction

Cancer Cell µTP:

-MCF7 Breast
Adenocarcinoma

Stromal Cell µTP

-Normal Fibroblasts (NF)
or
-Cancer Associated
Fibroblasts (CAF)

• Stroma activation at cellular and ECM level

Readouts • ECM composition and remodeling
• Transport properties

Gioella et al. Adv. Health. Mat. 2016

Phenotypic
Applica'on ofactivation
cell-seeded at cellular level
microcarriers strategy: organs and 'ssue on chip
AC-mTP 4 hours 8 hours 12 hours

MCF7-µTP

100 µm

Phenotypic ac9va9on from α-SMA PDGFRβ-r

healthy to ac9vated stroma

NF = NORMAL stroma
NF-µTP CAF = TUMOR stroma
AC = NORMAL stroma
ACTIVATED by 50 µm
100 µm
malignant cells

Negative control Positive control

NF-µTP α-SMA CAF-µTP α-SMA

50 µm

PDGFRβ-r PDGFRβ-r

100 µm
 MMPsmicrocarriers
Applica'on of cell-seeded modulation in activated
strategy: tissues
organs and 'ssue on chip

MMP-9
NF-µTP CAF-µTP AC-µTP
MMP-9

75 µm

MMP-2
MMP-2

@48h post device loading

 ECM ac-va-on
Application of cell-seeded microcarriers strategy: organs and tissue on chip
NF-µTP CAF-µTP AC-µTP Fibronec-n
Fibronectin

100 µm
Hyaluronic Acid
Hyaluronic Acid

75 µm
Collagen
Collagen

50 µm

@48h post device loading