human organs-on-chipsnas-sites.org/emergingscience/files/2014/07/bahinski.pdf · constituent cells...
TRANSCRIPT
Anthony Bahinski, Ph.D., MBA, FAHA
Lead Senior Staff Scientist
Advanced Technology Team,
Wyss Institute for Biologically Inspired Engineering at Harvard University
National Academy of Sciences, Tissue Chip Workshop
Washington, DC
July 21, 2014
Human Organs-on-Chips
Biomimetic Microsystems• Engineer microchips containing living human cells that
reconstitute organ-level functions for drug screening,
diagnostic, toxicology, and therapeutic applications
• ACCELERATE drug development & REPLACE animal testing
Biomimetic
Spleen
Don IngberKit Parker
George Whitesides
Ali Khademhosseini
Dave Weitz
Human Breathing Lung-on-a-Chip
Underlying Microsystem Challenge
Goal is to replicate human ORGAN-LEVEL functions in vitro,
but what defines an Organ?
ORGANS ARE:
• Composed of 2 or more tissues that exhibit unique functions when
they are interfaced
• Perfused by blood flowing through endothelium-lined vessels
• Controlled by chemical and molecular factors produced by
constituent cells or delivered through the vasculature
• Regulated by mechanical forces (e.g., due to motion, breathing,
peristalsis) and blood flow
• Structured to secrete or transport factors in specific directions
• Infiltrated with immune cells during inflammatory responses
• Physiologically coupled to other organs via factors transmitted in
blood flowing through linking vessels
We use Microengineering to:
• Recreate Tissue-Tissue Interface
- Analyze transport, absorption, transport, permeability, conductivity
• Provide Mechanical Cues necessary for relevant physiology
- Fluid flow, cyclical mechanical strain, Air-Liquid Interface, directional clearance
• Precisely Orient Cells for High-Resolution Real-Time Imaging
- Enables analysis of molecular and cellular mechanisms at critical tissue boundaries
• Place cells in separate channels to study different cell populations
- Harvest cells or medium independently for molecular genetic analysis
- Sample oriented (e.g., lumenal) secretions in real-time
• Control Fluid Flow through Microfluidic Channels
- Supports long-term culture
- Enables pharmacokinetic analysis
- Permits co-culture of Microbiome
• Create Endothelium-Lined Vascular Channels
- Permits real-time analysis of recruitment of circulating immune cells
- Potentially can use of blood or plasma to feed organ chips
- Enables physiological vascular coupling between different organ chips
A Human Breathing Lung-on-a-Chip(Dan Huh, Wyss Institute; Huh et al., Science 2010)
www.nucleusinc.com
Alveoli
Paton & Byron, Nat. Rev. Drug Discov. 2007
Air
Blood
BIODESIGN PRINCIPLES:• Tissue-Tissue Interface
• Dynamic Flow
• Cyclic Breathing Movements
Capillary endothelial cells
VE cadherin
Occludin
Endothelium
Epithelium
Alveolar epithelial cells
Capillary endothelial cells
Lee & Downey, Am J Respir Crit Care Med 2001
Lung inflammation
Pro-inflammatory cytokines
Endothelial activation
(express surface ICAM-1)
Leukocyte adhesion
Diapedesis & transmigration
Infiltration into alveoliPathogensAlveolus
Capillary lumen
EpitheliumTNF
Endothelium
Inflammatory Protein
White Blood
Cells (WBCs)
Bacteria
Mimicking the Immune Response
WBC Transmigrating WBC Killing Pathogens
Control Inflamed
Inflammatory Protein
White Blood
Cells
(WBCs)
Vascular leakage syndrome
Pulmonary edema
Interleukin-2 (IL-2)
Kidney cancerMelanoma
Chemo
Human Disease Model:Chemotherapy-Induced Pulmonary Edema
(Huh et al., Sci. Trans. Med. 2012)
Air
Liquid
IL-2
Membrane
Day 0
LiquidDay 4
Liquid
IL-2
Meniscus
Day 2 Day 3
AirLiq
uid
Liq
uid
IL-2
Human Disease Model:
Pulmonary Edema-on-a-Chip
(work of Dan Huh)
Day 0
Pro-thrombin
Fluorescent fibrinogen
IL-2Day 4
Fluid
Air
Fibrin
Alveolar epithelium
Fibrin clots
Alveolar Fibrin Deposition On-Chip
IL-2
Flow
FITC-inulin
Lun
gC
apill
ary
Modulating Lung Barrier Permeability with IL-2
Vascular Leakage Model
IL-2
Flow
FITC-inulin
Lun
gC
apill
ary
Epi
Endo
Control (10% strain)
Occludin
VE-cadherin
t = 3 days
IL-2 & 10% strain
Tissue Barrier Integrity
Ventilation
(FITC-inulin, IL-2)
Perfusion
IL-2
Flow
FITC-inulin
Lun
gC
apill
ary
Bronchoalveolarlavage (BAL)
Results Confirmed in Whole Lung
Lung-on-a-Chip
IL-2 & Drug A
Flow
FITC-inulin
Lun
gC
apill
ary
DRUG A
Predicting Drug Efficacy
DRUG A
IL2 + TRPV4
Inhibitor
IL2 alone
(Huh et al., Sci. Trans. Med. 2012)
TRPV4 Inhibitor
TRPV4 Inhibitor
(With GlaxoSmithKline)
Biomimetic Microsystems Technology Pipeline
Other Organ-on-Chip Devices in Development:
– Lung alveolus-on-chip
– Heart-on-chip
– Small Airway-on-chip
– Gut-on-chip
– Bone Marrow-on-chip
– Kidney-on-chip
– Liver-on-chip
– Skin-on-chip
– Muscle-on-chip
– Blood Brain Barrier-on-Chip
– Eye-on-chip
Peristaltic Human Gut-on-a-Chip(Kim et al., Lab on a Chip 2012 & Integrative Biology 2013)
Human Intestine Microfluidic Platform
Human Gut Epithelium (Caco-2 cell monolayer in Microfluidic)
24 hr after seeding + Peristaltic-like motions
PNAS, 2007, 104:10295
Intestinal Villi
Lumen
Capillary
Gut Chip
Gut-on-a-Chip Mimics
Normal Gut Morphology
Bar, 20 μm
Transwell
Gut Chip
58 h 130 h 170 h
Formation of Intestinal Villi in the Gut-on-a-Chip(Caco-2 cells/ 0.01 dyne/cm2 Flow + 10% Cyclic Strain/0.15 Hz)
58 h 130 h 170 h
Crypt
Formation of Intestinal Villi in the Gut-on-a-Chip(Caco-2 cells/ 0.01 dyne/cm2 Flow + 10% Cyclic Strain/0.15 Hz)
Restoration of Basal Proliferative Crypts(0-2 hr EdU pulse-chase labeling for DNA synthesis)
Drug MetabolismBarrier Function Differentiation
Restoration of Intestinal Physiology
Mucus Production
Co-culture of Epithelial Cells with Human Gut Microbes
Microcolony of Human Gut MicorbesStrategy of Co-culture
Increased Barrier Function
Villi
Villi
Villi
Probiotic Co-culture
(> 10 days)
Villi
Villi
Villi
Villi
Bacteria
VSL#3 1. Bifidobacterium breve2. B. longum3. B. infantis4. Lactobacillus acidophilus5. L. plantarum6. L. paracasei7. L. bulgaricus8. Streptococcus thermophilus
Microenvironment-Dependent Changes in Gene Expression Profiles
Bacteria
in Gut-on-a-Chip (+Shear, +Strain)
Gut-on-a-Chip(+Shear, +Strain)
Transwell(Static)
• GEDI: Gene Expression Dynamics Inspector
• ~23,000 genes
Applications
Crohn’s Disease on a Chip Drug Transport on a Chip
PDMS
1 mm
8 Weeks
Bone-inducing
Material (DMP+BMPs)
3 mm
Bone Marrow-on-a-Chip(work of Yusuke Torisawa and Katie Spina
Nature Methods – 2014 May 4 [Epub ahead of print].)
Perfused
Microfluidic
Channels
Bone Marrow
Transplantation
In Vitro Blood Cell
Manufacturing
Stem Cell Niche Model
Bone-inducing
material
2 mm
eBM at 4 wk
Muscle side
Skin side
eBM at 8 wk
2 mm
PDMS Device
1 mm
Bone Marrow-on a Chip (after 8 Weeks Implantation)
50 mm100 mm
Trabecular & Cortical Bone
Bone Marrow Formation
500 mm 50 mm
eBM at 8 wk (H&E section)
Femur (mBM)
eBM is nearly identical to natural mBM
mBM
eBM
(8 wk)
cKit
Sc
a1
0.09
0.61
0.14
cKit
Sc
a1
0.08
0.59
0.30
Hematopoietic Stem and Progenitor Cell Distribution
Flow Cytometric Analysis
Dis
trib
uti
on
(%
)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Sca-1
cKit
CD34
CD135
LSK
mPB
(-RBC)
eBM
4wk
eBM
8wk
mBM
Lin-Sca1
Lin-cKit
Lin-Sca1+cKit
Lin-CD34
Lin-CD135
(HSCs)
mBM
eBM
(8 wk)
Flow Cytometric Analysis
CD45
Te
r11
9
35
58
CD45
Te
r119
33
56
Dis
trib
uti
on
(%
)
0
10
20
30
40
50
60
70
80
90
100
CD3
CD19
MacGr
Gr-1
Mac-1
Ter119
mPB eBM
4wk
eBM
8wk
mBM
CD3
CD19
Mac1+Gr1
Gr1
Mac1
Ter119
Differentiated Blood Cell Distribution
eBM is nearly identical to mBM
Dexter culture
mBM cells are culture on a
2D stromal feeder cell layer
In Vitro Culture
Viability
no significant difference0
10
20
30
40
50
60
70
80
90
100
Via
bilit
y (
%)
mBM (Dexter) eBM (on-chip)
Day4 Day4Day7 Day7Harvest Harvest
eBM
on-chip
0
1
2
3
4
5
6
HSCs and progenitors
Dis
trib
uti
on
(%
)
D4 D7Harvest D4 D7Harvest
mBM (Dexter) eBM (on-chip)
Lin-Sca1
Lin-cKit
Lin-Sca1+cKit
Lin-CD34
Lin-CD135
HSCs
Progenitors
Lin-Sca1+cKit
Long-term HSCs
0
0.1
0.2
Lin
- CD
150
+C
D48
-(%
)
D4 D7Harvest D4 D7Harvest
mBM (Dexter) eBM (on-chip)
αγγ
γ
Myelodysplasia
General term for haematologic
defects involving deficiencies
in the myeloid blood lineages,
which in fact manifest
dysfunction in multiple blood
lineages. Myelodysplasias are
sometimes referred to as
‘preleukaemias’ because they
can transform over time.
a generalized model for stem cell regulation. However,
in certain cases, direct evidence that this model is appli-
cable is still lacking. Nonetheless, it is widely accepted
that niches exist in most, if not all, tissues, and that they
provide basic cellular necessities, such as mechani-
cal support, trophic factors and hospitable physical
and chemical conditions, as well as stem cell-specific
self-renewal and differentiation cues (FIG. 2).
Several new and elegant techniques and model sys-
tems have been applied to the study of HSC develop-
ment, permitting an improved functional and anatomical
dissection of HSC interactions with the niche. In par-
ticular, real-time in vivo imaging has enabled the direct
visual ization of HSCs and their niches, providing key
insights into the origins, dynamics and physiological
regulation of the anatomical compartments in which
Figure 1 | Hierarchical model of haematopoiesis in the adult bone marrow. All haematopoietic cells ultimately
derive from a small population of haematopoietic stem cells (HSCs), which is separable into at least two subsets:
long-term reconstituting HSCs (LT-HSCs) and short-term reconstituting HSCs (ST-HSCs). LT-HSCs maintain self-renewal
and multi-lineage differentiation potential throughout life (represented by the bold arrow). ST-HSCs derive from LT-HSCs
and, although they maintain multipotency, they exhibit more-limited self-renewal potential. Further differentiation of
ST-HSCs generates multipotent progenitors (MPPs) and then oligopotent progenitors, which are marked with asterisks.
Haematopoietic progenitor cells lose their differentiation potential in a stepwise fashion until they eventually generate
all of the mature cells of the blood system (these are depicted at the bottom of the schematic). Several potentially
distinct subsets of MPPs have been described, but MPPs are shown here as a condensed population for simplicity.
Lineage-committed oligopotent progenitors derived from MPPs include the common lymphoid progenitor (CLP),
common myeloid progenitor (CMP), megakaryocyte-erythrocyte progenitor (MEP) and granulocyte-monocyte progenitor
(GMP) populations. HSC and progenitor populations can be discriminated by flow cytometry, using antibodies that
recognize unique combinations of cell surface markers. Some commonly used profiles for identifying these cells are
shown adjacent to the HSC and progenitor populations. Dotted arrows denote a proposed lineal connection. CD135, also
known as FLK2 and FLT3; IL-7R, interleukin-7 receptor; lin, lineage markers (which are a combination of markers found on
REVIEWS
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© 2011 Macmillan Publishers Limited. All rights reserved
LT-HSC
ST-HSC
MEP CMP CLP
MPP
Composition of blood cells in cultured eBM maintains intact
whereas that in Dexter culture is completely different
Bone marrow-on-a-chip contains
a functional hematopoietic niche
In Vitro Culture
0.0
0.5
1.0
1.5
2.0D
istr
ibu
tio
n (
%)
with
cytokines
without
cytokines
D4 D7Harvest D4 D7
eBM
(on-chip)Lin-Sca1
Lin-cKit
Lin-Sca1+cKit
Lin-CD34
Lin-CD135
HSCs
Progenitors
Lin-Sca1+cKit
In Vitro Culture
Cytokines
SCF
IL-11
Flt-3
LDL
The microenvironment of eBM can function in
an autonomous fashion to support the hematopoietic cells
Without supplemental cytokines to support HSCs and progenitors
Bone Marrow Transplant
GFP mouseLethally irradiated
mouse (no GFP)GFP+ eBM
The cultured eBM cells engrafted and populated all blood lineages
Hematopoietic compartment of eBM retains fully functional
self-renewing, multi-potent HSCs
En
gra
ftm
en
t (G
FP
+%
)
6 wk 16 wk
mBM eBM D4
0
10
20
30
40
50
60
70
80
90
100
mBM
sBM
Post transplant
0
10
20
30
40
50
60
70
80
90
100
CD3
CD19
Mac1
Gr1D
istr
ibu
tio
n (
%)
in G
FP
+C
D4
5+
Cell
s
CD3
CD19
Mac1
Gr1
mBM mBMeBM
D4
eBM
D4
(6 wk) (16 wk)
(T cell)
(B cell)
(Myeloid)
0
0.5
1
1.5
2
2.5
0Gy 1Gy 4Gy
in vivo
eBM
Dexter
0
0.1
0.2
0.3
0.4
0.5
0Gy 1Gy 4Gy
in vivo
eBM
Dexter
Cell D
istr
ibu
tio
n (
%) in vivo
eBM
Dexter
HSCs (Lin-Sca1+cKit+) Progenitor Cells (Lin-CD34+)
Ce
ll D
istr
ibu
tio
n (
%)
Bone Marrow Chip Mimics Response to Radiation(Funded by FDA Medical Countermeasures Grant HHSF223201310079C)
Radiation Countermeasure
G-CSF (granulocyte colony-stimulating factor, 500 U/mL)
was added 1 day after exposure to g-radiation
0
0.2
0.4
0.6
0.8
1D
istr
ibu
tio
n (
%)
1 Gy
with
G-CSF
1 Gy 4 Gy
with
G-CSF
4 Gy
*
*
After 3 days in culture with G-CSFLin-CD34+
Lin-cKit+
HSCs
G-CSF induced proliferation of HSCs and hematopoietic progenitor
cells in the bone marrow chip in vitro, as previously reported in vivo
Organ-on-Chip Technology Pipeline
• Ongoing projects
– Lung Alveolus
– Lung Small Airway
– Heart
– Liver
– Small Intestine
– Large Intestine
– Kidney Proximal Tubule
– Kidney Glomerulus
– Bone marrow
– Skin
– Cancer
– Heart valve
– ……
Integrated Human Body-on-a-Chip
Organ-On-Chip
INTERROGATOR
Instrument
– Automated instrument
integrates multiple organs
– Designed for ease of use
“plug-and-play” approach
– Generate data to predict
human response
Universal
Chip Holder
DARPA Multiphysiological Systems Grant(D. Ingber, K. Parker, J. Wikswo & CFDRC/ G. Hamilton & D. Levner)
Enabling Human Trials Designs with Cells
From Different Genotypes on Chips?
wyss.harvard.edu