Bioreactors for Tissue EngineeringBioreactors for Tissue Engineering

Chris CannizzaroChris CannizzaroGordana VunjakGordana Vunjak--NovakovicNovakovic

Core 2: Resources and RepresentativeDesigns

Bioreactor platforms Bioreactors with perfusion and electrical stimulationcardiovascular

Bioreactors with perfusion and mechanical loadingosteochondral

Microfluidic bioreactorsstem cells, screening studies

Imaging compatibilityDesign Computer-aided design

SolidWorksComputer-aided modeling

MATLAB, FEMLAB

Fabrication Machining at MIT and TuftsSterile micromachining capability at MIT core lab

Microfabrication at the MTL lab at MIT (e.g., lithography, sputtering, e-beam)

Sensors Traditional and nontraditional sensing optical probes for on-line monitoring of oxygen and pH

How resource works…

• Initial meeting with Core 2 member(s) to assess user needs

• Design prototype bioreactor

• Computer modeling of important parameters– shear, diffusion…

• Build prototype

• Test and modify as needed

Partial list of ongoing projects

• Multi-well shear device– Vasculogenesis of hESC (Gerecht, MIT)

• Round perfusion bioreactor– Bone and cartilage engineering (Marolt, MIT)– Osteochondral plugs (Grayson an d Chao, Columbia)– Invertebral disc (Kandel, Mt. Sinai)– Vasculogenesis (Kang, Tufts)

• Removable rod bioreactor– Blood vessels (Lovett, Tufts)

• Bioreactors with electrical stimulation– Cardiac tissue engineering (H. Park, MIT)– Embryoid bodies (Figallo and Gerecht, MIT; Elvassorre, Padova)

• Microfluidics– Microbioreactor arrays (Elvassorre, Padova)– Gradient bioreactors (Moon, U. Washington, Cimetta, Columbia)

Multiwell shear device

• Designed for controlled shear studies in multiwell plate format

• Custom plunger design maintains laminar flow along well surface

• HUVEC and hESC differentiation

CIMIT

CFD modeling of plunger

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well surface

hESC response to shear

SMA

Phalloidin

Control24h

shear

Nuclei

Round perfusion bioreactor

• Our most popular bioreactor

• Simple design, easy to use– Essentially a functionalized Petri dish– Medium reservoir serves as both gas

exchanger and bubble trap

• Space for 6 constructs

• Imaging compatible:– Light and fluorescent microscopy

A. CAD model of perfusion bioreactor for six press-fit scaffolds; B. Cross-section view; C. Complete experimental setup showing perfusion loop and peristaltic pump; D. Distributed pressure drop through scaffolds and channels; E. Streamlines of velocity field.

BBAA

CC

Q

DD EE

Bone engineering with MSCs and silk scaffolds

Scaffolds seeding and positioning within bioreactor

In situ imaging of tissue constructs and cell growth at

scaffold periphery

Continuous scaffold perfusion (0.01 cm/s)

vs

intermittent scaffold perfusion (0.01 cm/s for 1 h/day)

Scaffold

Top surface ofTop surface ofbioreactor wellbioreactor well

Continuousperfusion

Top surface

Intermittentperfusion

Top surface

AA BB

CC DD

EE FF

– Rod Reactor• Well: 45 x 45 x 20 mm• Rod: ~1.6 mm

diameter

– Collagen gel seeded with hMSCs (P3-P5)

– Syringe pump to establish flow (~40 µm/s)

– Endothelial cells lining by perfusion (HUVECs)

Engineering vascularized tissuesin vitro

Engineering vascularized tissuesin vitro

Construct at day 15

500 μm

Construct at day 6

On line imaging

Bioreactors for branching vascularnetworks

• More advanced geometry• Co-culture• Application of mechanical forces

Bioreactors for application of electrical fields

• Functional assembly of engineered myocardium with combined electrical stimulation and perfusion

• Stem cell differentationand cardiac cell pacing using clear conductive electrodes

• Differentiation of embyroid bodies by application of DC electrical fields

Advanced culture systems for Advanced culture systems for controlled growth and differentiation of controlled growth and differentiation of human embryonic stem cellshuman embryonic stem cells

Sharon Gerecht & myself HST, MITGordana Vunjak-Novakovic Biomed Eng, Columbia UniversityElisa Figallo & Nicola Elvassore Chem Eng, University of PadovaJason Burdick Bioengineering, University of Pennsylvania

Chris Cannizzaro

Outline

• Introduction• 2D monolayer bioreactor system

– Bioreactor design– Image analysis– Differentiation of hES’s

• 3D hydrogel bioreactor system– Bioreactor design– Hyaluronic acid– Proliferation and differentiation of hES’s

• Conclusions

Advanced culture systems for controlled growth and differentiation of human embryonic stem cellshuman embryonic stem cells

• Why human embryonic stem cells?– Perpetual self-renewal in culture– Maintain undifferentiated phenotype and normal

karyotype– Ability to develop into all three primary germ

layer derivatives: ectoderm, mesoderm and endoderm

• Potential applications– Cell therapy (tissue engineering)– Pharmacological studies– Fundamental science, developmental biology etc.

Scientific American 2005

Advanced culture systems for controlled growth and controlled growth and differentiationdifferentiation of human embryonic stem cells

• Why controlled growth and differentiation?– Stem cells must be differentiated in vitro for

therapeutic applications– Growth must be well controlled to expand cells to

req’d large quantities– Would like to recapitulate stem cell “niche”

• micro-environment surrounding the stem cells

Advanced culture systemsAdvanced culture systems for controlled growth and differentiation of human embryonic stem cells

• Why advanced culture systems?– Rational understanding of the parameters that control the stem

cell differentiation pathways– Accurate spatial-temporal control of the micro-environment

surrounding the cells– High-throughput experiments with different stimulation conditions

• How can microscale technologies assist?– Low consumption of growth factors– Low consumption of antibodies for staining– Controlled reproducible studies– Quantitative analysis of differentiation pathways

2D monolayer bioreactor system

Bioreactor design

• 4 x 3 array of independentbioreactors– 102-103 number of cells per well– microliter volume (~20 μl)– well plate fit format

• 3 mm well diameter• 4.5 mm x 7 mm spacing

• Material and methods– Soft-lithography techniques – Continuous perfusion by syringe

pump (steady state conditions)

Bioreactor SchematicMBA-MIOMBA-BIO

2D Microbioreactor (MBA-MIO)

3D velocity field for bothconfigurations

Velocity field 100 micron above growth surface

Shear stress at growth surface

CFD modeling summary

3D velocity field 2D velocity field Shear stress

C2C12 myoblasts

Phase contrast after 7 days of culture with differentiativemedium (low serum concentration)A Homogenous distribution of cell in the entire wellB Differentiation of c2c12: myotube formationC Particular of the microbioreactor: inlet

A

B C

outlet

Immunostaining of a single well

Staining of c2c12 for Tropomyosin after 7 days of culture with low serum medium.

Cardiomyocyte: 4 days of dynamic culture

Homogeneous distribution of cardiomyocyte in the entire well after 4 days of culture

Staining of troponin in cardiomyocyte cultured for 4 days in microbioreactor

hESC: 4 days of dynamic culture

A

BPhase contrast of hESC after 4 days of colture in microbioreactor with : A dynamic condition B static condition

Immunostaining of hES’s after 4 days

SMAD

DAPI

high seeding density

low seeding density

Data acquisition and evaluation

• Evaluation of hES differentiation:– Down regulating markers: OCT4, SSEA-4, To-Pro3, etc.

– Up regulating markers: SMAD, CD31, etc.

• Requirements:– Quantitative evaluation– Automatic acquisition– Quick and cheap

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

iCyte® Automated Imaging Cytometer

Image analysis routine

DAPI Nuclei identification:1 coordinates2 geometry3 intensityImage

processing

FACS-like data

Extranucluearspace

Proteins

SMA

Transcriptionfactors

Intranucluearspace

OCT4

Cell seeding density correlates with differentiation

0.00

0.05

0.10

0.15

0.20

0.25

2.0 2.5 3.0 3.5

Log(Nc)

sma

posi

tive/

oct4

pos

itive

Low density

High density

SMA: smooth muscle actin

How do perfusion and growth factors influence differentiation?

• Experimental parameters:– Perfusion + and - with thermostated syringe pump:

• Flow rate of 0.3 μL/min– VEGF + and - medium– Slide precoated with collagen (IV)– 4 replicates for each condition– 4 day culture

• Immunofluorescence analysis:– Down regulating transcription factor: OCT4– Up regulating transcription factor: SMAD

Result: static condition

no growth factor with VEGF

OC

T4S

MA

D

Result: data analysis

OCT4

SMADOCT4 = 34.8SMAD = 31.7

SMAD/OCT4 = 0.9

OCT4S

MA

D

Result: static condition

no growth factor with VEGF

SM

AD

OCT4

SM

AD

OCT4

OCT4 = 30.7SMAD = 41.0

SMAD/OCT4 = 1.4

OCT4 = 34.8SMAD = 31.7

SMAD/OCT4 = 0.9

Result: static versus perfusionS

MA

D

OCT4

static condition perfusion condition

SM

AD

OCT4

OCT4 = 35.9SMAD = 27.7

SMAD/OCT4 = 0.8

OCT4 = 34.8SMAD = 31.7

SMAD/OCT4 = 0.9

Result: perfusion + growth factorS

MA

D

OCT4

SM

AD

OCT4

perfusion only perfusion + VEGF

Combination enhances differentiation significantlyOCT4 = 35.9SMAD = 27.7

SMAD/OCT4 = 0.8

OCT4 = 53.2SMAD = 98.1

SMAD/OCT4 = 1.8

3D hydrogel bioreactor system

Bioreactor design

Control and regulation of differentiation & Control and regulation of differentiation &

proliferation/selfproliferation/self--renewal processesrenewal processes

⇓⇓

Why culture cells in a hydrogel?

• Use chemically defined surroundings for human ESC cultures

• 3D cultures affect cellular response of mature cells• Mimic 3D setting of a developing blastocyst• Future tissue engineering applications

Why hyaluronic acid (HA) hydrogel?

• HA is involved in the growth of undifferentiated human ESCs and is therefore an excellent substrate for their propagation

• HA hydrogel maintains hESCs in their undifferentiated state in contrast to other 3D systems

• HA hydrogel is a chemically defined and controllable environment in contrast to Matrigel and MEF

Levels decrease at the onset of

differentiationToole BP. Semin Cell Dev Biol. 2001

Improves IVF embryo survival and

developmentFurnus et al., Theriogenology 1998 Kim et al., Theriogenology 2005

HA receptor expressions during early embryo

development Campbell et al., Hum Reprod. 1995 Furnus et al.., Theriogenology. 2003

High expressionlevels during

embryogenesis Toole BP. Nat Rev Cancer . 2004

HA

Role of HA in early development

++

MeHA

Cells

hν

MEF conditionedMEF conditionedmedium medium

++

initiator

Cell encapsulation in HA hydrogel

Oct4 SSEA-4 To-Pro3 Merge

Oct4 TRA-1-81 To-Pro3 Merge

100μm

Expression of undifferentiating markers after 15 days

Gerecht-Nir (Unpublished)

Cells do not differentiate in HA

18 h18 h

24 h24 h

Release from gel using Release from gel using HAseHAse

100μm

24 h24 h

Passage 3Passage 3

ReRe--culture on culture on MEFsMEFs

Gerecht-Nir (Unpublished)

Cell release and re-culture

Bioreactor design

Experimental setup for 3D system

A

B

C

Perfusion improves viability

Live + Dead cells

Live cells Dead cells

Live + Dead cells

Live cells Dead cells

Perfused Perfused 3D culture (4 3D culture (4 daysdays)) Static Static 3D culture (4 3D culture (4 daysdays))

Differentiation can be studied by adding growth factors to medium

DAPI + CD31

CD31 DAPI

Cellular morphology Cellular morphology &&sproutingsprouting

7 days, VEGF+ medium

Conclusions

• Stem cells– are needed to advance tissue engineering field– must be differentiated in vitro to be useful– we do not yet know how to do this and impractical to learn at

large scale

• Microfabricated bioreactors are useful because…– they are inexpensive to build– they are flexible– they consume very little medium/growth factor– culture conditions are well controlled, in particular flow regime– laminar flow at small scale can be exploited for mixing/gradients

Conclusions

• 2D monolayer bioreactors are useful for…– differentiation in the presence of growth factors and shear– gradient studies– spatiotemporal pulses and steps

• 3D hydrogel bioreactors are useful for…– maintaining cells in undifferentiated state– maintaining high cell viability– differentiation in the presence of growth factors

Acknowledgments

University of Padova

Fulbright Scholar Program

Juvenile Diabetes Research FoundationQuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Original Abstract

• The in situ environment of an embryonic cell in a developing blastocyte has a three-dimensional (3D) architecture, in contrast to the commonly used monolayer cultures. In vivo, cells are surrounded by other cells and a complex network of extracellular matrix (ECM) fibers, and subjected to cascades of regulatory signals (molecular and physical) which interact according to specific spatial and temporal patterns. Together, the factors associated with this environment regulate the self renewal and differentiation of human embryonic stem cells (hESCs). So far, ECM-cell interactions have not been extensively studied in hESCs. Furthermore, most advanced existing bioreactors can provide either local control of oxygen and pH or biophysical stimuli, but not the two sets of factors concurrently. Our goal was to develop culture systems that have the necessary cues and signals to provide microenvironmental control and biophysical regulation of cultured hESCs. First, a bioactive hydrogel based culture system was designed to provide a defined 3D environment for propagation of hESCs. This was followed by the development of a bioreactor for microenvironmental control and biophysical regulation. Both 2D and 3D settings were developed to study hESC growth and differentiation.

• The ECM polysaccharide hyaluronic acid (HA) was found to be a developmentally relevant material for the growth of hESCs. hESCs encapsulated in HA hydrogels propagated as undifferentiating cells and could be released and re-cultured, illustrating the importance of both the HA hydrogel structure and chemical specificity. HA internalization and gel remodeling by hESCs validated the specific bioactivity of the HA hydrogel culture system. This was followed by the development of microfluidic systems for cell culture in 2D and 3D settings, with perfusion and environmental control, consisting of an array of independent micro-bioreactors fabricated using soft lithography. This optically transparent microfluidic device integrates multi-parametric changes in culture conditions with non-destructive monitoring of events in individual living hESCs. First, the steady-state and reproducible culture conditions including mass transport (i.e. gas, metabolite, and soluble factor concentration) and flow rate (i.e. hydrodynamics) within the 2D microbioreactor were confirmed, and regulated Smad 2/3, Oct4 and actin during hESC mesodermal differentiation. Cell density, soluble factors and substrate components were all found to affect hESC shape and differentiation. Further steps were taken to study hESCs in a 3D setting, utilizing this highly defined and controllable culture system. Continuous perfusion of 3D scaffolding was found to improve hESCviability and survival within HA hydrogels. Experiments focusing on understanding developmental cues of 3D differentiating hESCs are ongoing. Thus far, the utilization of bio- materials and bioreactors facilitated control of cell behavior and can help improve our understanding of the signaling pathways involved in hESC growth and differentiation.