Biological Background & Motivation

Tyler DiStefano, B.Eng., Vasudha Surampudhi, Ph.D., Anand Swaroop, Ph.D.Neurobiology Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD 20892

Engineering Retinal Therapies via Bioreactors and 3D Gels

Dry Age-related macular degeneration (AMD) and Retinitis Pigmentosa (RP) are among the leading causes of blindness in developed countries1

Currently, no formal treatment exists for dry AMD or RP, which leads to retinal degeneration1

Both ocular conditions rapidly progress into permanent blindness without treatment

Electrospin collagen-coated PCL on top of the PDMS mold and obtainrheological data for biocompatible hydrogels

Determine optic cup morphology, viability, and expression in the rotatingwall bioreactor and encapsulating hydrogels

Finalize bioreactor operation in the incubators and compare retinal tissuedevelopment between static and dynamic culture conditions

Future Directions

We would like to gratefully acknowledge Dr. Nicole Morgan and Mr. Thomas Pohida from NIBIB and CIT,respectively, for their advice and collaboration with this project. Moreover, we would like to thank Dr. TizianaCogliati for her support and guidance throughout the duration of this project. This work was supported byIntramural Research Program of the National Eye Institute and National Institutes of Health.

Acknowledgements

References[1] Curcio, C. et al. “Structure, Function, and Pathology of Bruch’s Membrane” Anatomy and Physiology: Basic Science and translation to Therapy (2013) 1: 465-481[2] Placzek, M., et al. “Stem cell bioprocessing: fundamentals and principles” J. R. Soc. Interface (2009) 6: 209-232.[3] Ellis, M., et al. “Bioreactor systems for tissue engineering: a four-dimensional challenge.” Bioreactors for Tissue Engineering: Principles, Design, andOperation (2005) 1: 1-18[4] Kuo, S.M., et al. “Fabrication of aspherical SU-8 microlens array utilizing novel stamping process and electrostatic pulling method” Opt. Express18: 19114-19119[5] Nuansing, W., et al. “Electrospinning of peptide and protein fibres: approaching the molecular scale” Faraday Discuss. (2013) 166: 209-221

Results & Key Findings

Biomaterial Fabrication and Analysis Average diameter of

the PDMS cups is 254.3µm

At a glass transition temperature of 80°C, we observed a 1:1 mask to mold stamping ratio

Average depth of the PDMS cups is 37.6µm

Top-down view of the positive PDMS mold

Optical profile of a PDMS cup

Fluid Mechanics within the Rotating Wall Bioreactor Vessel

Consistent trend among analytical and computational results: azimuthal velocity increases as vessel rotation speed increases

Laminar flow (Re < 2000) and a low shear environment are maintained within the bioreactor for all allowable vessel rotation speeds

Static Culture Growth and Development

mESC-derived Optic Cup Day 11 mESC-derived Optic Cup Day 15

After cutting optic vesicles (OV) off mESC-derived embryoid bodies, OVs matured into a larger optic cup structure

Boundary Conditions

1

2

Analytical solution for the azimuthal velocity profile:

ANSYS Fluent 2D Axisymmetric Simulation

R&D Methodology: A Three-Staged Approach

Computational

Experimental

Analytical

Navier Stokes & Flow Profile

Nondimensionalized Numbers Computational Fluid Dynamics (CFD)

Analytical Model Validation

In vitro cell culture bioreactor experiments

Transport Modeling Approach

Aim 1 : Create composite biomaterial structure for 3D Retinal Pigment Epithelium/Neural Retina co-culture

Aim 3 : Temporally characterize and align retinal development stages between static and dynamic culture systems

Thiele Modulus

Compares cellular demand for oxygen and glucose versus experimental hydrogel diffusivity

Biot Number

Compares tunable environmental conditions (pump flow rate) versus experimental hydrogel diffusivity

Peclet Number

Compares fluid speed within the bioreactor vessel versus experimental hydrogel diffusivity

𝑪𝑪𝒐𝒐𝒐𝒐𝒐𝒐𝒐𝒐𝒐𝒐𝒐𝒐𝒐𝒐𝒐𝒐𝒐𝒐 𝑻𝑻𝒓𝒓𝒓𝒓𝒐𝒐𝒓𝒓𝒓𝒓𝒐𝒐𝒓𝒓𝒐𝒐𝑫𝑫𝒐𝒐𝒊𝒊𝒊𝒊𝒊𝒊𝒓𝒓𝒐𝒐𝒐𝒐𝒐𝒐 𝑻𝑻𝒓𝒓𝒓𝒓𝒐𝒐𝒓𝒓𝒓𝒓𝒐𝒐𝒓𝒓𝒐𝒐

Aim 2 : Characterize transport phenomena within the rotating wall bioreactor vessel and 3D cell aggregate

3D cell aggregate – Features layered materials and co-culture to support mechanical and chemical conditions necessary for development

D?? – CrxExpression

D35+

Degeneration

D7 – Optic Vesicle forms

D26 –Ciliogenesis

Static Culture Development

Dynamic Culture Development ?

D35+

???

D7 – Optic Vesicle forms

D9-10 – 3D cell aggregate is

“set”& placed in bioreactor

D18 –Rho Expression

D?? –CiliogenesisD?? –Rho Expression

D10 – Otx2+ Optic Cups

D12 – CrxExpression

D?? – Otx2+ Optic Cups

(Petri Dish)

(Bioreactor)

RWB System Model

𝐿𝐿 = 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝐿𝐿𝐶𝐶𝐿𝐿𝐿𝐿𝐶𝐶𝐶, 𝜌𝜌𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐∗ = 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝐷𝐷𝐶𝐶𝐿𝐿𝐶𝐶𝐶𝐶𝐶𝐶𝐷𝐷, 𝑉𝑉𝑚𝑚𝑚𝑚𝑚𝑚 = 𝑀𝑀𝐶𝐶𝑀𝑀𝐶𝐶𝑀𝑀𝑀𝑀𝑀𝑀 𝑈𝑈𝑈𝑈𝐶𝐶𝐶𝐶𝑈𝑈𝐶𝐶 𝑅𝑅𝐶𝐶𝐶𝐶𝐶𝐶,𝑈𝑈𝑔𝑔 = 𝐸𝐸𝑀𝑀𝐶𝐶𝐶𝐶𝐶𝐶𝐿𝐿𝐶𝐶𝐶𝐶 𝑀𝑀𝐶𝐶𝐶𝐶𝐶𝐶 𝑇𝑇𝐶𝐶𝐶𝐶𝐿𝐿𝐶𝐶𝑇𝑇𝐶𝐶𝐶𝐶 𝐶𝐶𝐶𝐶𝐶𝐶𝑇𝑇𝑇𝑇𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐿𝐿𝐶𝐶, 𝐶𝐶𝑏𝑏 = 𝐵𝐵𝑀𝑀𝐶𝐶𝑈𝑈 𝐶𝐶𝐶𝐶𝐿𝐿𝐶𝐶𝐶𝐶𝐿𝐿𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐿𝐿, 𝐷𝐷𝑐𝑐∗ = 𝐸𝐸𝑇𝑇𝑇𝑇𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐸𝐸𝐶𝐶 𝐷𝐷𝐶𝐶𝑇𝑇𝑇𝑇𝑀𝑀𝐶𝐶𝐶𝐶𝐶𝐶𝐿𝐿 𝐶𝐶𝐶𝐶𝐶𝐶𝑇𝑇𝑇𝑇𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐿𝐿𝐶𝐶, 𝐸𝐸𝜃𝜃 = 𝐴𝐴𝐴𝐴𝐶𝐶𝑀𝑀𝑀𝑀𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝐹𝐹𝐶𝐶𝑀𝑀𝐶𝐶𝐹𝐹 𝑉𝑉𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐷𝐷,

Which nondimensional transport parameters are important to consider?

RWB CFD Mesh

Hydrogel layer composed of a carbohydrate-based polymer:

Gellan Gum—[Glucose, Rhamnose, Glucuronic Acid] Optimize between 1.4wt% - 2.0wt%

HyStem HP—[Hyaluronic Acid, Heparin, PEG-DA, Gelin-S] Optimize between 0.5wt% - 2.0wt%

PDMS Base and PCL/ECM Scaffold Fabrication Methods 4,5

SU8 Photoresist Microlenses

Glass Slide held below Glass Transition Temperature

PDMS Mold

Collagen-coated PCL

Significant Goals & Applications Create a bioprocess system (bioreactor and biomaterial combination)

that guides the development of stem cell-derived retinal tissue to a fully mature state

Bioprocess Design Principles2

Process Component Objective: Create composite biomaterial structure for 3D Retinal Pigment Epithelium/Neural Retina co-culture Hypothesis: The contiguous juxtaposition between RPE and NR will

produce RPE-phagocytosed outer segment structures, which is necessary for photoreceptor viability

Process Requirement Objective: Characterize transport phenomena within a rotating wall bioreactor (RWB) vessel and 3D cell aggregate Hypothesis: The RWB will mechanically and chemically condition

cellularized microcarriers at magnitudes that sufficiently maintain retinal tissues over biological development

Process Function Objective: Temporally characterize and align retinal development stages between static and dynamic culture systems Hypothesis: Physical differences between dynamic and static culture

systems cause a different temporal outcome in retinal progenitor cell differentiation

Synthecon RCCMax Rotating Wall Perfusion-based Bioreactor

Dynamics within a bioreactor3

Primary Goals: Adapt this model for a potential human AMD and/or RP

treatment modality Utilize this bioprocess system as an in vitro small

molecule and/or drug screening platform

Dry Age-related Macular Degeneration leads to photoreceptor death and loss of visual function

Bioreactor Environment