thesis_karan sharma_final

Engineering Surfaces to Support Neural Stem Cells (hNSC’s) and Hepatocytes Adhesion and Growth.

By: Karan Sharma

August 9th, 2016

Advisor: Dr. Xuejun Wen M.D., Ph.D.

Committee Members: Dr. Daniel Conway, Dr. B. Frank Gupton

Masters Thesis Presentation

All Rights Reserved

The Plan

• Introduction

• Purpose of the thesis

• Goals

• Methods

• Results

• Conclusion

• Future Work

• Acknowledgements

• Need to develop xeno-free and pure synthetic biomedical devices– Nerve grafts: current using decellularized nerve grafts

from cadavers (possible immune response)

– Artificial livers: easier FDA approval.

• Wen lab has developed reproducible protocol to induce human induced pluripotent stem cells into functional mature hepatocytes.

• Limited cell attachment on hollow fibers.

Introduction

Mature hepatocyte differentiation

Direct Differentiation from Human Embryoid Body

• Most in vitro studies are conducted in 2D – 3D

culture settings.

• Human Neural Stem Cells (hNSCs)

• Isolated in the early 1990’s

• Generate mainly cells for nervous system.

• Differentiate into astrocytes, oligodendrocytes and

neurons

Introduction

Introduction (conti.)

• Hepatocyte

• Model hepatocyte cell line: Liver Hepatocellular

Carcinoma Cells (HepG2)

• Epithelial morphology

• Chosen for their application and robust nature


• Looped peptides (W-945 and 947 peptides)• Pure synthetic (xeno-free, non-bio-derived)

• Application here was to coat an artificial substrate

• Replacement of laminin and Matrigel®

Conventional peptides

Our looped peptides

Cell binding site

Cell Binding site

Day 1 Day 5 Day 10

Commercial

Peptide

Novel

PeptideLooped Peptides (W-945, 947)

Conventionalpeptides

Human neural stem cells


• Poly 4-vinylphenol (P4VP)• A polymeric substance similar to polystyrene

• Artificial synthetic commercially available

• Used mainly with electronics in the past

• Application here was to make an artificial substrate

• Replacement of laminin and Matrigel®

• Molecular weight Ranges from 11,000 to 25,000

• Effective ability to create a hydrophilic surface

• Observed to create an attractive surface for cell adhesion and growth


• Polyacrylonitrile (PAN) Membrane

• Synthetic, semi-crystalline organic polymer

• Thermoplastic, porous membrane, thermally stable,

commercially available, resistant to most organic

solvents

• Used for separation and purification processes

• Used currently in dialyzers

• Originally have hydrophobic surface

Flat PAN HF Membrane

Goals

• To develop different artificial substrates to supportcell adhesion and growth.

• Possible applications:

– Pure synthetic artificial nerve grafts

– Artificial Livers

• To develop biocompatible polymeric flatmembrane.

• Use the coatings and membrane to conduct cellculture experiments

Methods

• To develop Poly 4-vinylphenol (P4VP)

Coating(s) protocol

• To fabricate Polyacrylonitrile (PAN) Flat

Membrane

• Imaging cultures with different conditions

(Microscope, Immunofluorescence, SEM)

• Metabolic Testing

Methods: Poly 4-vinylphenol (P4VP)

• P4VP coating preparation (MW: 11,000 – 25,000)

• P4VP powders were dissolved in different

Ethanol%

• Sterilized using 0.22 µm syringe filter

• 0.0625, 0.125, 0.25, 0.5 and 1% of P4VP

concentrations

• Incubated for 4 hours minimum to be a working

coating for cell culture at 37°C under 5% CO2.

Methods: Peptide Coating

• Fabricated in our lab

• Artificial synthetic coating developed for use with hNSCs

• Uses a PAVAS as a precursor coating (0.01% PVAVS in PBS)

• Peptide W-945 & W-947 (0.04mg.mL peptide mixture)

• Incubation time of about 2-4 hours at 37°C under 5% CO2.

Methods: Polyacrylonitrile (PAN) Hollow

Fiber (HF) Flat Membrane

• PAN power and N, N-Dimethylformamide (DMF-anhydrous 99.8%)

• Put on shaker for 24 hours to complete dissolve

• Originally made solution was 15% reduced to 12%

• To attach to culture dish it was spin coated at 150RPMS, 30 seconds.

• Dissolved in Nano-pure water for 120 seconds

• Sterilized by 100% Ethanol for 4 hours minimum

Methods: Imaging cultures

• Many difficulties were faced for the purposes of

imaging.

• Good images for normal cell cultures through

microscope, confocal and SEM.

• Imaging cultures on membrane only through

SEM.


• Immunofluorescence

• Strict protocol was developed to fit needs for

different coatings and membrane.

• Stained with DAPI (4’, 6-diamidino-2-phenylindole )

and Alexa Fluor® 546 phalloidin (Actin)


• Scanning Electron Microscope• As Immunofluorescence did not work with

membrane conditions

• Serial dehydration was conducted on fixed cells

• Super Critical Drying was carried out to maintain cell morphology

• Sputter coating of Platinum and Gold

Methods: Metabolic Testing

• Made for testing cell expression and proliferation

• 1:9 dye – media ratio.

• Light sensitive

• Inncubation time of 4 hours at 37°C under 5% CO2.

• Inserted into to a 96 well plate with 9 samples for every condition

Results• Most studies were conducted in 2D culture setting

• Successful long term peptides and P4VP artificial

coating studies (hNSCs & HepG2 cell lines)

• Successful long term Membrane and artificial

coating cell studies (HepG2)

Results P4VP Coating (hNSCs)

hNSC cell line A,B: 50% 0.5EtOH P4VP, C: 75%0.5% EtOH P4VP, D: 75%1% EtOH P4VP, E,F: 100%0.5% EtOH P4VP,

G,H: 100%1% EtOH P4VP all in a 6 well plate

Results hNSCs P4VP Coating Study

hNSC Long term P4VPcoatings study where A-D is 75% EtOH Day 4: A1-A2 (0.25% P4VP), A3-A4 (0.5% P4VP), A5

(1% P4VP), A6 (matrigel) Day 7: B1 (0.25% P4VP), B2-B3 (0.5% P4VP), B4 (1% P4VP), B5 (laminin). Day 9: C1-C2

(0.25% P4VP), C3-C4 (0.5% P4VP), C5 (laminin). Day 12: D1-D2 (0.25% P4VP), D3-D4 (0.5% P4VP), D5(1%

P4VP), D6 (laminin).

Results hNSCs P4VP Coating Study

hNSC Long term P4VPcoatings study where E-H is 100% EtOH. Day 4: E1-E2 (0.25% P4VP), E4 (matrigel), E3

(laminin). Day 7: F1-F2 (0.25% P4VP), F3 (0.5% P4VP),F4-F5 (1% P4VP), F6 (matrigel). Day 9: G1-G2 (0.25% P4VP),

G3 (0.5% P4VP), G4-G5 (1% P4VP) G6 (laminin). Day 12: H1 (0.25% P4VP), H2-H3 (1% P4VP), H4 (matrigel).

Results HepG2 P4VP Coating Study

HepG2 Liver Cell cultures for a long term study on 75% 1% (EtOH, P4VP) coatings where A-

H is days (2, 5, 9, 14, 18, 26, 36, and 43)


HepG2 Liver Cell cultures for a long term study on 100% 1% (EtOH, P4VP) coatings where A-

D is days (2, 5, 9 and 14)


HepG2 Liver Cell cultures for a long term study on 100% 1% (EtOH, P4VP) coatings where E-

H is days (18, 26, 36 and 43)

Immunofluorescence Characterization of HepG2 Cells

Images of HepG2 taken through a confocal microscope where the stains are DAPI Nuclear Counterstains (blue), Actin (Red)

and Pink (DAPI + Actin).Where A1-D1 (1 week), A2-D2 (2 weeks) A is 75%1% EtOH P4VP coating, B is 100%1% EtOH

P4VP coating, C is control cells and D is Peptide coating.

Immunofluorescence Characterization of HepG2 Cells

Images of HepG2 taken through a confocal microscope where the stains are DAPI Nuclear Counterstains (blue), Actin (Red)

and Pink (DAPI + Actin). A3-D3 (4 weeks) and A4-D4 (5 weeks). A is 75%1% EtOH P4VP coating, B is 100%1% EtOH

P4VP coating, C is control cells and D is Peptide coating.

Immunofluorescence Characterization of

HepG2 Cells on PAN membrane

Images of HepG2 cells on a flat

Polyacrylonitrile (PAN) hollow fiber

membrane where A1-A5 is 1-5 weeks

taken through a confocal microscope

where the stains are DAPI Nuclear

Counterstains (blue), Actin (Red) and

Pink (DAPI + Actin).

HepG2 Cell culture (control)

Images taken through an SEM: Images of a HepG2 cell culture grown in a normal condition (control). A-G

are different magnifications of different regions of the culture taken at 15keV. A (300X, 100µm), B (800X,

50 µm), C (1kX, 50 µm), D (2kX, 20 µm), E (5kX, 10 µm), F (8kX, 5 µm) and G (20kX, 2 µm).

HepG2 Cell culture on Peptide coating

Images taken through an SEM: Images of a HepG2 cell culture grown in a peptide coating. A-F are different

magnifications of different regions of the culture taken at 15keV. A (300X, 100µm), B (900X, 50 µm), C (1kX,

50 µm), D (2kX, 20 µm), E (5kX, 10 µm), F (10kX, 5 µm).

HepG2 Cell culture on PAN membrane

Images of different regions taken through an SEM (15keV) of a HepG2 cell culture grown on a PAN flat hollow fiber

membrane. A-B (300X, 100 µm), C, G, K (500X, 1KX, 2.5KX), D, E, F, H, I, J (500X, 800X, 100KX, 2.5KX, 5KX, 10KX) and

L (20keV, 2KX, 10 µm).

HepG2 Cell culture on PAN membrane with Peptide coating

Images of different regions taken through an SEM (15keV) of a HepG2 cell culture grown on a PAN flat hollow fiber

membrane with a peptide coating. A-B (300X, 100 µm), C-D (400X, 100 µm), E (40X, 1mm), F (70X, 500 µm), G (500X, 100

µm), H (1KX, 50 µm), I (2KX, 20 µm), J (7KX, 5 µm), K (10KX, 5 µm), L (20KX, 2 µm).

Metabolic Assay Results

No Membrane(control)

Membrane Peptide Membrane Peptide 100% 1% P4VP100% 1% P4VP

Mem100% 0.5% P4VP

100% 0.5% P4VPMem

Day 2 80% 57% 57% 55% 48% 30% 48% 45%

Day 4 87% 64% 69% 81% 49% 32% 57% 49%

Day 8 93% 62% 65% 93% 84% 45% 63% 54%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

% R

EDU

CED

HepG2 Cell Growth Over Time

A cell viability test was conducted on all 8 conditions with HepG2 cell cultures to metabolically determine cell expression

and proliferation.

Conclusion

• Excellent adhesion and proliferation of HepG2

cell culture on Peptide (W-945) & P4VP coating

• Increased growth with PAN membrane with

most on Peptide (W-945) & 100% 0.5% EtOH

P4VP coating

• Membrane is observed to be biocompatible.

Future Work

• Test different molecular weight of P4VP coating

• Test with stem cell derived hepatocytes or primary liver cells and neural cells

• Develop hollow fiber tubular membranes of different morphologies and structures, identify optimal membranes to support neural cells and liver cells

• Construct artificial livers and nerve grafts

Future WorkA B C D

Acknowledgements

• I would like to thank my mentor and advisor Dr. Wen for all the experience, support and knowledge he has given me.

• My committee members Dr. Conway and Dr. Gupton.

• My lab coworkers Dr. Vasudha Surampudi, Dr. Pettinato, Dr. Bo Xue, Dr. Xiomei Li, Jessica Forrester, Debbie Campbell, Chenyang Jiang and the many interns

• My family and friends for all their support.

thesis_karan sharma_final

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