1. 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

2. The Plan
• Introduction
• Purpose of the thesis
• Goals
• Methods
• Results
• Conclusion
• Future Work
• Acknowledgements

3. • 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

4. Mature hepatocyte differentiation
Direct Differentiation from Human Embryoid
Body

5. • 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

6. Introduction (conti.)
• Hepatocyte
• Model hepatocyte cell line: Liver Hepatocellular
Carcinoma Cells (HepG2)
• Epithelial morphology
• Chosen for their application and robust nature

7. Introduction (conti.)
• 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
Peptide
Looped
Peptides
(W-945, 947)
Conventional
peptides
Human neural stem cells

8. Introduction (conti.)
• 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

9. Introduction (conti.)
• 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

10. Flat PAN HF Membrane

11. Goals
• To develop different artificial substrates to support
cell adhesion and growth.
• Possible applications:
– Pure synthetic artificial nerve grafts
– Artificial Livers
• To develop biocompatible polymeric flat
membrane.
• Use the coatings and membrane to conduct cell
culture experiments

12. 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

13. 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.

14. 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.

15. 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

16. 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.

17. Methods: Imaging cultures
• 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)

18. Methods: Imaging cultures
• 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

19. 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

20. 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)

21. 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

22. 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).

23. 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).

24. 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)

25. Results HepG2 P4VP Coating Study
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)

26. Results HepG2 P4VP Coating Study
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)

27. 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.

28. 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.

29. 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).

30. 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).

31. 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).

32. 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).

33. 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).

34. Metabolic Assay Results
No Membrane
(control)
Membrane Peptide Membrane Peptide 100% 1% P4VP
100% 1% P4VP
Mem
100% 0.5% P4VP
100% 0.5% P4VP
Mem
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%
%REDUCED
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.

35. 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.

36. 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

37. Future Work
A B C

38. 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.