BIOMATERIALS

Biological Phenomena of Neural Stem Cell Differentiation on Graphene

Sabrina Ghosh, Arvind Parthsarathy

2015/2016 Research Project

ABSTRACT

Recognizing an insufficiency in therapeutic strategies for repair and regeneration in

regard to neurodegenerative diseases, we focus on decreasing current controversial
animal model testing for drug efficacy and pharmacology. Robustly differentiating
neural stem cells (NSCs) and expanding their longevity after differentiation in culture
is a pressing issue. To better understand the exact mechanisms of how surface
substrates can induce faster cell differentiation, a triad of analyses were performed
on graphene. Our goal was to study the underlying forces and phenomena that affect
cell differentiation and their interaction with surface substrates, primarily the unique
surface of graphene. The study reveals distinctive and perplexing morphological
changes, quantitative progressions of cells, and marker expressions throughout the
development of NSCs to neurons. The research maintains a self-equilibrium obtained
through the contribution of actin filaments that are under tension and microtubules
that are under compression. The graphenes high density of cell bodies, processes,
and cell-to-cell interactions predict the paralleled thickening of nervous tissue
increasing with the generation of large numbers of neurons for tissue-based assays.
Most significantly, these analyses proved that the basal process of radial glial cells
elongate to retain attachment to the microscopic pillars that create point targets for
NSC neurites to sense. Thus, this proves that graphene provides a basis for the
development of new tools for studying mechanosensing and testing pharmaceuticals.
Because of the unanswered questions of the processes of differentiation of NSCs on
substrates, this research can suggest that growing neurons respond to the stiffness of
graphene and the sustained mechanical tension induces stretch growth in neurons.
Since damaged neurons dont self-renew, graphene has been shown to provide a
viable method to stimulate neurogenesis.
PRELIMINARY NOTES
Context of Research
Before beginning this specific project, I had been independently interning at Lehigh
University for five months under the guidance of graduate students and Dr. Sabrina
Jedlicka in 2015. During this period, I conducted experiments on the most effective
polyacrylamide gel stiffness for neural stem cell differentiation. After my classmate
Arvind Parthasarathy joined the lab in October, we decided to begin research on neural
stem cell differentiation on graphene and submit it for the 2016 Intel International
Science and Engineering Fair at Lehigh University. After these competitions, we began
to learn about and use Raman Spectroscopy to measure mechanical forces on our three
differentiation surfaces.

Assistance

In October 2015, Arvind Parthasarathy and I decided to collaborate on this research

project. Dr. Sabrina Jedlicka provided scientific guidance, lab space and materials, and
was a sounding board off of whom we could discuss obstacles, ideas, and discoveries.

Omitted Material

The results section of the "Appendix" exceeded an attachable file size and was therefore
excluded from the submitted plan. This omitted part contains labeled images that better
depict "Neural Stem Cell Marker Expression" and the image analyses conducted after
immunocytochemistry. Final conclusions drawn from these images are still included in
the paper; therefore, it does not affect the overall reading.

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Introduction
Main Issue
New research shows that neural stem cells (NSC) are used in testing new
pharmaceutical compounds for central nervous system disorders, such as Parkinsons,
Multiple Sclerosis, and Spinal cord injuries; however, research is limited because of the
difficulty in robustly differentiating the cells and expanding its longevity after
differentiation in culture. Currently, drug efficacy and pharmacology is best understood
using animal models. NSCs decrease the use of this controversial strategy by providing
cell and tissue based assays. They also show an increasingly promising therapeutic
strategy for repair and regeneration. Consequently, further development of neural stem
cell models is imperative for widespread use and adoption.

Research
NSCs have the ability to differentiate into neurons, oligodendrocytes, and
astrocytes. Researchers have previously recorded that, on polyacrylamide gels, 80-100%
of the stem cells differentiate into specified neurons after 21 days; and, on glass surfaces,
5-25% differentiate into neurons after 21 days. These results were obtained with the non-
specific serum-starvation method, which decreases certain growth factors in the stem
cells and forces them into a post-mitotic state. The data collected, however, indicates that
mechanical forces also play a large role in the dynamics of differentiation. Understanding
these mechanical forces and engineering systems will allow for robust cell activity and
precise cell model development with the ability to manipulate these forces.

Ongoing research efforts are showing promise in use of biomechanical stimulation

to propagate human neural stem cells to differentiate into oligodendrocytes, astrocytes,
and neurons. There is also evidence that substrates of various stiffness may have varying
effects. In this study, we investigated graphene, polyacrylamide gel, and glass to induce
differentiation of murine C17.2 neural stem cells. Murine C17.2 neural stem cells are well
characterized clone. They are self-renewing and differentiate into all neural lineages in in
vitro studies. Previous research has shown that human NSCs can replace depleted
neuronal population, and thus are ideal candidates to experiment with. Furthermore,
culturing NSCs in in vitro conditions is also well documented. Applying fluorescence
conjugated antibodies to the exoskeleton components provides a way to view
differentiated cells. Tubulin is a major component of microtubules, actin is a globular
multifunctional protein that forms microfilaments, and nestin is type VI intermediate
filament protein that is important for the radial growth of axons. Graphene is a thin layer
of tightly packed layer of carbon that is highly biocompatible. Investigation of
biomechanical induced neural stem cell differentiation can provide valuable insights into
making stem cell therapy a reality.

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 Mechanically induced stress has shown to cause progenitor, uncommitted neural
stem cells to differentiate into oligodendrocytes, astrocytes, and neurons. Recent
evidence is growing to support the use of graphene to induce neural stems cells to
develop longer neurite extensions. The varying stiffness of the substrate is thought to
impact the cell growth as well as neurite extension.

Mechanical Properties of Graphene

Graphene is one atom thick, made up of carbon atoms arranged in a repeated
hexagonal pattern. It provides mechanical stability and optical properties.

Substrate
Graphene
Cells
Glass

Premise
Our project delves into the differences in differentiation of a standard surface of
differentiation (glass), an optimized surface (polyacrylamide gels), and an unknown
surface (graphene). The underlying forces and phenomena that affect cell differentiation
and their interaction with surface substrates was observed and is discussed. Most
significantly, the project provides data for the development of new tools for the study of
mechanosensing.

Proposals/Aim
The aim of this project is to evaluate a graphene surface for cell differentiation as
well as enhance researchers understanding of the impact of a graphene substrate on the
dynamics of differentiation. Our primary goal is to investigate the fundamental,
biological phenomena that occur during neural stem cell differentiation.

We explored three ways to accomplish this. (1) A morphological analysis of C17.2

neural stem cells during induced differentiation provided a basis for understanding
evolution from an undifferentiated to a differentiated state. It also helped identify the
process of cell division from stem cell to radial glial cell to neural basal progenitor to a
neuron. (2) The efficiency of synaptogenesis on the surfaces was compared after
differentiation by observing protein cell markers and synaptic proteins. This determines
if the cells have reached a mature differentiation state as a neuron within the time period.
(3) Masses of nuclei on each of the materials surfaces were quantified and observed for

4
cell density. This is indicative of cell proliferation and post-mitotic behavior in reference
to neuronal differentiation.

Methods
An independent team was responsible for the acquiring of graphene and, most
significantly, the graphene transfer onto the polyacrylamide gel. The graphene transfer
protocol, electrodeposition, works through hydrolysis. C17.2 neural stem cells were set
on all of the coverslips of the control (glass and gel) and experimental (graphene) groups.
C17.2 neural stem cells were split from a stock plate into each of the wells of the control
and graphene samples. Over a period of 15 days, the cells were fed using the serum-
starvation method forcing the cells into a post-mitotic state. After this time,
immunocytochemistry and fluorescent microscopy were used for morphological
analysis, quantifying nuclei, and distinguishing protein markers. This involved fixation,
permeabilization, blocking, primary and secondary antibody solutions and nuclear
stains.

Graphene Manufacturing
The research and specific mechanical forces of graphene was not of interest to the
researchers, therefore an independent team at Lehigh University was responsible for the
acquiring of graphene and, most significantly, the graphene transfer onto the
polyacrylamide gel. The graphene transfer protocol, electrodeposition, works through
hydrolysis, which is splitting water molecules into hydrogen gas. Graphene, as
previously described, is a monolayer of one atom thickness and is, therefore, virtually
transparent on the copper that the graphene was acquired on. Once a current passes
through the copper, effervesces form between the copper and graphene. This peels off
the graphene, causing it to float to the top. However, a substrate is necessary for the
graphene to attach to or the effervesces will shred. When the polymer, or substrate, peels
with the graphene, the substance is placed on a glass coverslip into a solution of distilled
water. By capillary action, the substance adheres to the glass surface allowing the removal
of wrinkles and folds. Chloroform is then used to dissolve the polymer and gel while
maintaining the quality of graphene. The graphene on the polyacrylamide gel is then
directly used placed into glass wells and used accordingly in regard to the current project.

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Preparing the Gel Samples

Glass coverslips were sterilized and used as the glass control and the graphene on
glass treatment. Polyacrylamide gels were placed on activated coverslips from a
Aminopropyltrimethoxysilane and Glutaraldehyde solution. The coverslips were
flamed, covered in sodium hydroxide and silane, incubated in glutaraldehyde, and
coated with a solution of 40% Acrylamide, 2% Bis-Acrylamide, 0.5-M of HEPES, TEMED,
and distilled water. A range of stiffness values can be achieved simply by having a
constant total concentration of acrylamide while varying the concentration of
bisacrylamide cross-linker. Previous research has shown that too low of a concentration
of bisacrylamide will cause the surface of the gel to crack and it is possible that the gel
will not polymerize. Previous observation in the lab had shown that a 5-8 % acrylamide
to 0.1 -0.03 % bisacrylamide gel mixture produces an appropriate scaffold for measuring
cell traction forces. Equally as important to the overall concentrations of the gels
components, is maintaining an appropriate thickness. The thickness of a gel cannot be
calculated from the volume of solution used. The actual gel thickness tends to be up to
four times greater than the calculated value. This is due to the gels response to changes
in temperature, osmolarity, and hydration. It is therefore extremely important to monitor
the experimental atmosphere and constantly maintain suitable experimental conditions
when working with polyacrylamide gels. After being polymerized with ammonium
sulfate, which allows the polyacrylamide to begin to cross-link, the gels are activated by
Sulfo-Sanpah and then either coated with graphene or collagen. Collagen allows for cell
adhesion in later steps. Graphene is transferred to the surfaces of interest using
electrodeposition.

Examining Polyacrylamide
The researchers found from prior experimentation that a gel stiffness of 140 Pascal
would be most effective for the testing of this experiment. The following graph, with data

6
from previous experimentation, on the Area Shrinkage of Polyacrylamide explains this
choice:

Examining Graphene
Cells
Graphene
PA gel
Glass

The stars in the diagram above represent the points of contact between the NSCs
and the graphene. During neuronal differentiation, NSCs extend neurites, which are
known to interrogate the growth surface. These neurites are capable of both applying
forces to the surface and sensing specific features on the surface. Current methods rely
on microscopy techniques that use point targets for imaging to track material
deformation. Furthermore, they represent the points of contact between the NSCs and
the graphene. Graphene is a thin yet highly strong substrate material which is
biocompatible. It is highly conductive and has a fibrous topography that mimics the
extracellular matrix found in cells. This promotes the NSCs to propagate into neuronal
lineage expression. Growing neurons respond to the stiffness of graphene and the
sustained mechanical tension induces stretch growth in neurons. Since damaged neurons
do not self-renew, graphene provides a viable method to stimulate neurogenesis.

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 Differentiation Process
NSC cells were fed with serum free media for 14 days. Serum starvation reduces
basal cellular activity. In serum starved conditions, cells withdraw from the cell cycle and
enter the dormant G0 phase. After 14 days, the cells re-enter the G1 phase, resulting in
the differentiation into homogenous cells. Elimination of serum from culture medium
also removes any unknowns and thus improves the reproducibility of the experiment.

Figure 1
Figure 2

Immunocytochemistry
Mechanotransduction forces from cells influence differentiation and cytology
methods thus include immunocytochemistry and fluorescent microscopy. NSC cells were
stained with fluorescence conjugated antibodies to extracellular matrix proteins, B-
Tubulin 3, Actin, Nestin and GFAP. B-Tubulin 3 is a major component of microtubules.
It is found only in neurons and helps with many cellular processes including division.
Actin is a globular multifunctional protein that forms microfilaments. These
microfilaments allow cells to adapt to endogenous and exogenous stimuli and allow the
cells to rapidly remodel themselves. Nestin is an important intermediate filament that is
critical for the radial growth of axons in nerve cells. They also play a role in regulating
the assembly and disassembly of intermediate filaments. GFAP (Glial Fibrillary Acidic
Protein) is an intermediate filament protein that is present exclusively in astrocytes found
in the central nervous system. It helps maintain the shape of cells and promotes
mechanical strength. The expression of these proteins point to the growth of
differentiated nerve cells.

Figure 1 Source: http://www.sigmaaldrich.com/life-science/cell-biology/cell-biology-products.html?TablePage=20848687

Figure 2 Source: http://www.intechopen.com/books/neural-stem-cells-new-perspectives/systems-for-ex-vivo-isolation-and-culturing-of-neural-stem-cells
8
 Examples of Primary & Secondary Antibody Stains

Researchers Note
This project with all three experiments was conducted previously during Fall of
2015. However, all three experiments had to be terminated because of external
contamination. These results were not used for final data determinations of the second
round of this project. However, they were used as a reference and as guidance when
examining images for qualitative data.

Research Expertise
Expertise on biomaterials were received from mentor and professor Dr. Sabrina
Jedlicka at the Lehigh University Whitaker Lab.

Biosafety
Possible safety risks included the use of potentially hazardous biological agents and
chemicals as follows:
C17.2 mouse neural stem cells (Biosafety Level 1-mammalian immortalized
cells)
Acrylamide (for material synthesis)
NaOH, TEMED, Triton X-100, formaldehyde
All risks were minimized by the following:
Using gloves, safety glasses, Type A2 biosafety cabinet, chemical fume hood,
and the necessary equipment from the Whitaker Lab at Lehigh University
Sterilizing materials in a steam autoclave
Proper disposing of waste by the university

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 Proper training in chemical and biological safety to handle any unidentified
cells by the professor

Findings and Results

Morphological Analysis
After two days of differentiation, there was no noticeable difference in NSC
growth of morphology between the surfaces. At this period of time, the cells exhibited
embryonic NSC structures that were elongated, but, in regard to signaling pathways,
were independent and separated. Between Day 2 and Day 5, the neural stem cells
progressed into first stages of progenitors and briefly into radial glial cells. Few fully
formed radial glial cells were imaged at Day 5 on the graphene.

By Day 10, some bare regions of glass and gel coverslips were partially exposed
due to the detachment or retraction of NSCs during the differentiation process. After 10
days of differentiation, there was a clear difference in morphology between the cells on
the graphene and gel regions and those on the glass region. The graphene region was
fully occupied with confluent, elongated basal progenitors. By day 10, the gel regions had
this same confluent basal progenitor formation. However, the graphene regions exhibited
morphologically mature neurons with neurite-outgrowths between each other. Many
NSCs on glass at this time were only beginning elongation as basal progenitors. After 15
days of differentiation,, the cells on both of graphene and gel regions exhibited elongated
cell shapes with neurite outgrowths, leading to neural network formations. The glass
regions were not as mature as others by Day 15. Graphene NSCs progressed the fastest
morphologically exhibiting neurons and synaptic connections.

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 The following image shows the morphological progression of a neuron from a
dividing progenitor to a neuron. Glial cells, astrocytes, oligodendrocytes are possible
final morphologies, as well. They are not shown in the diagram.

Figure 3

Nuclei Quantification
Nuclei quantifications established the progression of differentiation regarding
increases in mass over time. The cell layer thickened on all three surfaces over 15 days, as
represented by the clear linear relationships on the graph pictured below. Graphene had
the greatest cell proliferation over the time period. NSCs on graphene, however,
exhibited a phenomenon on the edge.

To calculate the average for the nuclei in the the wells on the coverslips the
following measurements and calculations were made:
Length of Window: 0.4 millimeters or 400 micrometers
Area of Window: 0.16 millimeters sq. or 160000 micrometers sq.
Length of Coverslips: Gels: 22 millimeters or 22000 micrometers
Glass: Diameter- 22 millimeters or 22000 micrometers/Radius-11
millimeters or 11000 micrometers
Area of Coverslips: Gels: 484 millimeters sq. or 484000000 micrometers sq.
Glass: 380 millimeters sq. or 380000000 micrometers sq.

Figure 3 Source: http://www.sigmaaldrich.com/life-science/cell-biology/cell-biology-products.html?TablePage=20848687 11

The compared data is statistically significant and the differences between the number of
nuclei were not due to chance and occurred purposefully.

Progression of Nuclei Quantities on Graphene

Neural Stem Cell Marker Expression

NSC cells were stained with fluorescence conjugated antibodies to extracellular
matrix proteins, B-Tubulin 3, Actin, Nestin and GFAP. Nestin was a useful marker
because although it is expressed predominantly in stem cells of the central nervous
system (CNS), its expression is absent from most mature cells. Therefore, neurons on the
glass surface at Day 15, expressing neurons, proved to have not reached full maturity.
Glial Fibrillary Acidic Protein (GFAP) is a type III intermediate filament protein. GFAP
is the predominant component of astrocyte intermediate filaments in the central nervous
system and was used as an astrocytic and glial cell marker. Cell cycle and morphological

12
analyses showed that Actin was used as a marker for early phase untransformed cells;
however, more significantly, large increases in actin in currently transforming cells, such
as in graphene from Day 5 to Day 15, suggested major growth during the G2 + M phases
of the cell cycle as the NSCs progressed and divided. The actin content of dividing cells
was greater that of non-dividing cells. Beta-Tubulin III antibodies labelled neuronal cell
bodies, dendrites, axons, and axonal terminations and were commonly used for the
identification of newly committed neurons. In addition, beta-III Tubulin antibodies
served as a marker for neuronal development.

The results proved that cells grew and differentiated on all three surfaces. The cells
differentiated on the glass surface showed an expression for Beta-Tubulin, Actin, and a
low expression for GFAP on Day 5. By Day 15, the cells expressed an increased quantity
of Beta-Tubulin III and GFAP, indicating progression of neuronal development into glial
cells and/or astrocytes. NSCs on gels exhibited Beta-Tubulin III on Day 5. By Day 15, the
cells underwent major cytoskeletal growth. The NSCs on graphene exhibited Actin and
Nestin on Day 5, confirming the underdevelopment at that stage. However, by Day 15,
the cells contained increased Actin and Beta-Tubulin III. While all of the materials had an
increase of these markers, cells differentiated on graphene showed the greatest positive
overall marker expression and growth.

Discussion of the Results

The presence of an interconnected network of filaments, exemplified in this
research by actin filaments and beta-tubulin III, represent the mechanical force balance
of the cell on the substrate and predict the sustainability in the cytoskeleton. The research,
through NSC marker expression, maintains a self-equilibrium obtained through the
contribution of actin filaments that are under tension and microtubules that are under
compression. This provides a basis for the development of a tensegrity model to evaluate
the internal elastic energy changes as a function of cell interaction with the underlying
surface, while modifying its topography and composition.

The mechanical behavior of a cell mainly depends on the filamentous structure,

the cytoskeleton, by means of its components such as microtubules, microfilaments and
intermediate filaments that are in the form of an integrated network.

Developmental malformations such as a small or a large brain, or focal

abnormalities (focal cortical dysplasia) have been attributed to disturbed production of
neurons or glial cells during embryonic development and are commonly associated with
mental retardation and epilepsy. Cortical malformations that are attributed to disturbed
migration have been linked to genes that encode components of the extracellular
environment or molecules that control the cellular response to that environment. The
markers used in research were early-stage and embryonic and the markers were positive
for Beta-Tubulin, Nestin, Actin, and GFAP over all three materials.

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 NSCs start to express glial markers 50% into the differentiation process and
assume a more elongated morphology. Reflecting this transition, the emerging neural
stem cell/progenitor type is now named a radial glial cell (RG) and retains the bipolar
morphology. In humans, this would parallel the thickness of the nervous tissue
increasing with the generation of large numbers of neurons; the basal process of the radial
glial cell would then elongate in order to retain attachment to the surface substrate as
shown in the image labeled A below.

NSC pulling
polymer film

The graphene surface was characterized by a high density of cell bodies and
processes and cell to cell interactions and are another avenue for the regulation of
progenitor behavior. The graphene propagated the NSCs to express their neural lineage.
Growing neurons respond to the stiffness of graphene and the sustained mechanical
tension induces stretch growth in neurons. Since damaged neurons dont self-renew,
graphene provides a viable method to stimulate neurogenesis.

Conclusions

Review of Previous Research

This study builds on previous work that successfully demonstrated the
differentiation of mouse clone 17.2 neural stem cells on graphene. Both the NSC clone
and graphene are well characterized in literature. Snyder et al. showed that mouse NSCs
grafted into the spinal cord in adult rats proliferated and followed normal cell
development . Graphene is a well characterized synthetic material with unique
[1]

characteristics such as its tensile strength, topography, and electrical conductivity that
promotes stem cells into a differential pathway. The exact mechanism of how graphene
induces cell differentiation is still not completely understood.

Summary of Results
In this study, cell culture was used to test three different substrates, graphene,
glass, and polyacrylamide gels to induce neural stem cells (NSC C17.2) to differentiate in
special neuronal lineages. Immuno-histochemical methods using antibodies against
extracellular membrane proteins, Beta-Tubulin III, Actin, Nestin, and GFAP, showed that

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cells differentiated most effectively on graphene. The study showed the morphological
changes and quantity of cells for the 15 days of the experiment.

Field of Long-Term Importance

The results in this study are to provide the research necessary for understanding
how to manipulate and control neural stem cells to differentiation into a phenotype of
interest. Replenishment of neuronal cells can have significant implications for many
central nervous system disorders. Degenerative disorders such as Parkinsons disease,
multiple sclerosis, and spinal cord injuries are marked by a loss of neuron cells. A gap in
the population of neuron cells can show itself in many phenotypic symptoms involving
motor functions. Because of the unanswered questions of the processes of differentiation
of NSCs on substrates, this research leads us into a new era of tissue engineering and
regeneration.

Our research shows evidence that graphene most successfully induced

differentiation. Further work is needed to understand the exact manner of this
mechanical stimulus. This preliminary evidence is paving the way for quantifying cell
growth as well as documenting conditions on development of neurite length. These
findings point to a promising outlook for the use of stem cell therapy to treat debilitative
neurological conditions involving spinal cord injury.

Other fields of importance include Confirming C17.2 cells grow on graphene,

confirming cell force has ability to deform graphene, and confirming graphene transfer
process.

A long-term goal of this research also includes redefining the petri dish in order
to better facilitate differentiation to get to a certain cellular mass for tissue scaffolding.
Another goal is to achieve Raman mapping. To study mechanical forces, researchers
currently use traction force microscopy (TFM). TFM is a form of microscopy that allows
for tracking of discrete points that are deformed by adherent cells. These discrete points
are often larger than the individual contact points that a cell exhibits when attached to a
surface, and the resolution of this technique is limited by the wavelength of light.
Graphene is a flat monolayer of carbon atoms, arranged in a two-dimensional hexagonal
structure, and may provide a means to perform continuum analysis on adherent cell
force. In addition, graphene is a biocompatible material, with potential uses in
regenerative medicine. Graphene is being used because it allows continuum analysis
instead of analysis on point sources, which is a completely different approach compared
to traditional biomechanical method analyses. However, it is unclear how differentiation
dynamics will be impacted by a graphene substrate.

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Current and Further Research
Along with the applications and further research possibilities presented in the
preceding section, the researchers are currently working on further imaging and
analyzing the current data and possibly evaluating more data points of interest. The
researchers are also working on another set of experiments to evaluate more quantitative
data that would allow to further analyze cell mechano-transduction and signaling
pathways. The most indicative set of data would be to calculate the % neuron, % NSC,
and % other cells, a neurite length analysis from the nucleus to the dendrites, a stress
fiber thickness analysis and cell area with the Image J program.

Acknowledgements
Dr. Sabrina Jedlicka for her mentorship throughout the course of this research
and for use of the Whitaker Lab at Lehigh University.

Evan Snyder, PhD of the Burnham Institute for the gift of the C17.2 Neural Stem
Cells.

NSF-CBET #1014957, for funding the supply purchases.

Lehigh University Biosystems Dynamics Summer Institute for the graphene

specimens and funding necessary supplies.

Rat 401 antibody to nestin was provided through the Developmental Studies
Hybridoma Bank, as deposited to the DSHB by Hockfield, S. (DSHB Hybridoma
Product rat-401).

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Appendix

Protocols
Polyacrylamide Gel Protocol
Day One
1. Prepare NaOH solution:
- 0.1 g NaOH
- 25 ml dH2O
2. Flame coverslips, place in large petri dish
3. Use q-tip to swab NaOH immediately afterward
4. Add 30 l 3- Aminopropyltrimethoxysilane (Silane) onto each coverslip.
5. Wait 10 min.
6. Wash: 3 times for 10 minutes each using dH2O. (Approximately 5 ml for the
whole dish)
7. Prepare Glutaraldehyde solution:
- 19.6 ml PBS
- 400 l Glutaraldehyde
8. Place coverslips on Kim wipes (Trick: b/c glutaraldehyde sticks to petri dish)
9. Add 200 l of Glutaraldehyde on each coverslip.
10. Wait 30 min.
11. Place coverslips back into petri dish
12. Wash: 3 times for 10 minutes each using dH2O. (Approximately 5 ml for the
whole dish)
13. Allow the coverslips to dry
14. Store on bench with petri dish labelled until the following day

Day Two
1. Prepare solutions:
A. 0.5 M Hepes:
- 5.95 g Hepes
- 45 ml dH2O
- Check pH (adjust pH to 4.2)
- BTV 50 ml
B. 40% Acrylamide (w/v)
- 20 g Acrylamide
- 50 ml
C. 2% Bis-Acrylamide (w/v)
- 1 g Bis-Acrylamide
- 50 ml dH2O
2. Evenly coat another set of coverslips with Rain-X on Kim wipes. Allow to dry
for 10 minutes
3. Place coverslips on a sheet of parafilm

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 4. Polyacrylamide gel:
- 648.9 l dH2O
- 100 l Hepes
- 75 l Acrylamide
- 20 l Bis-acrylamide
- 0.5 l Temed
- 0.5 l Ammonium Persulfate
5. Add 5 l Ammonium Persulfate onto each coverslip
6. Immediately afterward, add 20 l PA gel onto each coverslip
7. Sandwich the gel placing the Rain-X slips on top
8. WAIT 45 min.
9. Add dH2O to the petri dish and remove Rain-X slips with forceps
10. Prepare Sulfo-SANPAH solution:
- 0.5 mg Sulfo-SANPAH
- 5.0 l DMSO
- 1 ml Hepes
11. Add 100 l Sulfo-SANPAH solution to each coverslip
12. UV coverslips for 15 min.
13. Aspirate pink liquid from each coverslip
14. WASH with Hepes 2 times for 10 minutes
15. Prepare Secondary Substrate:
- 750 l dH2O
- 172.4 l Collagen
16. Put the coverslips into a 6 well plate
17. Add 100 l of Secondary Substrate to each well
18. UV the plates overnight (4 hours minimum)

Splitting Protocol: (Transfer of C17.2 NSCs onto samples)

1. Splitting of stock-plate C17.2 neural stem cells on glass coverslips and
polyacrylamide gels on glass coverslips (control)
a. After following standard chemical fume hood procedure, set up new
experimental plate and cell stock-plate (cells should be confluent before
splitting)
b. Aspirate original media from the stock-plate
c. Add 1 mL of fresh media with serum into each well of plate
d. Aspirate all media from cell stock plate
e. Add half of trypsin( __ mL) in the tube into the cells stock plate using a
pipette
f. Pipette this first round of trypsin
g. Add the last half of the trypsin from the tube into the stock plate
h. Incubate plate for 2-3 minutes
i. Set up new petri dish for new stock plate following a sterile procedure

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 j. Fill the new petri dish with 10 mL of fresh media
k. Take out stock plate from incubation
l. One drop of stock-plate cells into the new petri dish
i. 15-25 microliters into each well of plate
m. Aspirate remaining cells of original stock-plate
n. Incubate all plates
2. Splitting of stock-plate C17.2 neural stem cells on graphene on glass coverslips
and graphene on polyacrylamide gels (experimental group)

Immunocytochemistry Protocol:
Fixation:
1. Remove media from wells. Add 10% formalin solution to wells in order to
completely submerge cover slips.
2. Leave on for 45 minutes at RT.
3. Rinse 3 times (2 min) with PBS
Permeabilization:
1. After removing the PBS from the samples, add 0.1% Triton X-100 to wells
2. Leave on for 30 minutes at RT
Blocking:
1. Submerge coverslips in 1% BSA solution.
2. Leave on for 45 minutes at RT
Primary Antibody Solutions:
1. For 1 mL solutions use 900 microliters of PBS, 100 microliters of blocking
solutions, and 0.3 microliters of appropriate antibody
2. Cover each coverslip with appropriate amount (100-200 microliters per well)
3. Leave on samples overnight at RT at 4 degrees Celsius
4. Rinse 3 times (2 minutes) with PBS
Secondary Antibody Solutions:
1. For samples that need a secondary antibody solution, use same concentration
as primary antibody solution
2. Cover coverslips entirely and let react for at least 1 hour at RT
3. Rinse 3 times (2 minutes) with PBS
Hoechst Nuclear Stain:
1. Counterstain nuclei with Hoechst dye (0.002 mg/mL in aq. solution)
2. Rinse 2 times (2 minutes) with distilled water

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