Name: preparation of tunable extracellular matrix microenvironments to evaluate schwann cell phenotype specification
Uploaded: 2020-06-02T14:30:00
Description: Watch this Scientific Journal Video about Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification at JoVE.com

The authors gratefully acknowledge funding support from the University of Cincinnati. The authors also thank Ron Flenniken of the University of Cincinnati Advanced Materials Characterization laboratory for support.

1. Tunable cell culture substrate preparation and characterization
<ol>
	<li>Substrate preparation
	<ol>
		<li>Mix the PDMS base elastomer and curing agents using a pipette tip vigorously at a ratio between 10:1 and 60:1 until bubbles are homogeneously dispersed within the mixture. Remove bubbles using vacuum desiccation until bubbles are dissipated. 
		NOTE: During PDMS polymerization, curing agent crosslinks with the base elastomer to provide final polymer desired mechanical properties. Crosslin...

SCs can promote nerve regeneration due to their phenotypic transformation and regenerative potential following nerve injury. However, how ECM cues regulate this regenerative capacity remains mostly unclear, potentially hindering not only the development of biomaterials that aim to promote nerve regeneration but also the understanding of the mechanisms involved in nerve regeneration. To begin to examine this interplay, cell culture substrates were created where ECM cues such as stiffness, protein coating, and adhesive top...

Peripheral nervous system (PNS) injuries remain a major clinical challenge in healthcare by compromising the quality of life for patients and creating a significant impact through a multitude of socioeconomic factors1,2. Schwann cells (SC), as the major glial cells in the PNS, provide necessary molecular and physical cues to induce PNS regeneration and aid in functional recoveries in short gap injuries. This is due to the remarkable ability of SCs to dedifferentiate into a &#8220;repair&#8221; cell phenotype from a myelinating or Remak phenotype3. The repair SC is a distinctive cell phenotype in several ways. Following injury, SCs increase their proliferation rate by re-entering the cell cycle and begin expression of several transcriptional factors to facilitate reinnervation. These factors, such as c-Jun and p75 NTR, are upregulated while myelinating SC markers, such as myelin basic protein (MBP), are downregulated4,5. In addition, SCs change morphology to become elongated and aligned with each other to form B&#252;ngner bands across the injury site6. This provides a physical guidance mechanism for the axons to extend to the correct distal target7. However, despite the ability that SCs possess to promote nerve regeneration in short gap injuries, the outcome of functional recovery remains poor in severe injuries. This is due in part to a loss of extracellular matrix (ECM) guidance cues, as well as the inability of SCs to maintain the regenerative phenotype over long periods of time8.
The nerve regeneration and recovery process are intimately tied to the state of the basal lamina following injury. The basal lamina is a layer of ECM around the nerve that facilitates guidance and provides ECM-bound cues for axons and SCs in cases where it remains intact following injury9. The state of the ECM and its ability to deliver matrix bound cues to cells is vitally important and has been previously explored in a variety of different contexts10,11,12,13,14. For example, it has been shown that the stiffness of the ECM can guide cell functions such as proliferation and differentiation11,15,16. Composition of the ECM can also lead to a distinct cellular response and regulate cell behaviors such as migration and differentiation through intracellular signaling pathways17,18. Furthermore, cell morphology, including spreading area and cellular elongation, play a major role in regulating the function and can be governed by ECM-bound cues19,20. Many previous studies have focused on stem cells differentiating into defined lineages, yet SCs possess a similar ability to alter phenotype from a homeostatic, adult SC within a healthy nerve, to a repair SC capable of secreting proteins and growth factors while remodeling the ECM following nerve injury5,21. Therefore, it is especially crucial to identify mechanisms underlying the relationship between the innate SC regenerative capacity and ECM bound cues for the insight to ultimately harness this capacity for nerve regeneration.
To address this, we have developed a detailed methodology to produce a cell culture substrate where mechanical stiffness and ligand type can be easily tuned in physiologically relevant ranges. Polydimethyl siloxane (PDMS) was chosen as a substrate due to its highly tunable mechanics as compared to polyacrylamide gel, where the maximum Young&#8217;s modulus is around 12 kPa contrasted to PDMS at around 1000 kPa22,23,24. This is beneficial to the work at hand, as recent studies have shown the Young&#8217;s modulus of a rabbit sciatic nerve can exceed 50 kPa during development, thereby suggesting that the range of stiffness of nerves within the PNS is wider than previously examined. Different proteins are capable of adsorption onto PDMS substrates to analyze the combinatorial regulation of mechanics and ligands on SC behavior. This allows for the investigation of multiple microenvironmental cues present in the PNS regeneration process and comparison of a high degree of tunability to the work focusing solely on stiffness of the substrate25. Further, these engineered cell culture substrates are compatible with a multitude of quantitative analysis methods such as immunohistochemistry, western blot, and quantitative polymerase chain reaction (q-PCR).
This engineered cell culture platform is highly suitable for analyzing mechanistic pathways due to the high level of individual tunability of each ECM-bound signal. In addition, popular methods for cell micropatterning, including microcontact printing, can be achieved on the substrates to allow for controlled cellular adhesion to analyze cell shape in relation to other ECM bound cues24. This is critical because line patterned substrates, which promote elongation in cell populations, provide a tool to mimic and study elongated and regenerative SCs within B&#252;ngner bands during nerve regeneration. Further, cellular morphology is a potent regulator of multiple cell functions and can potentially introduce confounding experimental results if not controlled26,27. Significant attention is now being provided to the mechanisms governing the SC regenerative phenotype as regulated by ECM cues28,29,30. This is essential to provide insight into the design of biomaterials that can be applied as nerve guidance conduits for aid in PNS nerve regeneration. These detailed protocols can ultimately be applied as a potential tool to decipher the mechanisms of SC and other cell type function as regulated by ECM bound cues.

<table>
	<tbody>
		<tr>
			<td>Albumin from Bovine Serum (BSA), Texas Red conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A23017</td>
			<td>BSA staining to show micropatterns</td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7076S</td>
			<td>Antibody used for western blot analysis</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP-linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7074S</td>
			<td>Antibody used for western blot analysis</td>
		</tr>
		<tr>
			<td>BrdU</td>
			<td>Thermo Fisher Scientific</td>
			<td>B23151</td>
			<td>Reagent used to measure cell proliferation</td>
		</tr>
		<tr>
			<td>BrdU primary antibody conjugated with Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>B35130</td>
			<td>Used to visualize BrdU in cell proliferation assays</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10483-01</td>
			<td>Protein used to coat coverslips</td>
		</tr>
		<tr>
			<td>Compression force test machine</td>
			<td>TestResources</td>
			<td></td>
			<td>Instrument to quantify mechanical properties of polymers</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000044</td>
			<td>Cell culture medium supplemental</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Thermo Fisher Scientific</td>
			<td>33010-018</td>
			<td>Protein used to coat coverslips</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti2</td>
			<td>Fluorescence microscope</td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>78440</td>
			<td>Protease and Phosphatase Inhibitor</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Thermo Fisher Scientific</td>
			<td>23017015</td>
			<td>Protein used to coat coverslips</td>
		</tr>
		<tr>
			<td>Mounting medium with DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36971</td>
			<td>Coverslip mountant and nuclei staining</td>
		</tr>
		<tr>
			<td>Mouse c-Jun primary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>711202</td>
			<td>Primary antibody to visualize c-Jun protein</td>
		</tr>
		<tr>
			<td>Mouse &#946;-Actin primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>3700S</td>
			<td>Loading control for western blot experiments</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Cell culture medium supplemental</td>
		</tr>
		<tr>
			<td>Photoresist SU 2010</td>
			<td>KAYAKU</td>
			<td>SU8-2010</td>
			<td>Photoresist</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma Aldrich</td>
			<td>P-2443</td>
			<td>Block non-specific protein binding</td>
		</tr>
		<tr>
			<td>Rabbit c-Jun primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9165S</td>
			<td>Primary antibody for visualization of c-Jun protein</td>
		</tr>
		<tr>
			<td>Rabbit myelin basic protein primary antibody</td>
			<td>Abcam</td>
			<td>ab40390</td>
			<td>Primary antibody for visualization of MBP</td>
		</tr>
		<tr>
			<td>Rabbit p75NTR primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>8238S</td>
			<td>Primary antibody for visualization of p75NTR</td>
		</tr>
		<tr>
			<td>Rhodamine phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>R415</td>
			<td>Visualization of cell cytoskeleton</td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>Abcam</td>
			<td>ab156034</td>
			<td>Cell lysis buffer</td>
		</tr>
		<tr>
			<td>RT4-D6P2T Schwann cell line</td>
			<td>ATCC</td>
			<td>CRL-2768</td>
			<td>Cell line used in experiments</td>
		</tr>
		<tr>
			<td>SYLGARD 184 PDMS base and curing agent</td>
			<td>Sigma Aldrich</td>
			<td>761036</td>
			<td>Tunable polymer used to coat coverslips</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15090-046</td>
			<td>Cell dissociation reagent</td>
		</tr>
		<tr>
			<td>UV-Ozone cleaner</td>
			<td>Novascan</td>
			<td></td>
			<td>Increase hydrophicility of PDMS</td>
		</tr>
		<tr>
			<td>Versene (1x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15040066</td>
			<td>Cell dissociation reagent</td>
		</tr>
	</tbody>
</table>

preparation of tunable extracellular matrix microenvironments to evaluate schwann cell phenotype specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

To analyze and quantify the interplay between substrate stiffness and protein composition on SC phenotype, a tunable PDMS cell culture substrate was developed (Figure 1A). Compression testing of the polymer at differing base: curing agent ratios was utilized to quantify the Young&#8217;s modulus (E) of the substrate (Figure 1B). The resulting range of modulus values represents physiologically relevant substrate conditions. Following preparation of substrates, SC...

Watch this Scientific Journal Video about Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification at JoVE.com

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

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