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1. Tunable cell culture substrate preparation and characterization
<ol>
	<li>Substrate preparation&#8203;
	<ol>
		<li>Mix the Polydimethylsiloxane (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. 
		&#8203;NOTE: During PDMS polymerization, the curing agent crosslinks with the base elastomer to provide the final polymer&#39;s desired mechanical properties. Crosslink ratios can be adjusted to alter PDMS stiffness.</li>
		<li>Place a drop (~0.2 mL) of desiccated PDMS mixture on a square or circular coverslip (e.g., 22 mm x 22 mm) and rotate the coverslip on a spin coater at 2500 rpm for 30 s.</li>
		<li>Incubate the coverslip in either an oven at 60 &#176;C for 1-2 h or at room temperature overnight for PDMS to solidify.</li>
		<li>Treat the coverslip using UV-Ozone cleaner for 7 min (UV wavelength: 185 nm and 254 nm) to increase the surface hydrophilicity. Place it into a sterilized 6-well plate.</li>
		<li>Before using it for cell culture, incubate substrates in 70% ethanol for at least 30 minutes. 
		&#8203;CAUTION: UV-Ozone cleaner can generate Ozone that is harmful to humans. Work in a chemical fume hood or with some form of ventilation.</li>
	</ol>
	</li>
	<li>Micropatterned substrate preparation
	<ol>
		<li>Draw the desired geometry and cell adhesive areas (900 &#181;m2, 1,600 &#181;m2 and 2,500 &#181;m2) using computer-aided design (CAD) software. Create a chrome photomask based on those patterns from a commercial supplier.</li>
		<li>In a clean room or dust-free environment, use standard photolithography techniques to fabricate silicon wafers. Critical parameters for this particular application are as follows: Photoresist: SU-8 2010; Spin profile to disperse the photoresist: 500 rpm for 10 s with an acceleration of 100 rpm/s, then 3500 rpm for 30 s with an acceleration of 300 rpm/s; Exposure energy of UV light: 130 mJ/cm2. 
		&#8203;NOTE: The height of patterns on the silicon wafers is approximately 10 &#181;m following these parameters. Potential cracks around the edge outside the rectangular or triangular patterns can be seen using a light microscope after step 1.2.2. Baking the silicon wafer at 190 &#176;C for 30 min helps to eliminate the cracks.</li>
		<li>Place the patterned silicon wafer inside a circular 150 mm diameter x 15 mm height Petri dish and pour de-gassed PDMS (mixing ratio 10:1) as prepared in step 1.1.1 onto the silicon wafer. 
		&#8203;NOTE: Ensure the thickness of PDMS is at least 5 mm for ease of handling during microcontact printing steps.</li>
		<li>Solidify PDMS on a silicon wafer in an oven at 60 &#176;C overnight. Allow PDMS to cool to room temperature. Precisely cut 30 mm x 30 mm square stamps containing the correct patterns from the silicon wafer using a surgical scalpel. Do not damage the silicon wafer. 
		&#8203;NOTE: Silicon wafers can be reused many times at this point to produce more stamps following cleaning with isopropanol.</li>
		<li>Sterilize PDMS stamps and the tunable coverslips (prepared in steps 1.1.1 to 1.1.3) by immersing them into 70% ethanol for 30 min.</li>
		<li>To confirm the efficacy of micropattern by PDMS stamps after microcontact printing, dry the surface of PDMS stamps using a filtered air stream and pipette 50 &#181;g/mL Bovine serum albumin (BSA) (Texas Red conjugated) solution to cover the entire patterned side of the PDMS stamp.</li>
		<li>Incubate PDMS stamps with BSA solution for 1 h at room temperature to allow for protein adsorption.</li>
		<li>Dry the surface of tunable coverslips using a filtered air stream and increase surface hydrophilicity as described in step 1.1.5.</li>
		<li>Air dry the PDMS stamps to remove the remaining BSA solution. 
		&#8203;NOTE: Take care that the BSA solution is completely removed from the stamp because any remaining solution will cause stamps to slide on the coverslip during microcontact printing.</li>
		<li>Bring the patterned side of the stamp into conformal contact with the tunable coverslip for BSA adsorption on the coverslip surface. Gently press the stamp against the coverslip for 5 min. 
		NOTE: Do not apply excessive force on the stamp since it will bend and cause non-specific contact between the stamp and the coverslip. The appropriate amount of force applied to the stamp is essential for successful microcontact printing.</li>
		<li>Examine the micropattern using a fluorescence microscope with a FITC (Fluorescein isothiocyanate) filter.</li>
		<li>To print cell adhesive areas rather than fluorescent patterns, substitute laminin for BSA protein and repeat steps 1.2.5 to 1.2.10.</li>
		<li>Remove stamps from coverslips and transfer coverslips into a sterilized 6-well plate. Add 2 mL of 0.2% w/v Pluronic F-127 solution into each well to cover the surface of the coverslip and incubate for 1 h at room temperature. 
		&#8203;NOTE: Pluronic F-127 can be adsorbed to the PDMS surface, increasing the hydrophobicity of the PDMS surface to block cells from adhesion.</li>
		<li>Aspirate Pluronic F-127 solution and wash 5x with Phosphate buffer saline (PBS) and 1x with the cell culture medium before seeding cells. A typical seeding density for SCs is 1,000 cells/cm2.</li>
		<li>45 min following cell seeding, remove the cell culture medium and wash coverslips with PBS 2x to prevent multiple SCs from adhering to the same pattern. Maintain cells in the desired cell culture environment for 48 h before quantification.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>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>Laminin</td>
			<td>Thermo Fisher Scientific</td>
			<td>23017015</td>
			<td>Protein used to coat coverslips</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Cell culture medium supplemental</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>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>UV-Ozone cleaner</td>
			<td>Novascan</td>
			<td></td>
			<td>Increase hydrophicility of PDMS</td>
		</tr>
	</tbody>
</table>

preparation of a micropatterned substrate to study schwann cell phenotypes

Source: Xu, Z., Orkwis, et al. <a href="https://app.jove.com/v/61496">Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification.</a> J. Vis. Exp. (2020).
The video describes the preparation of a micropatterned substrate to study cell phenotype specification. A protein-coated micropatterned PDMS stamp is used to transfer the micropattern on a PDMS-coated coverslip. The coverslip is then treated with a surfactant to hinder cell adhesion at the unwanted surfaces. Cells are then added to settle in the pattern and incubated for further analysis.

Preparation of a Micropatterned Substrate to Study Schwann Cell Phenotypes

1. Tunable cell culture substrate preparation and characterization

	Substrate preparation&#8203;
	
		Mix the Polydimethylsiloxane (PDMS) base elastomer ...

Take a silicon wafer with micropatterns of the desired geometry to grow cells in a defined shape.
Pour a polymer mixture on it and allow it to solidify.
Cut a square stamp of the polymer.
Coat the stamp&#39;s patterned surface with a protein layer, which facilitates cell attachment.
Using an air stream, remove unbounded proteins.
Take a polymer-coated coverslip.
Place the patterned side of the stamp on it.
Press the stamp to transfer the pattern. Remove the stamp.
Transfer the coverslip into a well containing a surfactant to coat the bare surfaces. This increases surface hydrophobicity, preventing unwanted cell adhesion.
Remove the surfactant and wash with a phosphate buffer.
Add the Schwann cells and allow them to attach to the micropattern.
Wash with a buffer to remove unattached cells.
Add culture medium and incubate.
As the cells grow along the surface, they take the micropattern shape, enabling a study of Schwann cell phenotype.

preparation-micropatterned-substrate-to-study-schwann-cell

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

23 Aufrufe. Quelle: Xu, Z., Orkwis, et al. Vorbereitung von einstellbaren extrazellulären Matrix-Mikroumgebungen zur Bewertung der Spezifizierung des Schwann-Zell-Phänotyps. J. Vis. exp. (2020). Das Video beschreibt die Vorbereitung eines mikrostrukturierten Substrats, um die Spezifikation des Zellphänotyps zu untersuchen. Ein proteinbeschichteter PDMS-Stempel mit Mikrostruktur wird verwendet, um das Mikromuster auf ein PDMS-beschichtetes Deckglas zu übertragen. Das Deckglas wird dann mit einem Tensid ...

Serie: Vorbereitung eines mikrostrukturierten Substrats zur Untersuchung von Schwann-Zell-Phänotypen - Konzept

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

Herstellung von Zunge extrazellulären Matrix und Rekonstitution der Zunge Squamous Zelle Krebsgeschwür In-vitro-

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Forschung

JoVE Journal

Krebsforschung

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Synthese von Hydrogelen aus dezellularisiertem Knorpel mit extrazellulärer Matrix

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

Biotechnik

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

Preparation of a Micropatterned Substrate to Study Schwann Cell Phenotypes

Transkript

Preparation of a Micropatterned Substrate to Study Schwann Cell Phenotypes