Name: fabricating complex culture substrates using robotic microcontact printing (r-&#181;cp) and sequential nucleophilic substitution
Uploaded: 2014-10-31T15:00:00
Description: Watch this Scientific Journal Video about Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R-ÂµCP and Sequential Nucleophilic Substitution at JoVE.com

Funding for this work, GTK, TK, and JDM were provided by the Wisconsin Institute for Discovery and the Wisconsin Alumni Research Foundation.

1. Generating Elastomeric Stamps
<ol style="margin-left: 40px;">
	<li>To generate the PDMS stamp&#8217;s silicon masters, design the photomask&#8217;s feature patterns using computer-aided design software.
	<ol>
		<li>Design the first pattern as a 20 x 20 array of annuli with 300 &#956;m inner diameter (ID) and 600 &#956;m OD with 1,200 &#956;m center-to-center spacing.</li>
		<li>Design the second pattern as a 20 x 20 array of annuli with 600 &#956;m ID and 900 &#956;m OD with 1,200 &#956;m center-to-center spacing.</...

<ol>
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M., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Fabrication+of+Antibody+Microarrays+on+Nonfouling+Polymer+Brushes+with+Femtomolar+Sensitivity+for+Protein+Analytes+in+Serum+and+Blood.">Simple Fabrication of Antibody Microarrays on Nonfouling Polymer Brushes with Femtomolar Sensitivity for Protein Analytes in Serum and Blood.</a> Advanced Materials. 21 (19), 1968-1971 (2009).</li><li>Hucknall, A., Rangarajan, S., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Pursuit+of+Zero:+Polymer+Brushes+that+Resist+the+Adsorption+of+Proteins.">In Pursuit of Zero: Polymer Brushes that Resist the Adsorption of Proteins.</a> Advanced Materials. 21 (23), 2441-2446 (2009).</li><li>Rozkiewicz, D. I., Ja&#324;czewski, D., Verboom, W., Ravoo, B. J., Reinhoudt, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click&#8221;+Chemistry+by+Microcontact+Printing.">Click&#8221; Chemistry by Microcontact Printing.</a> Angewandte Chemie International Edition. 45 (32), 5292-5296 (2006).</li><li>Jewett, J. C., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cu-free+click+cycloaddition+reactions+in+chemical+biology.">Cu-free click cycloaddition reactions in chemical biology.</a> Chemical Society Reviews. 39 (4), 1272-1279 (2010).</li><li>Sha, J., Lippmann, E. S., McNulty, J., Ma, Y., Ashton, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+Nucleophilic+Substitutions+Permit+Orthogonal+Click+Functionalization+of+Multicomponent+PEG+Brushes.">Sequential Nucleophilic Substitutions Permit Orthogonal Click Functionalization of Multicomponent PEG Brushes.</a> Biomacromolecules. 14 (9), 3294-3303 (2013).</li><li>Tugulu, S., Silacci, P., Stergiopulos, N., Klok, H. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGD&#8212;Functionalized+polymer+brushes+as+substrates+for+the+integrin+specific+adhesion+of+human+umbilical+vein+endothelial+cells.">RGD&#8212;Functionalized polymer brushes as substrates for the integrin specific adhesion of human umbilical vein endothelial cells.</a> Biomaterials. 28 (16), 2536-2546 (2007).</li><li>Ashton, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Screening+of+Gene+Function+in+Stem+Cells+Using+Clonal+Microarrays.">High-Throughput Screening of Gene Function in Stem Cells Using Clonal Microarrays.</a> Stem Cells. 25 (11), 2928-2935 (2007).</li><li>Koepsel, J. T., Murphy, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+Self-Assembled+Monolayers:+Efficient,+Chemically+Defined+Tools+for+Cell+Biology.">Patterned Self-Assembled Monolayers: Efficient, Chemically Defined Tools for Cell Biology.</a> ChemBioChem. 13 (12), 1717-1724 (2012).</li><li>Mrksich, M., Dike, L. E., Tien, J., Ingber, D. E., Whitesides, G. 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C., Cabrera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+instrument+for+automated+microcontact+printing+with+stamp+load+adjustment.">A new instrument for automated microcontact printing with stamp load adjustment.</a> Review of Scientific Instruments. 79 (6), (2008).</li><li>Trinkle, C. A., Lee, L. 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P., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-Initiated+Atom+Transfer+Radical+Polymerization+of+Oligo(ethylene+glycol)+Methyl+Methacrylate+from+a+Mixed+Self-Assembled+Monolayer+on+Gold.">Surface-Initiated Atom Transfer Radical Polymerization of Oligo(ethylene glycol) Methyl Methacrylate from a Mixed Self-Assembled Monolayer on Gold.</a> Advanced Functional Materials. 16 (5), 640-648 (2006).</li><li>Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stem-cell+niche+as+an+entity+of+action.">The stem-cell niche as an entity of action.</a> Nature. 441 (7097), (2006).</li><li>Codelli, J. A., Baskin, J. M., Agard, N. J., Bertozzi, C. 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Ideal substrates for tissue engineering would be bioinspired and thereby recapitulate the spatial distribution of critical bioactive ligands found within the native tissues. They would also possess dynamic properties that enable temporal adjustments of the ligands and the spatial patterns in which they are presented to permit directed tissue morphogenesis and spatially restricted induction of cell fate. Fabrication of such substrates requires the immobilization of multiple biochemical cues in complex and highly ordered o...

The ability of PEG-grafted surfaces to display covalently bound biochemical ligands while simultaneously maintaining inherent non-fouling properties make them an ideal choice for engineering custom microscale environments on culture substrates1,2,3. The biospecific interactions mediated by ligand conjugated PEG brushes enables reductionistic analysis of the effects of biochemical cues found within complex in vivo tissue microenvironments on individual cell phenotypes. Furthermore, bio-orthogonal &#8220;click&#8221; chemistries can be used to facilitate directional immobilization of ligands so that they are presented in native conformations4-6. Thus, microscale spatial patterning of PEG brushes is a versatile tool to create designer in vitro niches to investigate cell signaling induced by immobilized biochemical cues6,7.
A common method for generating spatial patterns of biochemical cues entails microcontact printing (&#956;CP) gold-coated substrates with patterns of PEG conjugated alkanethiols. Then, the micropatterned self-assembled monolayers (SAMs) of PEG-ylated alkanethiols restricts physical adsorption of biochemical molecules, e.g., proteins, only to non-patterned regions of the substrate8,9. However, the SAMs generated by this technique are sensitive to oxidation in long term cell culture media. Thus, &#956;CP&#8217;d alkanethiol SAMs are often further grafted with PEG polymer brushes using surface-initiated atom transfer radical polymerization (SI-ATRP) to increase the region&#8217;s non-fouling stability10. Specifically, &#956;CP of the alkanethiol polymerization initiator, &#969;-meraptoundecyl bromoisobutyrate, on gold-coated surfaces followed by SI-ATRP of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) monomers generates surfaces with micropatterned long-term, stable, and non-fouling PEG brushes. Moreover, these are capable of being further modified to present diverse chemical moieties11.
Taking advantage of this property, Sha et. al. developed a method to engineer culture substrates with multicomponent PEGMEMA brushes presenting orthogonal &#8220;click&#8221; chemistries. In this method, they use a series of &#956;CP/SI-ATRP steps interspersed with sequential sodium azide, ethanolamine, and propargylamine nucleophilic substitutions to create culture substrates presenting microscale patterns of multiple immobilized ligands6. While the potential of using such chemistries in conjunction with manual &#956;CP to engineer novel culture substrates is immense, it is limited by the precision and accuracy with which multiple &#956;CP steps can be aligned on a single substrate. A high level of precision and accuracy would be required to reproducibly manufacture complex in vitro niches using these versatile techniques.
To address this limitation, several automated and semi-automated &#956;CP systems have been generated. Chakra et. al. developed a &#956;CP system in which custom stamps are placed on a rail system and brought into conformal contact with gold-coated slides using a computer-controlled pneumatic actuator. However, this method requires the precise fabrication of custom stamp designs and reports a 10 &#956;m precision with no report of the accuracy achieved when performing multiple &#956;CP steps12. More recently, a method utilizing an integrated kinematic coupling system reported precision below 1 &#956;m using a single pattern, but were unable to accurately align multiple patterns due to a lack of precise control of stamp features from mold to mold13. Additionally, both of the previous methods require the substrate to remain fixed between patterning steps, thereby significantly limiting the diversity of surface modification chemistries that can be utilized. Here, we describe an automated R-&#956;CP system capable of accurate and precise alignment of multiple &#956;CP steps while allowing maximal flexibility in stamp design and fabrication. Furthermore, the patterned substrates can be repeatedly removed from the system between stampings, thereby permitting the use of diverse substrate modification chemistries, including sequential nucleophilic substitutions. Substrates engineered using such chemistries have been used for cell culture previously by both us6,14 and others7. Thus, we have merged R-&#956;CP and sequential nucleophilic substitution reactions to develop a method for scalable manufacture of culture substrates with complex and micropatterned biochemical cues.

<table border="0" cellpadding="0" cellspacing="0" width="740">
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			<td height="21" style="height:21px;width:185px;">Name</td>
			<td style="width:185px;">Company</td>
			<td style="width:185px;">Catalog Number</td>
			<td style="width:185px;">Comments</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">SCARA&#160;</td>
			<td style="width:185px;">Epson</td>
			<td style="width:185px;">LS3-401ST</td>
			<td style="width:185px;">Higher end models with increased precision are available if desired.&#160;</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:185px;">(TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL)TRICHLOROSILANE</td>
			<td style="width:185px;">Gelest</td>
			<td style="width:185px;">SIT8174.0</td>
			<td style="width:185px;">CAUTION, Should only be handled in a chemical fume hood. When silanizing wafers no one should enter the hood until all silane has been evaporated.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:185px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:185px;">Ellsworth Adhesive Co</td>
			<td style="width:185px;">NC9020938</td>
			<td style="width:185px;">Thouroughly degass solutions via vacuum exposure before use. Alternative kits such as Kit 182 are acceptable.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:185px;">24mm X 50 mm #1 Cover Glass Slides</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">48393106</td>
			<td style="width:185px;">These can be purchased from a number of suppliers with varying dimensions to suit need.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">CHA-600 Telemark Electron Beam Evaporator</td>
			<td style="width:185px;">Telemark</td>
			<td style="width:185px;">SEC-600-RAP</td>
			<td style="width:185px;">Requries specialized training.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">EPSON LS3 SCARA</td>
			<td style="width:185px;">EPSON</td>
			<td style="width:185px;">LS3-401ST</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:185px;">&#969;-mertcaptoundecyl bromoisobutyrate</td>
			<td style="width:185px;">Prochimia</td>
			<td style="width:185px;">FT 015-m11-0.2</td>
			<td style="width:185px;">Store at -20&#176;C. Other ATRP initiators may be used as this R-&#956;CP platform is applicable to all micropatterning modalities.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Schlenk Tube Flask 50 mL</td>
			<td style="width:185px;">Synthware</td>
			<td style="width:185px;">60003-078</td>
			<td style="width:185px;">Requires rubber stoppers with diaphram.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">Poly(ethylene glycol) methyl ether methacrylate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">447943</td>
			<td style="width:185px;">Shipped containing MEHQ and BHT free readical inhibitors.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Methanol (Certified ACS)</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">A412-4</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Copper(II) Bromide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">437867</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">2&#39;,2-Bipyridine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">D216305</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Sodium L-Ascorbate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">A4034</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">20mL Borosilicate Glass Scintillation Vials</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:185px;">03-340-4E</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Sodium Azide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">S2002</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">N,N-dimethyformamide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">227056</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Ethanolamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">398136</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Triethylamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">T0886</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Dimethylsulfoxide</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">276855</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Propargylamine</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">P50900</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">200 Proof Ethanol</td>
			<td style="width:185px;">University of Wisconsin Material Distribution Services</td>
			<td style="width:185px;">2292</td>
			<td style="width:185px;">CAUTION, only handle in chemical fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Azide-PEG3-Biotin</td>
			<td style="width:185px;">ClickChemistryTools</td>
			<td style="width:185px;">AZ104-100</td>
			<td style="width:185px;">Solubilized in DMF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Copper(II) Sulfate</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">C1297</td>
			<td style="width:185px;">CAUTION, limit exposure with surgical mask.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">678937</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">L-Ascorbic Acid</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">A7506</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">Phosphate Buffer Saline</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:185px;">14190144</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:185px;">Donkey Serum</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:185px;">D9663</td>
			<td style="width:185px;">Donkey serum contaminated items are considered bio-hazardous material and should be disposed of accordingly. Various other compounds (e.g. BSA) are available and serve this purpose.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:185px;">12-Well Polystyrene Plate</td>
			<td style="width:185px;">Thermo Scientifit - NUNC</td>
			<td style="width:185px;">07-200-81</td>
			<td style="width:185px;">Plates can be purchased form a number of suppliers with varying dimensions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:185px;">DBCO-PEG4-Biotin</td>
			<td style="width:185px;">Clickchemistytools</td>
			<td style="width:185px;">A105P4-10</td>
			<td style="width:185px;">Solubilized in DMF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Streptavidin, Alexa Fluor 488 Conjugate</td>
			<td style="width:185px;">Life Technologies</td>
			<td style="width:185px;">S-11223</td>
			<td style="width:185px;">Solubilized in PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:185px;">Streptavidin, Alexa Fluor 546 conjugate</td>
			<td style="width:185px;">Life Technologies</td>
			<td style="width:185px;">S-11225</td>
			<td style="width:185px;">Solubilized in PBS</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:185px;">Nikon A1-R Confocal Microscope</td>
			<td style="width:185px;">Nikon</td>
			<td style="width:185px;">Nikon Eclipse Ti, A1R</td>
			<td style="width:185px;">An epifluorescent microscope is sufficient to image functionalized micropatterned substrates.</td>
		</tr>
	</tbody>
</table>

fabricating complex culture substrates using robotic microcontact printing (r-&#181;cp) and sequential nucleophilic substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

The use of manual alignment &#956;CP techniques to engineer culture substrates with arrays of PEG-grafted brushes functionalized with orthogonal &#8220;click&#8221; chemistries has been demonstrated in previous work6. However, this offers minimal control of pattern orientation and often results in overlap of functionalized areas. Here, a novel R-&#956;CP system is used to overcome this limitation, and its ability to accurately pattern an array of PEG brush annuli with 300 &#956;m ID and 600 &#956;m OD presenti...

Watch this Scientific Journal Video about Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R-ÂµCP and Sequential Nucleophilic Substitution at JoVE.com

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &amp;#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

fabricating-complex-culture-substrates-using-robotic-microcontact

Research

JoVE Journal

Bioengineering

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Four-Dimensional CT Analysis Using Sequential 3D-3D Registration

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data remains difficult because segmentation in all volumetric frames is time-consuming. We aimed to analyze joint kinematics using a sequential 3D-3D registration technique to provide the kinematics of the moving bone with respect to the fixed bone semiautomatically using 4DCT DICOM data and existing software. Surface data of the source bones are reconstructed from 3DCT. The trimmed surface data are respectively matched with surface data from the first frame in 4DCT. These trimmed surfaces are sequentially matched until the last frame. These processes provide positional information for target bones in all frames of the 4DCT. Once the coordinate systems of the target bones are decided, translation and rotation angles between any two bones can be calculated. This 4DCT analysis offers advantages in kinematic analyses of complex structures such as carpal or tarsal bones. However, fast or large-scale motions cannot be traced because of motion artifacts.

We analyzed joint kinematics from four-dimensional computed tomography data. The sequential 3D-3D registration method semiautomatically provides the kinematics of the moving bone with respect to the subject bone from four-dimensional computed tomography data.

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...