Name: microfluidic genipin deposition technique for extended culture of micropatterned vascular muscular thin films
Uploaded: 2015-06-26T14:00:00
Description: Watch this Scientific Journal Video about Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films at JoVE.com

We acknowledge financial support from the American Heart Association Scientist Development Grant, 13SDG14670062 (PWA) and the University of Minnesota Doctoral Dissertation Fellowship (ESH). We also acknowledge the microfabrication resources of the Minnesota Nano Center (MNC) and the image processing resources of the University Imaging Centers (UIC), both at the University of Minnesota. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRS program.

Note: The goal of this protocol is to construct and utilize a vascular muscular thin film (vMTF) with the structure shown in Figure 1 to assess contractility during extended culture of vascular smooth muscle cells (VSMCs) on PDMS substrates. To prolong VSMC viability, we utilize the crosslinker compound genipin. The substrates for these vMTFs are designed to analyze tissue contractility as developed by Grosberg et al.8 Other vMTF methods5 may also be used, with subtle chang...

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Here, we present a protocol that builds upon previously developed vMTF technology, allowing extended experiment times more typical of chronic vascular disease pathways1,23,24. To accomplish this, we micropattern genipin, which has previously been shown to provide long-term functionalization of PDMS substrates11, using a microfluidic deposition technique to yield engineered arterial lamellae with improved vascular tissue viability for use in MTF contractility experiments. McCain et al. devel...

Vascular diseases, such as cerebral vasospasm1,2, hypertension3, and atherosclerosis4, develop slowly, are typically chronic in nature, and involve dysfunctional force-generation by vascular smooth muscle cells (VSMCs). We aim to study these slow-progressing vascular dysfunctions using in vitro methods with finer control of experimental conditions than in in vivo models. We have previously developed vascular muscular thin films (vMTFs) for measuring functional contractility of in vitro engineered cardiovascular tissues5, but this method has been limited to relatively short-term studies. Here, we present a substrate modification technique that expands our previous vMTF technique for long-term measurements.
While the endothelium is also critical in overall vascular function, engineered arterial lamellae provide a useful model system for assessing changes in vascular contractility during disease progression. To engineer a functional vascular disease tissue model, both the structure and function of the arterial lamella, the basic contractile unit of the vessel, must be recapitulated with high fidelity. Arterial lamellae are concentric, circumferentially-aligned sheets of contractile VSMCs separated by sheets of elastin6. Microcontact printing of extracellular matrix (ECM) proteins onto polydimethylsiloxane (PDMS) substrates has been previously used to provide guidance cues for tissue organization to mimic aligned cardiovascular tissue5,7-10. However, tissues patterned using microcontact printing can lose integrity after 3-4 days in culture, limiting their applicability in chronic studies. This protocol provides a solution to this issue by replacing previous microcontact printing techniques with a new microfluidic deposition technique.
Genchi et al. modified PDMS substrates with genipin and found prolonged viability of myocytes up to one month in culture11. Here, we use a similar approach to extend culture of patterned vascular smooth muscle cells on PDMS. Genipin, a natural hydrolytic derivative of the gardenia fruit, is a desirable candidate for substrate modification due to its relatively low toxicity compared to similar crosslinking agents and its increasing use as a biomaterial in the fields of tissue repair12,13 and ECM modification14,15. In this protocol, fibronectin is utilized as a cell guidance cue, as in previous microcontact printing methods; however, genipin is deposited onto PDMS substrates prior to fibronectin patterning. Thus, as cells degrade the patterned matrix, newly synthesized ECM from attached VSMCs can bind to the genipin-coated PDMS substrate.
This protocol utilizes a microfluidic delivery device for two-step genipin and ECM deposition. The design of the microfluidic device mimics microcontact printing patterns used for engineered arterial lamellae in previous studies16. Thus, we expect this protocol to yield arterial lamellae mimics that successfully recapitulate the highly-aligned in vivo structure and contractile function of arterial lamellae. We also evaluate tissue contractility to confirm that genipin is a suitable substrate modification compound for long-term in vitro vascular disease models.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Coverslip staining rack</td>
			<td>Electron Microscopy Sciences</td>
			<td><a href="http://www.emsdiasum.com/">www.emsdiasum.com/</a></td>
			<td>72239-04</td>
			<td>Alternative coverslip rack may be used</td>
		</tr>
		<tr>
			<td>Microscope cover glass - 25 mm</td>
			<td>Fisher Scientific, Inc.</td>
			<td><a href="http://www.fishersci.com/">www.fishersci.com</a></td>
			<td>12-545-102</td>
			<td>Alternative brand and size may be used; Microscope slides may also be substituted as substrate base</td>
		</tr>
		<tr>
			<td>Poly(N-iso-propylacrylamide) (PIPAAm)</td>
			<td>Polysciences, Inc.</td>
			<td><a href="http://www.polysciences.com/">www.polysciences.com/</a></td>
			<td>#21458</td>
			<td>Sigma-Aldrich makes an alternate compound, but we have not tested it for use with this protocol; Compound gives strong odor, use proper ventilation</td>
		</tr>
		<tr>
			<td>1-butanol</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td align="right">360465</td>
			<td>Hazard: flammable (store stock solution in flammable cabinet); flash point is 37 &#176;C, avoid heating; alternative product may be used</td>
		</tr>
		<tr>
			<td>Spincoater</td>
			<td>Specialty Coating Systems, Inc.</td>
			<td><a href="http://www.scscoatings.com/">www.scscoatings.com</a></td>
			<td></td>
			<td>SCS G3P8 Model; Alternative brand and/or model may be used</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Ellsworth Adhesives (Dow Corning)</td>
			<td><a href="http://www.ellsworth.com/">www.ellsworth.com</a></td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>Alternative distributor may be used</td>
		</tr>
		<tr>
			<td>Fluorescent microbeads</td>
			<td>Polysciences, Inc.</td>
			<td><a href="http://www.polysciences.com/">www.polysciences.com/</a></td>
			<td align="right">17151</td>
			<td>Alternative brand and/or larger size may be used</td>
		</tr>
		<tr>
			<td>Silicon wafers</td>
			<td>Wafer World, Inc.</td>
			<td><a href="http://www.waferworld.com/">www.waferworld.com</a></td>
			<td align="right">2398</td>
			<td>Alternative brand and/or size may be used</td>
		</tr>
		<tr>
			<td>Photoresist&#160;</td>
			<td>MicroChem Corp.</td>
			<td><a href="http://www.microchem.com/">www.microchem.com</a></td>
			<td></td>
			<td>SU-8 3025 allows 20-25-&#181;m feature height</td>
		</tr>
		<tr>
			<td>Contact mask aligner</td>
			<td>Suss MicroTec</td>
			<td><a href="http://www.suss.com/">www.suss.com</a></td>
			<td></td>
			<td>MA6 contact mask aligner; alternative brand and/or model may be used for wafer exposure</td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>MicroChem Corp.</td>
			<td><a href="http://www.microchem.com/">www.microchem.com</a></td>
			<td></td>
			<td>SU-8 Developer; Hazard: flammable</td>
		</tr>
		<tr>
			<td>Tridecafluro-trichlorosilane</td>
			<td>UCT Specialties, Inc.</td>
			<td><a href="http://www.unitedchem.com/">www.unitedchem.com</a></td>
			<td>T2492</td>
			<td>Silane for non-stick coating of patterned silicon wafers (CAUTION: Tridecafluro-trichlorosilane is a flammable and corrosive liquid. Proper personal protective equipment and local exhaust is necessary for use. )</td>
		</tr>
		<tr>
			<td>Surgical biopsy punch</td>
			<td>Integra LifeSciences Corp.</td>
			<td><a href="http://www.miltex.com/">www.miltex.com</a></td>
			<td>33-31AA-P/25</td>
			<td>Alternative brand and/or size may be used</td>
		</tr>
		<tr>
			<td>Genipin</td>
			<td>Cayman Chemical</td>
			<td><a href="http://www.caymanchem.com/">www.caymanchem.com</a></td>
			<td align="right">10010622</td>
			<td>Sigma-Aldrich (G4796-25MG) makes an alternate compound, but we have not tested it for use with this protocol</td>
		</tr>
		<tr>
			<td>1X phosphate buffered saline</td>
			<td>Mediatech, Inc.</td>
			<td><a href="http://www.cellgro.com/">www.cellgro.com</a></td>
			<td>21-031-CV</td>
			<td>Alternative brand may be used</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning, Inc.</td>
			<td><a href="http://www.corning.com/">www.corning.com</a></td>
			<td align="right">356008</td>
			<td>Sigma-Aldrich (F1056) makes an alternate compound, but we have not tested it for use with this protocol</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies, Inc.</td>
			<td><a href="http://www.lifetechnologies.com/">www.lifetechnologies.com</a></td>
			<td>15140-122</td>
			<td>Alternative brand and/or size may be used, as long as concentration is the same</td>
		</tr>
		<tr>
			<td>Umbillical artery smooth muscle cells</td>
			<td>Lonza</td>
			<td><a href="http://www.lonza.com/">www.lonza.com</a></td>
			<td>CC-2579</td>
			<td>Alternative cell types may be used for alternative applications. Media should be modified accordingly</td>
		</tr>
		<tr>
			<td>Tyrode&#39;s solution components</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td>various</td>
			<td>Alternative brand may be used for mixing solution</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Zeiss</td>
			<td><a href="http://www.zeiss.com/">www.zeiss.com</a></td>
			<td>4350020000000000</td>
			<td>SteREOLumar V12; Alternative brand/type of stereomicroscope may be used</td>
		</tr>
		<tr>
			<td>Temperature-controlled platform</td>
			<td>Warner Instruments</td>
			<td><a href="http://www.warneronline.com/">www.warneronline.com</a></td>
			<td>641659; 640352; 641922</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelin-1</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td>E7764-50UG</td>
			<td>Alternative amount may be purchased, as long as treatment concentration is maintained</td>
		</tr>
		<tr>
			<td>HA-1077</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td>H139-10MG</td>
			<td>Alternative amount may be purchased, as long as treatment concentration is maintained</td>
		</tr>
	</tbody>
</table>

microfluidic genipin deposition technique for extended culture of micropatterned vascular muscular thin films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

The primary goal of this work was to extend the viability of micropatterned VSMCs on hydrophobic PDMS substrates. This was accomplished by incorporating a microfluidic delivery system to deposit patterned genipin and fibronectin on PDMS (Figure 1). Deposition of ECM proteins using microfluidic delivery yielded high fidelity transfer of the channel pattern with bare PDMS between lines of genipin and fibronectin (Figure 1D). The attached cells (Figure 1E) form confluent mo...

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Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

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Research

JoVE Journal

Bioengineering

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...