The authors would like to thank Jeremy Garcia for testing this protocol and Bhushan Kharbikar for providing training on the equipment at the UCSF Biomedical Micro and Nanotechnology Core. This research was supported in part by grants from the Department of Defense Breast Cancer Research Program (W81XWH-10-1-1023 and W81XWH-13-1-0221), NIH (U01CA199315, DP2 HD080351-01, 1R01CA190843-01, 1R21EB019181-01A, and 1R21CA182375-01A1), the NSF (MCB1330864), and the UCSF Center for Cellular Construction (DBI-1548297), an NSF Science and Technology Center. O.J.S was funded by an NSF Graduate Research Fellowship, a Siebel scholarship, and a P.E.O. Scholarship. Z.J.G and A.R.A. are Chan-Zuckerberg BioHub Investigators.

<ol>
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Z.J.G. is an advisor and equity holder in Provenance Biosciences.

In this article, we present a detailed protocol for high-resolution patterning of cells in 2D and 3D for in vitro cell culture experiments. Unlike previously published versions of this method, the protocol presented here focuses on usability: it does not require highly specialized equipment and all reagents can be purchased from vendors instead of requiring custom synthesis. Unlike other cell micropatterning methods, this method is rapid and cell-type agnostic: it does not require specific adhesion to extracellular matrix proteins15. Cells patterned by CMO-DPAC can be embedded within an extracellular matrix such as Matrigel or collagen, resulting in 3D cultures with much higher spatial resolution than is currently possible with extrusion printing-based methods22. CMO-DPAC can be used to create hundreds to thousands of microscopic features per slide, allowing for many replicates to be performed at the same time.
One of the most important parameters in the success of this protocol is the density of cells added to the flow cells on top of the patterned slide. Ideally, the density should be at least 25 million cells/mL. When loaded into the flow cells, this density of cells results in a nearly close-packed monolayer of cells above the pattern (Supplemental Figure 2B). These high cell densities maximize the probability that a cell will settle directly on top of a DNA spot and adhere. Reducing the cell density will decrease the overall patterning efficiency. Another critical step in this protocol is thoroughly re-suspending the cells in PBS or serum-free media before adding the CMO solution. The CMOs partition very rapidly into cell membranes and adding the CMO solution directly to a cell pellet will result in heterogeneous labeling of cells. After adding the CMO solution to the cell suspension, it is important to mix thoroughly by pipetting so that the cells are uniformly labeled with the CMOs. After the incubations, it is necessary to thoroughly wash out the excess CMOs through multiple centrifugation and wash steps. Excess free CMO present in the cell suspension will bind to the patterned amine-modified DNA on the glass slide, blocking hybridization and adhesion of the CMO-modified cells in suspension. Time is also a key consideration for this protocol. It is important to work as quickly as possible when using CMOs and to keep the cells on ice in order to minimize internalization of the CMOs and maximize cell viability. Flow cytometry experiments have shown that CMOs do not persist as long on the cell surface as LMOs, with 25% loss of CMO complexes over two hours of incubation on ice36. Furthermore, the viability of cells will decrease as the cell handling time increases. Viability can be maximized by working quickly, keeping cells on ice, using ice-cold reagents, and using serum-free media to provide some nutrients. 
Although CMO-DPAC can be a powerful way of studying cell biology by patterning cells with high precision, it does have its limitations. CMO-DPAC experiments can be challenging, particularly as the experimental complexity is added with multiple cell types, layers, or 3D cell culture (Supplemental File 1). Experimental failures can be common when starting this protocol, as described in Table 1. Therefore, we recommend that users institute quality control checks (confirming that DNA is present on the slide, confirming that cells are sufficiently labeled with DNA (Step 8), confirming that excess cells have been thoroughly washed away, etc.) to make sure that the experiment succeeds and to identify steps that may require further optimization. We hope that the information provided in this manuscript and its supplemental files will help facilitate any required troubleshooting.
Cholesterol is a bioactive molecule whose internalization may influence cell metabolism, gene expression, and membrane fluidity37,38. A previous study compared the effects on gene expression of CMO- and LMO-labeled cells using single cell RNA sequencing. CMO-labeled HEK cells had altered gene expression compared to unlabeled and LMO-labeled cells36. Labeling cells with CMOs resulted in the differential expression (&#62; 1.5-fold) of eight genes relative to unlabeled controls, including AP2B1, which has been linked to cholesterol and sphingolipid transport (GeneCards), and MALAT1, a long non-coding RNA that regulates cholesterol accumulation39. While minor, these transcriptional responses may nevertheless be of concern if the experiment in question is studying metabolism, membrane dynamics, or other cholesterol-associated pathways in cells.
This protocol is flexible and can be adjusted to meet the needs of each experiment. Because the CMO inserts itself into the lipid membrane instead of using any specific receptor, the method is cell type agnostic (HUVECs, MCF10As, HEKs, and MDCKs have been demonstrated here). Although cholesterol is a different hydrophobic anchor than our previously published LMOs, we have thus far found them to behave similarly. Thus, we would expect the CMOs to work with any of the wide variety of cell types that we have previously published with LMOs, including but not limited to neural stem cells, fibroblasts, peripheral blood mononuclear cells, tumor cells, and primary mammary epithelial cells6,23,27,29,36. CMO labeling does not stimulate TLR9, suggesting that the protocol is compatible with immune cells. Membrane incorporation of the CMO is a function of total cell size and the degree of negative charge in the cell glycocalyx35. Thus, we have included a protocol (Step 8) for testing the extent of membrane incorporation that is amenable to rapid optimization. The specific features of each cell pattern will inevitably vary based on the experimental design (see Supplemental File 1 for more guidance). Although the photopatterning protocol described above for patterning the DNA is recommended, any method of spatially confining droplets of amine-DNA solution should work, such as the use of high-resolution droplet printers. The pattern resolution and minimum feature spacing will vary based on the method used. It is also theoretically possible to combine the DNA-photopatterning sections of this protocol with other methods that have been used to label cells with DNA, such as with DNA hybridized to membrane-expressed zinc fingers40, using NHS-conjugated DNA41, and reacting azido sialic acid residues on the cell surface with phosphine-conjugated DNA42. CMO-DPAC can be applied to a variety of experiments that require tight control over cell-cell spacing, including studies of the interactions between pairs of cells, co-culture experiments looking at the transfer of signals from &#34;sender&#34; cells to &#34;receiver&#34; cells, and investigations of the effect of nearby extracellular cues on stem cell differentiation6,29. The method can also be used to create microtissues that can be used to study cell migration in three dimensions, the self-organization of cells into tissues23,27, and the dynamic interplay between cells and the ECM27. We hope that this protocol will provide researchers with an accessible platform to explore new applications of high-resolution DNA-based cell patterning in their own labs.

The positioning of cells with respect to one another in a tissue is an important feature of the microenvironment1,2,3,4. Techniques used to pattern live cells into spatially controlled arrangements are valuable experimental tools for studying differentiation4,5,6,7,8, cell motility9, morphogenesis10,11,12, metabolism13, and cell-cell interactions7,14. A variety of methods exist for patterning cells, each with their own advantages and drawbacks3,4. Methods that create adhesive islands of extracellular matrix (ECM) proteins, such as microcontact printing and laser-cut stencils, are simple and scalable. However, it is difficult to pattern more than one or two cell types at a time because the adhesive properties of different cell types to different ECM molecules are often similar15,16,17. More complex micropatterns can be created with light-induced molecular adsorption (LIMAP), a technique that uses UV light to ablate PEG-coated regions and allow for subsequent protein adsorption18,19. This process can be repeated to create high-resolution micropatterns with multiple cell types. However, cross-binding of cells to the different protein patches can occur, resulting in poor pattern specificity19. Physical methods such as seeding cells onto micromechanical reconfigurable culture devices can create structured co-cultures with dynamic control, but without the flexibility in pattern design of microcontact printing or LIMAP14,8. Unlike the other techniques, bioprinting can create three-dimensional arrangements of cells within hydrogels20,21. However, bioprinted constructs have much lower resolution than other micropatterning techniques, with an average feature size on the order of hundreds of microns22. An ideal cell patterning method would have high resolution, pattern multiple cell types, use equipment and reagents that are easily accessible, and have the ability to embed successful patterns into a hydrogel for three-dimensional (3D) cell culture. In this article, we present CMO-DPAC, a cell micropatterning technique that uses the flexibility and speed of DNA hybridization to target cell adhesion to a substrate. This method has been adapted from our previous protocols23, 24 to make it more affordable, modular, and accessible. Using the current protocol, any lab should be able to set up a fully functional system without any specialized equipment or expertise.
DNA Programmed Assembly of Cells (DPAC) is a powerful tissue engineering technique that patterns cells at single-cell resolution with precise control over cell-cell spacing and tissue geometry. In DPAC, cell membranes are decorated with DNA oligonucleotides (oligos) using two lipid-modified oligos designed to hybridize on the cell membrane. Because the oligos are conjugated to hydrophobic lipids, they rapidly partition to the cell membrane25 where they hybridize, increasing the net hydrophobicity of the non-covalently bound molecules, and thereby enhancing their lifetime at the cell surface26. The oligos are presented on the cell surface in a manner where they can hybridize with complementary oligos on other cells or DNA-functionalized glass slides to create defined 2D or 3D cell patterns with prescribed composition, cell-cell spacing, and geometry23, 24. The patterned microtissues can be cleaved off of the surface enzymatically and embedded into a hydrogel for prolonged 3D culture. When used in combination with primary cells or stem cells, the resulting collections of cells can undergo morphogenesis and form into organoids23,27,28. DPAC has been applied to investigate the dynamics of adult neural stem cell fate in response to competing signals6,29, to study self-organization of mammary epithelial cells23,28, and to generate &#34;tissue origami&#34; through mesenchymal condensation27.
DPAC allows for the precise placement of multiple cell populations and has substantially better resolution than extrusion-based bioprinters (on the order of microns)22,23. In addition, unlike ECM-based patterning methods such as microcontact printing, DPAC does not require differential adhesion of the different cell types to an ECM-coated surface15,23. It is ideal for answering questions about how the composition of a tissue affects its behavior, how cells integrate multiple cellular and microenvironmental cues when making decisions6,29, and how pairs of cells interact with each other. An advantage of this method over other micropatterning methods is that it can be used for 3D cell culture in a single imaging plane, facilitating time-lapse studies of tissue self-organization and organoid morphogenesis23, 27,30.
Despite these advantages, successful implementation of DPAC has required the synthesis of custom oligonucleotide reagents and access to specialized equipment for DNA patterning23,24, limiting widespread adoption. For example, the optimal lipid-modified oligos (LMOs) used in the original protocol must be custom synthesized, modified with&#160;lignoceric acid or palmitic acid, and purified26. This process requires the use of a DNA synthesizer and a high-performance liquid chromatography instrument, as well as the purchasing of the associated reagents such as methylamine, a controlled substance that is subject to both institutional and federal regulations. As an alternative, LMOs can be custom purchased in bulk, but this requires a significant up-front investment in the technology.
To overcome these limitations, we have developed a revised version of DPAC that uses commercially available cholesterol-modified oligos (CMOs) in place of the custom-synthesized LMOs. To further reduce costs and to increase the flexibility of the platform, we have changed to a modular, three-oligo system. Instead of ordering a new cholesterol-modified oligo for each unique cell population, a user of this protocol can instead use the same cholesterol-modified oligos (&#34;Universal Anchor&#34; and &#34;Universal Co-Anchor&#34;) for every cell population and then employ an inexpensive, unmodified oligo (&#34;Adapter Strand&#34;) that hybridizes with both the Universal Anchor and either the amine-functionalized DNA on the surface or the Adapter Strand of another cell type.
Another limitation of the original DPAC protocol was that it created the DNA-patterned slides by using a high-resolution liquid printer (e.g., Nano eNabler, BioForce Nanosciences)23,24. While this instrument boasts extraordinary resolution and low reagent requirements, it is not available to most institutions and has a relatively low printing rate (approximately 1 feature patterned per second). Recently, two photolithographic methods have been developed to pattern DNA features onto surfaces. Viola and colleagues used a polyacrylamide and benzophenone coating that covalently bound single-stranded DNA oligos upon exposure to UV light30. Using this method, they were able to create tissue scaffolds that underwent large-scale, programmed shape changes as a result of cell contractility and self-organization. Scheideler et al. developed a method that uses UV exposure of a positive photoresist to selectively expose amine-modified DNA oligos to an aldehyde-functionalized slide29. After baking and reductive amination, the amine-modified DNA is covalently bound to the surface. This method was used to investigate the response of adult neural stem cells to spatially presented self-renewal and differentiation cues. This article adapts Scheideler et al.&#39;s protocol to create the DNA patterns that will capture CMO-labeled cells. This photopatterning protocol can be performed without using a clean room. It uses inexpensive and commercially available equipment that is easily deployed on a benchtop or fume hood. The use of inexpensive or DIY (do-it-yourself) photolithography equipment increases accessibility to researchers without access to clean room facilities and allows researchers to try the technique without a large investment of time or resources31,32. However, better resolution and the alignment of multiple DNA features can be achieved by using the commercial spin coater and mask aligner commonly found in cleanroom facilities.
Here, we describe a method to pattern cells at single-cell resolution using DNA-based adhesion. First, photopatterning with a positive photoresist is used to create high-resolution patterns of amine-modified DNA onto an aldehyde-modified glass substrate. Next, the slide is treated to reduce non-specific cell attachment and PDMS flow cells are created to confine cells over patterned regions. Cells are then labeled with short DNA oligonucleotides that are functionalized with cholesterol and as a result insert into the cell membrane. The cells are then flowed over the DNA micropatterns. Hybridization between the cell-surface DNA and the DNA on the glass surface results in specific adhesion of the cells to the DNA pattern. Non-adherent cells are washed away, revealing the adherent cell pattern. This process can be repeated to pattern multiple cell types or to create multi-layered structures. If desired, the cells can be fully embedded into an ECM for 3D cell culture.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-well Chambered Coverglass w/ non-removable wells</td>
			<td>Thermo Fisher Scientific</td>
			<td>155379</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Adapter with External SM1 Threads and Internal SM3 Thread</td>
			<td>ThorLabs</td>
			<td>SM3A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Aldehyde Functionalized Slides</td>
			<td>Schott</td>
			<td>Nexterion Slide AL</td>
			<td>Store under dry conditions after opening.</td>
		</tr>
		<tr>
			<td>All Plastic Syringes, 1 mL</td>
			<td>Fisher Scientific</td>
			<td>14-817-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine-Modified DNA Oligo</td>
			<td>IDT</td>
			<td>n/a</td>
			<td>See Supplemental File 1 for suggested sequences.</td>
		</tr>
		<tr>
			<td>Aspheric Condenser Lens</td>
			<td>ThorLabs</td>
			<td>ACL7560&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Disc, 6in Diameter X 1/2in Thick&#160;</td>
			<td>Chemglass</td>
			<td>CG-1906-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dishes 60x15 mm style</td>
			<td>Corning</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol-Modified Oligo</td>
			<td>IDT</td>
			<td>n/a</td>
			<td>See Supplemental File 1 for suggested sequences.</td>
		</tr>
		<tr>
			<td>Diamond Scribe</td>
			<td>Excelta</td>
			<td>475B</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Oligonucleotide</td>
			<td>IDT</td>
			<td>n/a</td>
			<td>See Supplemental File 1 for suggested sequences.</td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>278475</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix, Growth Factor Reduced</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene Chloride (Stabilized/Certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>D37-4</td>
			<td></td>
		</tr>
		<tr>
			<td>MF-321 Developer</td>
			<td>Kayaku Advanced Materials</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Microposit S1813 Positive Photoresist</td>
			<td>Kayaku Advanced Materials</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>&#216;3&#34; Adjustable Lens Tube, 0.81&#34; Travel&#160;</td>
			<td>ThorLabs</td>
			<td>SM3V10</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Thermo Scientific</td>
			<td>51-028-112H</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-50 Compact Benchtop Plasma Cleaning System</td>
			<td>Plasma Etch</td>
			<td>PE-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Photomask (custom)</td>
			<td>CAD/Art Services</td>
			<td>n/a</td>
			<td>Minimum feature size guaranteed by CAD/Art Services is 10 microns.</td>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>RCT Basic Hot Plate</td>
			<td>IKA</td>
			<td>3810001</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon Wafer (100 mm)</td>
			<td>University Wafer</td>
			<td>590</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Borohydride, 98%, granules</td>
			<td>Acros Organics</td>
			<td>419471000</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin Coater Kit</td>
			<td>Instras</td>
			<td>SCK-200</td>
			<td>This is a low cost option, but any spin coater that can maintain a speed of 3000 rpm will suffice.</td>
		</tr>
		<tr>
			<td>SU-8 2075</td>
			<td>Microchem</td>
			<td>Y111074 0500L1GL</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>Microchem</td>
			<td>Y020100 4000L1PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow</td>
			<td>2646340</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Needles</td>
			<td>Sigma-Aldrich</td>
			<td>Z192341</td>
			<td></td>
		</tr>
		<tr>
			<td>T-Cube LED Driver, 1200 mA Max Drive Current</td>
			<td>ThorLabs</td>
			<td>LEDD1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Tridecafluoro-1,1,2,2-tetrahydrooctyl dimethylchlorosilane</td>
			<td>Gelest</td>
			<td>SIT8170.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma-Aldrich</td>
			<td>90335</td>
			<td></td>
		</tr>
		<tr>
			<td>Turbo DNase</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM2238</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers Style N7</td>
			<td>VWR</td>
			<td>100488-324</td>
			<td>The curved shape of these tweezers is essential for delicately picking up the PDMS flow cells containing patterned tissues.</td>
		</tr>
		<tr>
			<td>UV LED (365 nm, 190 mW (Min) Mounted LED, 700 mA)</td>
			<td>ThorLabs</td>
			<td>M365L2</td>
			<td></td>
		</tr>
		<tr>
			<td>Wafer Tweezers</td>
			<td>Agar Scientific</td>
			<td>T5063</td>
			<td></td>
		</tr>
		<tr>
			<td>WHEATON Dry-Seal vacuum desiccator</td>
			<td>Millipore Sigma</td>
			<td>W365885</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple, affordable, and modular patterning of cells using dna

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Watch this Scientific Journal Video about Simple, Affordable, and Modular Patterning of Cells using DNA at JoVE.com

Simple, Affordable, and Modular Patterning of Cells using DNA

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

simple-affordable-and-modular-patterning-of-cells-using-dna

Research

JoVE Journal

Bioengineering

4.0K Views.  University of California San Francisco and University of California Berkeley. Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

Video: Simple, Affordable, and Modular Patterning of Cells using DNA

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

High-resolution Patterning Using Two Modes of Electrohydrodynamic Jet: Drop on Demand and Near-field Electrospinning

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning tool. EHD printing uses a fluidic supplier to maintain the extruded meniscus by pushing the ink out of the nozzle tip. The electric field is then used to pull the meniscus down to the substrate to produce high-resolution patterns. Two modes of EHD printing have been used for fine patterning: continuous near-field electrospinning (NFES) and dot-based drop-on-demand (DOD) EHD printing. According to the printing modes, the requirements for the printing equipment and ink viscosity will differ. Even though two different modes can be implemented with a single EHD printer, the realization methods significantly differ in terms of ink, fluidic system, and driving voltage. Consequently, without a proper understanding of the jetting requirements and limitations, it is difficult to obtain the desired results. The purpose of this paper is to present a guideline so that inexperienced researchers can reduce the trial and error efforts to use the EHD jet for their specific research and development purposes. To demonstrate the fine-patterning implementation, we use Ag nanoparticle ink for the conductive patterning in the protocol. In addition, we also present the generalized printing guidelines that can be used for other types of ink for various fine-patterning applications.

Here, we present a protocol to produce high-resolution conductive patterns using electrohydrodynamic (EHD) jet printing. The protocol includes two modes of EHD jet printing: the continuous near-field electrospinning (NFES) and the dot-based drop-on-demand (DOD) EHD printing.

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...