Name: simple, affordable, and modular patterning of cells using dna
Uploaded: 2021-02-24T13:30:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about Simple, Affordable, and Modular Patterning of Cells using DNA at JoVE.com

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.

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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 ...

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