This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.
Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.
The advent of hiPSCs and the subsequent development of protocols for their directed differentiation into different cell types have made it possible to study development and disease at a molecular and patient-specific level, specifically using patient-derived iPSC cardiomyocytes (iPSC-CMs) to model cardiomyopathies1,2. However, a major limitation to studying development and physiology using the iPSC system and other in vitro models is the absence of a structured microenvironment. In situ, cells are subjected to the constraints of the extracellular matrix (ECM), as well as neighboring cells. The particular biochemical composition and stiffness of these microenvironments dictate the spatial distribution of cells as well as factors available for engaging in cell adhesion. This, in turn, influences intracellular signaling pathways, gene expression, and cell fate determination. For example, micropatterned iPSC-CM in an adult-like rod shape has a significantly better contractile ability, calcium flow, mitochondrial organization, electrophysiology, and transverse-tubule formation3. Thus, the properties of the microenvironment are integral in the regulation of cellular functions.
Previous micropatterning techniques heavily relied on photolithography (Figure 1A). In this technique, a layer of photosensitive polymer, or photoresist, is spun on a flat substrate from solution to form a thin film about 1 μm thick. Next, ultraviolet (UV) light is applied onto the photoresist through a mask containing the desired pattern. Exposure to ultraviolet (UV) light chemically alters the photoresist by modifying its solubility in its respective developer solution, transferring the desired pattern from the mask onto the substrate. Many micropatterning methods incorporate photolithography, as it confers nanometer to micrometer-level control over the design of the cell patterns. However, the spinning of the photoresist is highly sensitive to impurities, because the smallest dust particles will disrupt the spreading of the solution into a thin film. Photolithography must therefore be carried out in uncontaminated facilities, which are costly to maintain and require special expertise to utilize. In addition, the chemicals used in photolithography are often toxic to cells and can denature important biomolecules. Thus, photolithography poses significant obstacles to the fabrication of micropatterns for convenient biological applications.
In 1994, Whitesides and colleagues4 overcame some of the challenges associated with photolithography by pioneering a collection of techniques called soft lithography. In soft lithography, a microstructured surface made with polydimethylsiloxane (PDMS), a transparent, rubber-like material, is used to generate a pattern of ECM proteins4. Common soft lithographic techniques include microcontact printing and microfluidic patterning. In microcontact printing, currently the most popular soft lithographic method, a PDMS stamp coated with ECM proteins transfers the material onto a surface at the areas contacted by the stamp (Figure 1B). In microfluidic patterning, microstructures are designed on a PDMS surface such that when the stamp is pressed to a substrate, a network of microchannels, through which fluids can be delivered to desired areas, is created (Figure 1C)5. Soft lithography offers several benefits over photolithography. Once a master wafer is microfabricated, the PDMS stamps can easily be replicated without further employment of clean-room facilities. In addition, the absence of organic solvents in the process of soft lithography allows for utilization of polymeric materials such as polystyrene, typically used in cell culture. Finally, micropatterning using soft lithographic methods is not restricted to flat surfaces. Thus, soft lithography increases the accessibility and functionality of micropattern fabrication over photolithography6. However, soft lithography has significant drawbacks. For example, an initial etching step, using photolithography, is still required to microfabricate the stamp. In addition, micropatterning using a PDMS stamp is subject to variations in the quality of protein transfer onto the substrate6. Avoiding these discrepancies requires optimization and consistency in the pressure applied to the PDMS stamp during protein transfer, otherwise deformation and distortion of the feature sizes of the PDMS molds can occur6. There is also a major concern of repeatedly using the PDMS due to small molecule absorption7.
To avoid using soft photolithography and PDMS stamps, we describe a stencil-based, lithography-free single cell micropatterning method that overcomes many of the obstacles associated with photolithography and soft lithography. In this method, a polyacrylamide hydrogel is used as a substrate for stencil-based ECM protein incorporation, allowing for selective plating of single hiPSC-CMs. This technique is highly compatible with polymeric materials used in classic cell culture conditions. Moreover, with proper cleaning and maintenance, the stencils are reusable and resistant to degradation and protein absorption during the microfabrication process. Finally, the patterning process is technically robust, inexpensive, customizable, and accessible to those with no specialized bioengineering skills. This stencil-based micropatterning technique has been broadly utilized in our recent publications modeling varied cardiomyopathies8,9,10.
1. Fabrication of negative pattern polyimide-based stencils
2. Preparation of sulfo-SANPAH aliquots
NOTE: The protocol is modified from Fischer et al.11.
3. Sterilization of tools
4. Preparation of polyacrylamide hydrogel precursor solution
NOTE: The protocol is modified from Lee et al.12.
5. Preparation of photoinitiator solution
6. Preparation of basement membrane matrix protein solution
NOTE: Basement membrane matrix (Table of Materials) is a temperature-sensitive material. Make sure to work on ice with chilled pipette tips and tubes as well as ice-cold solutions. A new stock of basement membrane matrix should be thawed slowly on ice at 4 °C overnight.
7. Hydrogel fabrication
8. Protein conjugation
9. Cleaning of the used stencils
Fabrication of stencils containing an array of squares or rectangles has been demonstrated (Figure 4A). Following this protocol, we obtained patterned matrix protein islands (Figure 4B and Figure 5A) and cells (Figure 4C). Suboptimal matrix protein solution concentration led to suboptimal patterning (Figure 5B). It is critical to use the front side of the stencil. If the back side of the stencil is used, the size of the matrix protein islands increases due to the direction of the laser cutting (Figure 6A,B). While the width of the rectangular patterns did not significantly change (Figure 6C), the height significantly increased when using the back side of the stencil, leading to a reduction in the intended aspect ratio (Figure 6D,E). We applied stencil-based patterning to silicon elastomer substrates (Figure 7). The patterned cardiomyocytes on silicon elastomer substrates were visualized by immunocytochemistry of cardiac troponin T at low magnification (Figure 7A) and sarcomeric protein α-actinin at high magnification (Figure 7B). The stencil-based patterning could also be applied to pattern two cardiomyocytes side-by-side on hydrogels with different stiffnesses. As a demonstration, we show the cardiomyocyte patterning on physiologically-relevant stiffness (Figure 8A) and pathologically-relevant stiffness (Figure 8B).
Figure 1: Schematic of three gold-standard micropatterning strategies. (A) Photolithography. (B) Microcontact printing. (C) Microfluidic patterning. Please click here to view a larger version of this figure.
Figure 2: Workflow of designing stencils. (A) The computer-assisted design of the stencil. (B) A representative photograph of a laser-cut polyimide stencil. (C) A representative microscopic image of a stencil. The yellow area represents the polyimide stencil, while the light blue area represents an array of laser-cut holes. Please click here to view a larger version of this figure.
Figure 3: Photographs of hydrogel fabrication steps. (A) Polyacrylamide hydrogel fabrication by sandwiching the precursor solution in between two coverslips. (B) UV photo-crosslinking of polyacrylamide hydrogels under the UV bench lamp. The UV lamp is covered with aluminum foil for user protection. (C) The photo-crosslinked hydrogel is retrieved by detaching a coverslip on one side. (D) The hydrogel construct is in contact with sulfo-SANPAH solution to modify the hydrogel for ECM protein incorporation. Before UV activation, the sulfo-SANPAH is orange. (E) After UV activation, sulfo-SANPAH turns brown, indicating that it is activated and incorporated onto the hydrogel surface. (F) After dabbing the hydrogel with sterile lint-free wipes to remove excess water, the stencil is placed on the hydrogel. (G) Basement membrane matrix protein solution is pipetted over the stencil-hydrogel constructs. (H) If the stencil-hydrogel contact is successfully tight, the basement membrane matrix protein solution will stay on top and will not seep through. Please click here to view a larger version of this figure.
Figure 4: Micropatterning of basement membrane matrix proteins and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with different rectangular shapes. (A) Microscopic images of stencils containing laser-cut rectangles with different aspect ratios (1:1, 4:1, 11:1). (B) Representative images of immunostained matrix protein islands on hydrogel substrates. (C) Representative images of hiPSC-CMs stained for nuclei (cyan) and cardiac troponin T (magenta). Scale bar = 20 µm for panels B and C. Please click here to view a larger version of this figure.
Figure 5: The ECM protein concentration plays an essential role in generating proper patterns. (A) Representative image of an optimal concentration with very homogenous distribution of basement membrane matrix proteins in the single patterns. (B) Suboptimal matrix protein patterns due to low concentration, which caused leakage of matrix protein outside the patterns and low amounts of matrix proteins within the patterns as determined by wheat germ agglutinin staining. Scale bar = 20 µm for panels A and B. Please click here to view a larger version of this figure.
Figure 6: Stencil side effect. (A) Basement membrane matrix protein patterns when the front side of the stencil was used. (B) Basement membrane matrix protein patterns when the back side of the stencil was used. Quantification of (C) width, (D) height, (E) aspect ratio of rectangular basement membrane matrix protein islands. Graph is plotted mean ± SEM. Statistics are calculated by unpaired student T test. n.s. = not significant. **** = p < 0.0001. Scale bar = 20 µm. Please click here to view a larger version of this figure.
Figure 7: Stencil-based patterning on silicon elastomer substrate. (A) A representative image of single cell patterned cardiomyocytes on silicon elastomer substrates stained by the cardiac marker, cardiac troponin T (cTnT), at low magnification. Scale bar = 300 µm. (B) A representative single-cell-patterned cardiomyocyte stained by sarcomeric protein α-actinin at high magnification. Scale bar = 10 µm. Please click here to view a larger version of this figure.
Figure 8: Micropatterning of hiPSC-CMs on polyacrylamide hydrogels with different stiffnesses. (A) 10 kPa, (B) 60 kPa. Please click here to view a larger version of this figure.
Step | Problem observed | Possible reason | Solution |
1.1 | The pattern size is bigger than expected | Stencil is flipped | Include a chiral letter/shape in the stencil design to indicate the front- and back-side of stencils |
8.1 | Hydrogel fractures when peeling stencils | Hydrogel sticks to stencils | Cleaning of stencils |
Do not dry hydrogel too much before putting stencils | |||
8.13 | Cell attachment is suboptimal | Degraded sulfo-SANPAH | Use new sulfo-SANPAH |
Inefficient protein incorporation | Prepare new ECM protein solution | ||
Old ECM protein solution | |||
8.13 | Cell patterning is suboptimal | Basement membrane matrix protein solution spills over | Perform immunostaining using fluorophore-conjugated anti mouse IgG antibody or Wheat germ agglutinine to examine the distribution of basement membrane matrix protein solution on hydrogel |
8.13 | More than one cell on one slot | Not in single cell suspension | Use 40−70 µm cell strainer to have single cell suspension |
Seeding density not optimal | Optimize the seeding density from 30 k to 90 k per mL |
Table 1: Troubleshooting guidelines. Frequent problems, possible reasons, and potential solutions are discussed.
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We describe a lithography-free stencil-based micropatterning method that enables effective patterning of adherent cells. In this protocol, we demonstrate patterning of hiPSC-CMs in different length-to-width ratios by micropatterning basement membrane matrix protein islands on polyacrylamide hydrogels with physiologically- or pathologically-relevant tissue stiffnesses or silicon-based elastomer substrates. This method is relatively simple and highly accessible to any researchers, including those who have little background in photolithography and microfabrication techniques. The described method circumvents significant challenges associated with generating stamps using soft lithography and transferring proteins from stamp molds to substrates of interest involved in the microcontact printing technique. This protocol provides a workflow to 1) create custom-designed stencils with an area and shape of interest in a cost-effective manner; 2) fabricate a hydrogel substrate with the stiffness of interest; and 3) efficiently conjugate ECM proteins on the hydrogel substrate.
Traditional soft lithography methods have been used for patterning various shapes of ECM proteins including triangles and stars on substrates of different materials such as silicon-based materials and glass coverslips13,14. In this study, we only demonstrate rectangular patterns given the physiologically- and pathologically-relevant shape of cardiomyocytes. We believe stencil-based patterning of other complex shapes can work as long as the smallest feature of the shape does not go beyond the resolution (~10 µm). With regards to substrate materials, we demonstrate the stencil-based patterning can be applied on hydrogel (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 8) or silicon-based elastomer substrate (Figure 7). The presented method does not work on substrates with minimal adhesiveness to polyimide-materials such as tissue culture plastics and glass coverslips because the tight sealing between the polyimide-based stencil and these substrates is not achieved. To enable tight sealing, further surface modification is required. Lastly, we show that the method can be used to reliably produce cell-pair patterns on hydrogels with different stiffnesses (i.e., 10 kPa, 60 kPa) (Figure 8).
In the protocol, the most critical step is optimization of ECM protein concentration (protocol section 6). The goal of this step is to find the optimal concentration of ECM protein that is viscous enough to inhibit the protein solution to seep through the holes of the stencil, but not too concentrated, to avoid gelation (Figure 5). This stencil-based method does not restrict the type of ECM protein. Although viscous ECM protein solution is more likely to stay on the top of the stencil, a less viscous protein can also be used. One requirement is making sure that the surfaces of the stencil and PA hydrogel are dry enough to form a relative stable hydrophobic interface. In this demonstration, basement membrane matrix protein was used because it is viscous and suitable for culturing hiPSC-CMs. However, other ECM proteins can likely be used (e.g., fibronectin, gelatin, collagen, laminin).
In addition to optimization of the ECM protein solution for each given application, generating a proper seal between the stencil and the hydrogel surface is crucial to obtain a precise pattern with the ECM protein. Because excessive drying can lead to rupturing of the hydrogel, it is essential to not overdry the stencil. These steps (i.e., placing the stencil on the hydrogel as well as gently pealing it off afterwards) require some practice. Establishing an optimal handling procedure for each given condition will also avoid having the hydrogel stick to the stencils. With this optimization, submicron scale laser burns caused by flaws in the laser cutting will not substantially affect the technique in regards to hydrogel damage.
Another important step is to use the front of the stencil when placing the stencil on the hydrogel substrate. We found that use of each side has an effect on the substrate, presumably due to laser cutting (Figure 6). One recommendation when patterning cells in specific size or shape for the first time is to generate an optimization stencil. Stencil-design features do not necessarily match the final patterned cell sizes. We recommend generating an optimization stencil that contains a gradient of feature sizes. We prepared a table listing potential problems and possible solutions to help with troubleshooting (Table 1).
It is not required to use the same microfabrication company mentioned in this manuscript. The keys to precise generation of the stencil are the beam diameter of the laser and the thickness of the stencil film, in our case, polyimide (50 µm). We believe a laser lab equipped with an appropriate laser could generate the stencil with a few steps, optimizing laser intensity, alignment, and other variables. Although we have only worked with one company, there are many available companies who perform precision laser cutting.
A limitation to this method is resolution. While photolithography can achieve nanoscale resolution, stencil-based patterning resolution is limited to the resolution of the laser-cutting of the polyimide stencils, which to date is usually on the scale of a few microns (~10 µm). Nevertheless, given that the average cell size is over 10 µm, the resolution is generally applicable to any single cell patterning.
In summary, this method provides versatile platforms for studying cell-ECM interactions, examining cell structure-function correlation, and many other applications. We believe this protocol will benefit many biomedical researchers and facilitate single cell studies.
The authors have nothing to disclose.
This work was supported by postdoctoral fellowship from Stanford Child Health Research Institute (CHRI) and National Institute of Health (1F32HL142205-01) to S.L, the NIH Office of Director’s Pioneer Award (LM012179-03), the American Heart Association Established Investigator Award (17EIA33410923), the Stanford Cardiovascular Institute, the Hoffmann and Schroepfer Foundation, and the Stanford Division of Cardiovascular Medicine, Department of Medicine to S.M.W, awards from National Institute of Health (UG3 TR002588, P01 HL141084, R01 HL126527, R01 HL113006, R01 HL123968) and Tobacco-related Disease Research Program (TRDRP 27IR-0012) to J.C.W, the American Heart Association (AHA) Postdoctoral Fellowship Award (18POST34030106) to H.Y, and the Hengstberger fellowship to T.S. We thank Dr. Andrew Olsen from Stanford Neuroscience Microscopy Service on the support of confocal imaging of the micropatterned hiPSC-CM. We thank H.Y. for first stencil design, fabrication, single cell micropatterning of iPSC-CM on the polyacrylamide hydrogel coated coverslip, and preliminary confocal imaging of the sarcomere structure of single cell micropatterned iPSC-CMs.
2-Aminoethyl methacrylate hydrochloride (powder) | Sigma-Aldrich | 516155 | |
Acrylamide solution 40% (solution) | Sigma-Aldrich | A-4058-100mL | |
Bench UV lamp 365 nm | UVP | UVP 95-0042-07, XX-15L | |
BioFlex culture plate | FLEXCELL INTERNATIONAL CORPORATION, Burlington, NC | 6-well plate with silicon elastomer substrate | |
Bis-acrylamide solution 2% (solution) | Sigma-Aldrich | M1533-25mL | |
Corning cover glasses square, No. 2, W × L 22 mm × 22 mm | Sigma | CLS285522 | |
Irgacure (2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone) (powder) | Sigma-Aldrich | 410896 | |
Matrigel | Corning | 356231 | basement membrane matrix protein solution |
Methyl sulfoxide, 99.7+%, Extra Dry, AcroSeal, ACROS Organics | Acros Organics | 326881000 | |
Millex (13mm) filter unit with Durapore Membrane | Millipore | SLGV013SL | |
Millipore 50mL Steriflip (0.22 µm) | Fisher Scientific | SCGP00525 | |
Stencils | Potomac | custom design | |
Sulfo-SANPAH | ThermoFisher Scientific | 22589 | |
TrypLE Select 10x | ThermoFisher Scientific | A1217702 | Enzyme used for stencil cleaning |