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* These authors contributed equally
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 bioch....
1. Fabrication of negative pattern polyimide-based 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 ba.......
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.......
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 ....
Name | Company | Catalog Number | Comments |
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 |
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