A Micropatterning Assay for Measuring Cell Chirality

Summary

We present a protocol for determining multicellular chirality in vitro, using the micropatterning technique. This assay allows for automatic quantification of the left-right biases of various types of cells and can be used for screening purposes.

Abstract

Chirality is an intrinsic cellular property, which depicts the asymmetry in terms of polarization along the left-right axis of the cell. As this unique property attracts increasing attention due to its important roles in both development and disease, a standardized quantification method for characterizing cell chirality would advance research and potential applications. In this protocol, we describe a multicellular chirality characterization assay that utilizes micropatterned arrays of cells. Cellular micropatterns are fabricated on titanium/gold-coated glass slides via microcontact printing. After seeding on the geometrically defined (e.g., ring-shaped), protein-coated islands, cells directionally migrate and form a biased alignment toward either the clockwise or the counterclockwise direction, which can be automatically analyzed and quantified by a custom-written MATLAB program. Here we describe in detail the fabrication of micropatterned substrates, cell seeding, image collection, and data analysis and show representative results obtained using the NIH/3T3 cells. This protocol has previously been validated in multiple published studies and is an efficient and reliable tool for studying cell chirality in vitro.

Introduction

Left-right (LR) asymmetry of the cell, also known as cellular handedness or chirality, describes the cell polarity in the LR axis and is recognized to be a fundamental, conserved, biophysical property^1,2,3,4,5. Cell chirality has been observed both in vivo and in vitro at multiple scales. Previous findings revealed chiral swirling of actin cytoskeleton in single cells seeded on circular islands⁶, biased migration and alignment of cells within confined boundaries

Protocol

1. Fabrication of polydimethylsiloxane (PDMS) stamps¹⁶

1. Draw an array of microscale rings using CAD software, with an inner diameter of 250 µm and an outer diameter of 450 µm. The pattern used in this protocol is a 10 x 10 array with an 850 µm distance between rings.
2. Print a transparency mask of the pattern at the desired resolution using a microfabrication company's mask printing service (see Table of Materials).
   NOTE: The provided dimensions of the ring have been proven to work for many cell types⁷.
3. Conduct ultraviolet (UV) photolithography using....

Results

Fifteen minutes after the seeding of NIH/3T3 cells, cell adhesion on the ring pattern was visually confirmed by phase-contrast imaging. After subsequent culture of 24 h, cells on the patterns became confluent and elongated with clearly asymmetrical alignments, biased towards the clockwise direction (Figure 2). Directional migration of attached cells is recorded by time-lapse imaging, cell motility and morphogenesis can be quantified with further analyses of the video. To conduct chirality an.......

Discussion

The ring-shaped patterning assay described here provides an easy-to-use tool for quantitative characterization of multicellular chirality, capable of producing highly reliable and repeatable results. Rapid generation of identical defined microenvironments and unbiased analysis enables automated high-throughput processing of large size of samples. This protocol discusses the fabrication of the ring micropatterns, cell patterning, and automatic analysis of the biased cell alignment and directional motion. This method is co.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
200 proof ethanol	Koptec	DSP-MD-43
BZX microscope system	Keyence	BZX-600
Dulbecco's modified eagle medium (DMEM), high glucose	Gibco	11965092
Electron beam evaporator	Temscal	BJD-1800	Gold-titanum film coating
Fetal bovine serum	VWR	89510-186
Fibronectin from bovine plasma	Sigma	F1141-5MG
Glass microscope slides	VWR	10024-048
Glass tweezers	Exelta	390BSAPI
Gold evaporation pellets	International Advanced Materials	AU18
HS-(CH2)11-EG3-OH (EG3)	Prochimia	TH 001-m11.n3-0.2
MATLAB	Mathworks	MATLAB_R2020b
NIH/3T3 cells	ATCC	CRL-1658
OAI contact aligner	OAI	200	UV photolithography
Octadecanethiol (C18)	Sigma	O1858-25ML
Orbital shaker	VWR	89032-088
Phosphate buffered saline (PBS)	Research product international	P32080-100T
Polydimethylsiloxane Sylgard 184	Dow Corning	DC4019862
Silicon Wafer	University Wafer	ID#809
Sodium pyruvate	Thermo fisher scientific	11360-070
SU-8 3050 photoresist	MicroChem	Y311075 0500L1GL
Titanium evaporation pellets	International Advanced Materials	TI14
Transparency mask (with feature)	Outputicity.com	N/A	Mask printing service
Trypsin-EDTA (0.25%)	Thermo fisher scientific	25200-072