Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Summary

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Abstract

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

Introduction

Human pluripotent stem cells (hPSCs)^1,2 self-renew unlimitedly and differentiate into various tissue lineages, which could revolutionize drug development, cell-based therapies, tissue engineering, and regenerative medicine^3,4,5,6. General culture dishes and microtiter plates, however, are not designed to enable precise physical and chemical cell manipulation at the cellular level with the range of nano- to micro-meters, which is a critical factor for cellular expansion, self-renewal,....

Protocol

1. Fabrication of MACME Array

Note: All materials and equipment are listed in the Materials Table.

1. Preparation of masks for a nanofiber array and a mold for a microfluidic structure
   1. Create three-dimensional (3D) images of masks used for the nanofiber arrays and molds for microfluidic structures using 3D-computer graphics software packages (Table 1).
      Note: The 3D images are read and printed by a 3D printer. The printed masks and mold have the same dimensions with 3D images defined using the graphics software at step 1.1.1.
   2. Print masks and a mold bas....

Results

MACME arrays: Design and fabrication: In combination with nanofiber technology, we used microfluidic cell culture and screening techniques employed previously to identify optimal conditions for hPSC self-renewal or differentiation^35,36 (Figure 1). This is well suited for establishing robust high-throughput cell-based assays because the cell culture chambers and conditions are precisel.......

Discussion

This protocol demonstrates the first screening method to establish a robust culture system for the maintenance of qualified hPSCs. First, we described how to prepare a platform featuring diverse artificial ECMs and cell seeding densities by using a microfluidic device integrated with a nanofiber array, the MACME array. Second, quantitative image-based single-cell phenotyping was performed⁵⁰ to evaluate individual cellular outcomes and behaviors altered by distinct biochemical and biophysical featu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polystyrene (PS)	Sigma	#182435	Average Mw: 290,000, average Mn: 130,000
Polymethylglutarimide (PMGI)	MicroChem	G113113
Gelatin (GT)	Sigma	G2625	From porcine skin, type A
Sylgard 184 silicone elastomer kit	Doe Corning Toray	#1064291	PDMS curing agent and silicone elastomer base are components of this kit.
OpenSCAD			This is a free 3D computer graphics software (http://www.openscad.org/) used for designing the mold of the microfluidic device.
AutoCAD 2014	Autodesk		This is a 3D computer graphics software (https://www.autodesk.com/products/autocad/overview) used for design of the mask used on nanofiber-array preparation.
3D printer, AGILISTA-3000	Keyence
UV-curable resin, AR-M2	Keyence		This is used for 3D printing by Agilista.
Acetic acid	Sigma	#338826	≥99.99%
Ethyl acetate	Sigma	#270989	Anhydrous, 99.8%
Tetrahydrofuran (THF)	Sigma	#401757
MSP-30T	Vacuum Device		Magnetron sputtering machine
Nunc OmniTray	Thermo Fisher Scientific	#242811	This is a polystyrene baseplate on which the nanofiber array is created. This plate size is typically 127.7 x 85.5 mm.
Gun-type corona discharge machine	Shinko Electric & Instrumentation	CFG-500	This handy device is used to generate corona for activation of the bottom surface of the PDMS layer at step 1.5 "Assembly of the MACME arrays" in the protocol.
5 mL syringe	Terumo	SS-05SZ
Stainless-steel blunt needle (23-gauge)	Nipro	#2166	Outside diameter and length are 0.6 and 32 mm, respectively.
High-voltage power supply	TechDempaz
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride	Dojindo	W001
N-Hydroxysuccinimide	Sigma	#56480
Matrigel hESC-Qualified Matrix	Corning	#354277	This protein is refered as basement membrane gel matrix in the protocol.
CellAdhere Vitronectin, Human, Solution	STEMCELL Technologies	#07004
TeSR-E8	STEMCELL Technologies	#05940	Feeder-free, xeno-free culture medium for maintenance of human ES and iPS cells
Y-27632	Wako Pure Chemical Industries	#253-00513
TrypLE Express Enzyme (1X), phenol red	Thermo Fisher Scientific	#12605028	This ia a recombinant trypsin-like protease for dissociation of adherant mammalian cells.
Click-iT EdU Imaging Kit with Alexa Fluor 647 Azides	Thermo Fisher Scientific	C10086	The fluorescent labeling of proliferating cells in on-plate fluorescent staining was performed along the product manual of this kit.
Annexin V, Alexa Fluor 594 conjugate	Thermo Fisher Scientific	A13203
4',6-diamidino-2-phenylindole (DAPI)	Thermo Fisher Scientific	D1306
Oct-3/4 Antibody (C-10)	Santa Cruz Biotechnology	sc-5279
Donkey Anti-Mouse IgG H&L (DyLight 488)	abcam	ab96875	This is a secondary antibody used in on-plate fluorescent cell staining.
ECLIPSE Ti-E	Nikon		This is an inverted fluorescence microscope equipped with a CFI Plan Fluor 4×/0.13 N.A. objective lens (Nikon), CCD camera (ORCA-R2, Hamamatsu), mercury lamp (Intensilight, Nikon), XYZ automated stage (Ti-S-ER motorized stage with encoders, Nikon), and filter cubes for four fluorescence channels (DAPI, GFP HYQ, TRITC, Cy5; Nikon)
NIS-Elements Advanced Research	Nikon		This is a microscope imaging software used for automatic image acquisition.
CellProfiler, Version 2.1.0			This is a free open software for cell image analysis (http://cellprofiler.org/).
R			SOM analysis is performed by kohonen package of this software. This is freely available (https://www.r-project.org/).
Cluster 3.0			This is the open source clustering software (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm). Unsupervised hierarchical clustering is performed with this software.
Java TreeView			This open source software (http://jtreeview.sourceforge.net/) is used to visualize clustering data as a heatmap and a dendrogram.
H9 human embryonic stem cell	WiCell Stem Cell Bank	WA09