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, and differentiation. To address this drawback, studies have investigated the roles of cellular microenvironments in regulating cell-fate decisions and cell functions⁴. In recent years, an increasing number of studies have been conducted to reconstruct cellular microenvironments in vitro^7,8. Nano- and micro-fabrication processes have established these microenvironments through the manipulation of chemical^{9,10,11,12,13,14,15,16,17} and physical^18,19,20 environmental cues. Until now, there were no reports to systematically investigate the underlying mechanisms of chemical and physical environmental cues on cell-fate decisions and functions within a single platform.

Here, we introduce a strategy based on simple design principles to establish a robust screening platform (Figure 1). First, we describe the development procedure of an integrated platform for creating versatile, artificial cellular microenvironments by using a nanofiber array and a microfluidic structure: The Multiplexed Artificial Cellular MicroEnvironment (MACME) array (Figure 1A and 2A). The nanofiber array has 12 different microenvironments in varying combinations of nanofiber materials and densities. Electrospinning was used to fabricate nanofibers. The nanofiber materials, such as polystyrene (PS)²¹, polymethylglutarimide (PMGI)²², and gelatin (GT)²³, were designed to test their chemical properties, which might affect cell adhesion and maintenance of pluripotency (Figure 2B). Nanofiber densities were varied by changing electrospinning time and the generated nanofibers were defined according to their densities (DNF, with D = XLow/Low/Mid/High). The microfluidic structure is composed of polydimethylsiloxane (PDMS) harboring 48 cell-culture chambers, which can be positioned along the standard dimensions of the 96-well microplate. PDMS is a biocompatible and gas-exchangeable polymer generally used to fabricate microfluidic devices²⁴. Each microfluidic channel was designed to be 700-µm wide and 8.4-mm long and had two inlets at its edges (Table 1). The chambers had different heights (250, 500, and 1000 µm) to manipulate the initial cell-seeding densities (0.3, 0.6, and 1.2 × 10⁵ cells/cm²), which might correlate with survival, proliferation, and differentiation of hPSCs²⁵ (Figure 2C). The number of cells seeded into a chamber is proportional to the column density above the chamber floor, and thus initial cell seeding density was controlled by introducing the same cell suspension into culture chambers with different heights. All channels were designed to be ≥ 250-µm-high²⁶ to minimize the effects of low-oxygen tension²⁷ and shear stress²⁸ on the cells. Channel heights of 250, 500, and 1000 µm are abbreviated here as XCD with X = Low, Mid, and High, respectively. The environments with distinct nanofiber densities and initial cell-seeding densities were shortened as "Material_NF density_Cell density" (e.g., GT_HighNF_HighCD: an environment characterized by high-density GT nanofibers and high initial cell-seeding density).

Subsequently, we describe how to perform single-cell analyses to systematically investigate cell behavior in response to environmental factors (Figure 1B). As a proof-of-concept, we identified the optimal cellular environment for hPSC self-renewal, which is a key function for hPSC maintenance (Figure 1B)²⁹. Image-based cytometry, followed by statistical analyses, allows for quantitative interpretation of individual cellular phenotypic responses to cellular environments. Among a variety of cellular functions, this paper provides a detailed procedure to identify the optimal conditions for maintaining hPSC 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 based on these designs using a 3D printer.
      Note: In this protocol, an ink-jet-like 3D printer with UV-curable resin was used³⁰. The printer resolution was 635 × 400 dpi and 15 µm in X, Y and Z, respectively, however, the actual X, Y resolution was approximately four times lower.
2. Nanofiber-array preparation (Figure 3A)
   1. Prepare polymer solutions for electrospinning.
      1. Dilute 13% PMGI liquid with tetrahydrofuran by 9% (w/v)²².
      2. Dissolve 0.08 g of PS (Number-average molecular weight (Mn): 130,000) in THF:dimethylformamide (1:1 volume ratio)²¹, and add THF:dimethylformamide to bring the volume up to the final 1 mL (8% (w/v) PS solution).
      3. Dissolve 0.1 g of GT in water:acetic acid:ethyl acetate (1:1.6:2.1 volume ratio), and add water:acetic acid:ethyl acetate to bring the volume up to the final 1 mL (10% (w/v) GT solution) as described^23,31.
   2. Use a magnetron sputtering machine, deposit a thin platinum layer with a 5-nm thickness on a polystyrene (PS) baseplate (127.7 × 85.5 mm), which serves as a cathode in the electrospinning setup.
   3. Load each polymer solution into a 5-mL syringe equipped with a 23-G stainless-steel blunt needle.
   4. Place and anchor the syringe with the needle on a syringe pump with 12-cm apart from the collector of the electrospinning device.
   5. Connect the syringe needle to a high-voltage power supply, and set to apply to 11 kV.
   6. Set the pumping rate of 0.2 mL/h.
   7. Keep temperature and humidity at 30 °C and < 30% (v/v).
   8. Put the mask on the baseplate prepared at step 1.2.2.
   9. Fabricate nanofibers on the baseplate through the holes of mask with distinct densities by changing the electrospinning time (e.g., 20, 60, 90, and 180 s).
   10. Remove the mask from the baseplate.
       Note. Ethanol can be sprayed on the electrospun GT nanofibers before removing the mask to avoid peeling off GT nanofibers together with the mask.
   11. Repeat step 1.2.4-1.2.10 until the completion of nanofiber-array fabrication.
   12. Place the nanofiber array in a desiccator at 25 °C for 16 h to evaporate the remaining solvent.
   13. Crosslink GT nanofibers with 0.2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.2 M N-hydroxysuccinimide (NHS) in ethanol at 25 °C for 4 h²³.
   14. Rinse GT nanofibers twice with 99.5% (v/v) ethanol and vacuum-dry at 25 °C for 16 h.
3. Fabrication of microfluidic structures (Figure 3B)
   1. Mix 2 g of PDMS curing agent and 20 g of PDMS base (1:10 weight ratio; Pre-PDMS). ²⁴
   2. Pour the pre-PDMS mixture onto the mold fabricated in step 1.2.
   3. De-gas the pre-PDMS mixture in a desiccator for 30 min.
   4. Cure the pre-PDMS mixture in an oven at 65 °C for 16 h.
4. Assembly of MACME arrays (Figure 3B)
   1. Peel off the PDMS structure cured at step 1.3.4 from the mold and clean with 70% (v/v) ethanol.
   2. Treat atmospheric corona discharge on the bottom side of the PDMS structure³².
      Note: In this protocol, the corona is discharged on the entire surface of a layer 3 or 4 times with a "Handy" corona discharge apparatus. Oxygen plasma treatment was replaced with atmospheric corona discharge to alter the surface adhesiveness.
   3. Assemble the PDMS structure with the nanofiber array quickly.
   4. Oven-bake at 65°C for 2 days.

2. Loading hESCs into MACME Array

1. For the routine culture of H9 human embryonic stem cells (hESCs), maintain cells in xeno-free chemically defined culture medium for hPSC maintenance³³ supplemented with 10 µM Y-27632 ROCK inhibitor³⁴ (named hPSC medium) in a 35-mm cell culture dish coated with basement membrane gel matrix (MG).
2. Passage cells every 4 to 7 days using a recombinant trypsin-like protease.
3. Sterilize a MACME array with 99.5% ethanol, by loading into the chambers for 10 min, and then remove the ethanol. Repeat this step 3 times.
4. Introduce 12 µL of MG, 10 µg/mL vitronectin (VN), and 0.1% (w/v) GT into control culture chambers, and incubate the chambers at 37 °C for 1 h to coat them on the chamber surfaces.
   Note: In this protocol, MG, VN, and GT were selected as reference proteins for cell adhesion and culture. Because MG and VN are standard cell-culture matrices used for maintaining hPSC self-renewal, they are used as positive controls; two-dimensional GT coating (2DGT) or non-coating (NC) were used as negative controls.
5. Introduce pre-warmed hPSC medium into all chambers of MACME array. Incubate the chambers for 30 min at 37 °C.
6. Wash H9 hESC cultured on a 35-mm dish with DPBS, add 0.5 mL of a recombinant trypsin-like protease to the dish and incubate at 37 °C for 1 min and carefully aspirate only the supernatants mixed with protease.
   Note: At the aspiration step, the cells are not detached.
7. Immediately add hPSC medium, gently dispense the medium against the dish surface repeatedly to dissociate cells and transfer the detached cells to a 15-mL conical tube.
8. Centrifuge at 200 x g for 3 min. Aspirate the supernatant and suspend cells in a pre-warmed hPSC medium.
9. Introduce 12 µL of the cell suspension (1.2 × 10⁶ cells/mL) into each microfluidic chamber.
   Note: Introduce cells to one chamber from another using a micropipette. Because the number of cells seeded to chambers is proportional to the column density above the chamber floor, the initial cell-seeding density could be controlled even though samples from same cell suspension were used. Ensure pipetting is done gently. Remove excess medium from the chambers using a cotton-tipped stick after loading. The MACME array is compatible with both commonly used laboratory pipettes and automated pipette systems, and it does not require any special equipment.
10. Change hPSC medium every 12 h and culture for 4 days.
    Note: Constant volumes of the medium (1.6, 3.2, and 6.4 µL) are maintained by using chambers of distinct sizes. Culture cells under a static condition to minimize shear stress except for the exchanging of the cell culture medium^35,36.

3. Quantitative Single-cell Profiling

1. On-plate fluorescent cell staining
   Note: To analyze phenotypic changes of self-renewing hPSCs, this protocol selected three key phenotypic markers for this protocol: OCT4, 5-ethynyl-2'-deoxyuridine (EdU), and Annexin V, to measure the status of pluripotency, proliferation, and apoptosis, respectively (Figure 2b). 4',6-diamidino-2-phenylindole (DAPI) is used to identify cell nuclei. OCT4 is one of the critical transcription factors of pluripotency.
   1. Incubate H9 hESCs in MACME arrays in hPSC medium supplemented with 10 µM EdU alkyne at 37 °C for 30 min.
   2. After washing with annexin-binding buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, 2.5 mM CaCl₂), stain the cells with an orange-fluorescent dye-Annexin V conjugate at 20 °C for 15 min.
   3. Fix cells in PBS with 4% (v/v) paraformaldehyde at 20 °C for 15 min, and then rinse cells with PBS twice.
   4. Permeabilize cells using PBS with 0.3% (v/v) Triton X-100 at 20 °C for 16 h.
      Note: Paraformaldehyde weakens the bond between a plastic base plate and a PDMS microfluidic structure. Thus, the detachment of the PDMS layer from the array at step 3.2.1 can also be performed right after this step.
   5. Incubate cells in Tris-based reaction buffer with a red-fluorescent dye azide at 20 °C for 30 min.
   6. Incubate cells in blocking buffer (PBS with 5% (v/v) normal goat serum, 5% (v/v) normal donkey serum, 3% (w/v) bovine serum albumin, 0.1% (v/v) Tween-20, and 0.1% (w/v) N-dodecyl-β-D-maltoside) at 4 °C for 16 h.
   7. Incubate in PBS with 0.5% (v/v) Triton X-100 solution and human OCT3/4 antibody (mouse IgG) at 4 °C for 16 h.
   8. Incubate in blocking buffer with green-fluorescent dye-labeled donkey anti-mouse IgG (H+L) at 37°C for 60 min.
   9. Incubate in PBS with DAPI at 20 °C for 30 min.
   10. Replace PBS-DAPI with PBS.
   11. Preserve the MACME array at 4 °C until image acquisition.
2. Image acquisition (Figure 4)
   1. Peel off the PDMS microfluidic structure from the MACME array.
   2. Remove the residual PBS from the MACME array, apply 90%(v/v) glycerol in PBS to the cells and place coverslips over the stained cells.
   3. Set the plate upside down on the stage of an inverted fluorescence microscope.
   4. Acquire 12-bit color images automatically using all motorized components (stage, filters, shutter, and focus systems) that were controlled by a microscope imaging software.
      Note. In this protocol, exposure times are adjusted to reach near maximum intensity values (highest pixel intensity: 4096), but no saturation, for each image, DAPI, OCT4, Annexin V, and EdU.
3. Computer-guided image processing and fluorescence signal quantification (Figure 5)
   Note: The following cell image analysis is performed based on a previously described method³⁷.
   1. Convert color images to grayscale.
   2. Calculate a single threshold value for each DAPI image with the Otsu method and classify pixels above the threshold as foreground and below as background. Ensure that the foreground value corresponds to the fluorescent intensity of DAPI.
      Note: The image analysis process contains 5 steps; identification of primary objects in DAPI images, identification of secondary objects in OCT4, Annexin V, and EdU images, respectively, and measurement of object intensity. Figure 3 provides the images of graphical user interface (GUI) on the setting of image processing.
   3. Measure the fluorescent intensities of OCT4 and EdU on each image.
   4. Identify the region stained with Annexin V with the propagation method, and quantify the fluorescent intensity.
4. 3.4. Statistical analysis to visualize individual cellular phenotypes on single-cell imaging
   1. Normalize the input fluorescent intensities of each phenotypic marker by centering and scaling these variables to give them equal weight.
   2. Perform self-organizing map (SOM) analysis by using a software environment for statistical computing and graphics as described by previous studies^38,39.
      1. Create 25 SOM nodes with distinctive four "codebook vectors" corresponding to the fluorescent intensities of four markers, DAPI, EdU, Annexin V, and OCT4.
      2. Assign individual cells in each microenvironment to a "winning" (most similar) node, and plot as cell frequency, according to which the codebook vectors of the winning node are updated using a weighted average, where the weight is the learning rate (here, 0.05 as default).
         Note: Omit data from chambers containing less than 1,000 cells because datasets containing a few cells did not provide statistically relevant SOM results.
   3. Perform unsupervised hierarchical clustering with the SOM-node values using the average-linkage method based on Pearson correlation by a clustering software⁴⁰.
   4. Render the clustering data into dendrogram and heatmap views⁴¹.

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