Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids

Summary

This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids.

Abstract

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and scalability. In this paper, we describe a strategy for the robust and reproducible formation of hPSC spheroids where a co-axial flow focusing device is utilized to entrap hPSCs inside core-shell microcapsules. The core solution contained single cell suspension of hPSCs and was made viscous by the incorporation of high molecular weight poly(ethylene glycol) (PEG) and density gradient media. The shell stream comprised of PEG-4 arm-maleimide or PEG-4-Mal and flowed alongside the core stream toward two consecutive oil junctions. Droplet formation occurred at the first oil junction with shell solution wrapping itself around the core. Chemical crosslinking of the shell occurred at the second oil junction by introducing a di-thiol crosslinker (1,4-dithiothreitol or DTT) to these droplets. The crosslinker reacts with maleimide functional groups via click chemistry, resulting in the formation of a hydrogel shell around the microcapsules. Our encapsulation technology produced 400 µm diameter capsules at a rate of 10 capsules per second. The resultant capsules had a hydrogel shell and an aqueous core that allowed single cells to rapidly assemble into aggregates and form spheroids. The process of encapsulation did not adversely affect the viability of hPSCs, with >95% viability observed 3 days post-encapsulation. For comparison, hPSCs encapsulated in solid gel microparticles (without an aqueous core) did not form spheroids and had <50% viability 3 days after encapsulation. Spheroid formation of hPSCs inside core-shell microcapsules occurred within 48 h after encapsulation, with the spheroid diameter being a function of cell inoculation density. Overall, the microfluidic encapsulation technology described in this protocol was well-suited for hPSCs encapsulation and spheroid formation.

Introduction

There is considerable interest in 3D cultures of human pluripotent stem cells (hPSCs) due to the improved pluripotency and differentiation potential afforded by this culture format^1,2,3. hPSCs are typically formed into spheroids or other 3D culture formats by means of bioreactors, microwells, hydrogels, and polymeric scaffolds^4,5,6. Encapsulation offers another means for organizing single hPSCs into spheroids. Once encapsulated hPSC spheroids may be handled with ease and transfe....

Protocol

1. Device fabrication

1. Make the designs for the microencapsulation device and dissociation device using CAD software^10,11.
2. Spin-coat the three layers of SU-8 photoresist sequentially on a silicon wafer (Figure 2A) to achieve structures with the desired heights: 60, 100, and 150 µm.
   NOTE: The process for the top and bottom molds is identical.
   1. Spin coat a clean 10 cm silicon wafer with SU-8 .......

Discussion

The encapsulation process described here results in reproducible formation of hPSC spheroids. The microcapsule format makes it easy to dispense spheroids into wells of a microtiter plate for experiments aimed at improving/optimizing differentiation protocols or testing therapies. Encapsulated stem cell spheroids may also be used in suspensions cultures where hydrogel shell protects cells against shear-induced damage⁷.

There are several critical steps within the protocol.......

Materials

Name	Company	Catalog Number	Comments
0.22 µm Syringe Filters	Genesee Scientific	25-244
1 ml syringe luer-lock tip	BD	309628
1x DPBS	Corning	23220003
4-arm PEG maleimide, 10kDa	Laysan Inc.	164-68
5 ml syringe luer-lock tip	BD	309646
6-WELL NON-TREATED PLATE	USA Scientific	CC7672-7506
Aquapel Applicator Pack	Aquapel Glass Treatment	47100
CAD software	Autodesk	AutoCAD v2020
CELL STRAINER 100 µm pore size	cardinal	335583
Chlorotrimethylsilane	Aldrich	386529-100mL
Countess II FL Automated Cell Counter	Life technology	A27974
Digital hot plate	Dataplate
Digital vortex mixer	Fisher Scientific	215370
Distilled water	Gibco	15230-162
Dithiotheritol (DTT)	Sigma	D0632-10G
DMEM/F12 media	gibco	11320-033
Falcon 15 mL Conical Centrifuge Tubes	Fisher scientific	14-959-53A
Fisherbrand accuSpin Micro 17 Microcentrifuge	live	13-100-675
HERACELL VIOS 160i CO2 Incubator	Thermo Scientific	50144906
Inverted Fluorescence Motorized Microscope	Olympus	Olympus IX83
Laurell Spin Coaters	Laurell Technologies	WS-650MZ-23NPPB
Live/Dead mammalian staining kit	Fisher	L3224
Magic tape	Staples	483535
Micro Medical Tubing (0.015" I.D. x 0.043" O.D.)	Scientific Commodities, Inc	BB31695-PE/2
Micro stir bar	Daigger Scientific	EF3288E
MilliporeSigma Filter Forceps	Fisher scientific	XX6200006P
Mineral oil	Sigma	M8410-1L
mTeSR 1 Basal Medium	STEMCELL TECHNOLOGY	85850
Needles-Stainless Steel  14 Gauge	CML supply	901-14-025
Needles-Stainless Steel  15 Gauge	CML supply	901-15-050
OptiPrep	STEMCELL TECHNOLOGY	7820
Oven	Thermo Scientific	HERA THERM Oven
Penicillin:Streptomycin (10,000 U/mL Penicillin G, 10mg/mL Streptomycin)	Gemini	400-109
Petri Dish 150X20 Sterile Vent	Sarstedt, Inc.	82.1184.500
Plasma Cleaning System	Yield Engineering System, Inc.	YES-G500
Pluronic F-127	Sigma	P2443-250G
Poly(ethylene glycol) 35kDa	Sigma	94646-250G-F
PrecisionGlide Needle 27G	BD	305109
Rock inhibitor Y-27632 dihydrocloride	SELLECK CHEM	S1049-10mg
Silicon wafer 100mm	University Wafer	452
Slide glass (75mm ´ 25mm)	CardinalHealth	M6146
Span 80	Sigma	S6760-250ML
SpeedMixer	Thinky	ARE-310
Spin-X Centrifuge Tube Filter (0.22 µm)	Costar	8160
SU-8 2025	Kayaku Advanced Materials	Y111069 0500L1GL
SU-8 developer	Kayaku Advanced Materials	Y020100 4000L1PE
Surgical Design Royaltek Stainless Steel Surgical Scalpel Blades	fisher scientific	22-079-684
SYLGARD TM 184 Silicone Elastomer Kit (PDMS)	Dow Corning	2065622
Syringe pump	New Era Pump System, Inc	NE-4000
Triethanolamine	Sigma-aldrich	T58300-25G
TrypLE Express	Gibco	12604-013
Tygon Tubing (0.02" I.D. x 0.06" O.D.)	Cole-Parmer	06419-01
Tygon Tubing (0.04" I.D. x 0.07" O.D.)	Cole-Parmer	06419-04
Ultrasonic cleaner FS20D	Fisher Scientific	CPN-962-152R
Vacuum desiccator	Bel-Art	F42025-0000
Zeiss Stemi DV4 Stereo Microscope 8x-32x	ZEISS	435421-0000-000
μPG 101 laser writer	Heidelberg Instruments	HI 1128