Harmonic Nanoparticles for Regenerative Research

Summary

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

Abstract

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.

Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Introduction

Nonlinear microscopy systems, thanks to their inherent three dimensional sectioning capabilities, have increasingly triggered the demand for photo-stable fluorophores with two-photon absorption bands in the near-infrared. Only in the last couple of years, to complement the development of fluorescence-based labels (dyes, quantum dots, up-converting nanoparticles), a different imaging methodology has been exploiting the use of a novel family of inherently nonlinear nanoparticles as labels, i.e. harmonic nanoparticles (HNPs) which have been specifically developed for multi-photon microscopy. These labels, based on inorganic noncentrosymmetric crystals, exert opt....

Protocol

1. Culture and Expansion of Human ESC

1. Prepare the cell propagation medium (called PM) containing Knockout DMEM supplemented with 20% Knockout Serum, 1% MEM Non-Essential Amino Acids, 1% L-glutamine 200 mM, 1% penicillin-streptomycin, and 3.5 µl β-mercaptoethanol.
2. Thaw human ESC (hESC) in 8 ml PM medium and centrifuge them 5 min at 115 x g to discard DMSO-supplemented freezing medium.
3. Add 1 ml of PM medium containing 10 mM Rock inhibitor to prevent apoptosis and improve cell survival upon thawing (24 hr incubation only).
4. Add basic fibroblast growth factor (bFGF) to the PM at the desired concentration (4-10 ng/ml)....

Results

Prior to assessing the beating activity by confocal imaging, a careful characterization of the nonlinear optical response of the PTFE filters was performed, either alone or in the presence of HNPs at high concentration (1 mg/ml). It was ensured that: i) the bare substrate two-photon excited fluorescence is very weak and cannot prevent measuring the relevant biological samples, and ii) the SH emission from isolated HNPs can be easily acquired by imaging through the substrate in epi-detection mode (Figure 2

Discussion

The application of nanotechnology to stem cell research is a relatively new but rapidly expanding field. As pointed out by various review articles on the subject, the use of nanoparticles can be applied to accomplish different research tasks, ranging from cell tracking (both in vitro and in vivo), to intracellular delivery of proteins and genes, not least the creation of biomimetic cellular environments for preferential stimulation/inhibition of specific differentiation pathways^19,20. Th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microscope Incubator	OKO LAB	UNO package (top stage)	37 °C, 5% CO2, moisturized
Multiphoton microscope	Nikon	AR1-MP
Fast scanning, four nonphotomultiplier descanned detectors
Filters SHG and autofluorescence	Semrock	FF01-360/12-25
FF01-395/11-25
FF02-485/20-25
Microscope objectives	Nikon
CFI Plan Fluor 10X			NA 0.30, WD 16 mm
CFI Plan Apo 20X			NA 0.75, WD 1.0 mm, VC
CFI Apo 40X			NA 1.25, WD 0.18 mm λS, Nano-Crystal Coat
Rhock inhibitor	Sigma	Y-27632
Knockout DMEM	Invitrogen	10829
Knockout Serum	Invitrogen	10828
MEM Non-Essential Amino Acids	Invitrogen	1140
L-glutamine 200 mM	Invitrogen	25030
Penicillin-streptomycin	Invitrogen	15140
β-mercaptoethanol	Sigma	M7522
Collagenase IV	Gibco	17104-019
Roller scraper tool	StemPro EZPassage, Invitrogen	23181-010
StemPro Accutase	Gibco	S11105-01
Aggrewell system	StemCell Technologies	27845
Hyclone serum	Thermo Scientific	SH30070.03
Gelatin	Sigma	G9391
6-well plates	Falcon	353046
24-well plates	Nunclon	142475
Polytetrafluoroethylene (PTFE) filters	Millipore	NA76/25
Inserts	Millipore	PICM03050