Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

Summary

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.

Abstract

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Introduction

Most knowledge about migrating cells comes from 2D experiments^1,2,3, which are normally conducted in a glass or plastic surface of a culture/imaging dish. However, a physiological scenario requires, in most cases, a 3D microenvironment, in which the extracellular matrix (ECM) plays a decisive role. ECM not only provides the 3D structure essential to maintain proper cell morphology but also offers survival signals or directional cues for an optimal functioning of many cells^4,5 . Therefore, a 3D environment is required ....

Protocol

Research carried out for this study with the human material (leukocyte reduction system chambers from human blood donors) is authorized by the local ethics committee (declaration from 16.4.2015 (84/15; Prof. Dr. Rettig-Stürmer)) and follows the corresponding guidelines.

1. Preparation of Neutralized Collagen Solution (500 µL)

1. Transfer 400 µL of chilled collagen stock solution (10.4 mg/mL) to a sterile 1.5 mL tube under the cell culture hood. Slowly add 50 µL of chilled 10x PBS (pH 7.0 - 7.3) to 400 µL of chilled collagen stock solution. Mix the solution by gently tiling the tube.
   Note: All st....

Results

Protrusion formation during T cell migration is a highly dynamic process, which is actin dependent. To visualize protrusion formation of primary human CTL, we transiently transfected a mEGFP fused protein to label the actin cytoskeleton in CTL as described before¹¹. One day after transfection, the cells were embedded in the collagen matrix. Image stacks were acquired every 40 s with a step-size of 1 µm at 37 °C using light-sheet microscopy. As shown in

Discussion

Most in vitro assays are carried out on a 2D surface, for example in cell-culture plates, Petri-dishes or on coverslips, whereas in vivo cells, especially immune cells, experience mostly a 3D microenvironment. Emerging evidence shows that migration patterns of immune cells differ between 2D and 3D scenarios¹⁷. Moreover, the expression profiles of tumor cells are also different in 2D- and 3D-cultured tissues^18,19,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fibricol, bovine collagen solution	Advanced Biomatrix	#5133-20ML	Collagen matrix
0.5 M NaOH Solution	Merck	1091381000	for neutralizing Fibricol solution
Ultra-Low melting agarose	Affymetrix	32821-10GM	Sample preparation in low c[Col]
Dynabeads Untouched Human CD8 T Cells Kit	Thermo Fisher	11348D	Isolation of primary human CD8+ T cells from PBMC
Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation	Thermo Fisher	11132D	Activation of CTL populations
Human recombinant interleukin-2	Thermo Fisher	PHC0023	Stimulation of cultured CTL
P3 Primary solution kit	Lonza	V4XP-30XX	Transfection
α-PFN1 antibody, rabbit, IgG	Abcam	ab124904	IF
Alexa Fluor 633 Phalloidin	Thermo Fisher	A22284	IF
CellMask Orange Plasma membrane Stain	Thermo Fisher	C10045	Fluorescent cell label
Tween 20	Sigma	P1379-250mL	IF
Triton X-100	Eurobio	018774	IF
DPBS Dulbecco's phopsphate buffered saline	Thermo Fisher	14190250	IF
Bovine serum albumin	Sigma	A9418-100G	IF
Goat α Rabbit 568, IgG, rabbit	Thermo Fisher	A-11011	IF
Lightsheet Z.1 (Light-sheet microscopy)	Zeiss	N.A.
Cell culture hood	Thermo Fisher	HeraSafe KS
Cell culture incubator HERACell 150i	Thermo Fisher	N.A.
Centrifuge 5418 and 5452	Eppendorf	N.A.
Pippettes	Eppendorf	3123000039, 3123000020, 3123000063
Pippette tips	VWR	89079-444, 89079-436, 89079-452
15 mL tubes	Sarstedt	62.554.002
Capillaries 50 µL	VWR (Brand)	613-3373	Zeiss LSFM sample preparation
Plunger for capillaries	VWR (Brand)	BRND701934	"Stamps with Teflon tip" LSFM sample preparation
MColorPhast pH stips	Merck	1095430001	to test pH of neutralized Fibricol
BD Plastipak 1mL syringes	BD	Z230723 ALDRICH	Alternative sample preparation
Modeling clay (Hasbro Play-Doh A5417EU7)	Play-Doh	N.A.
Imaris file converter	Bitplane	available at http://www.bitplane.com	Convert imaging files to Imaris file format
Imaris 8.1.2 (MeasurementPro, Track, Vantage)	Bitplane	available at http://www.bitplane.com	Analysis of 3D and 4D imaging data