Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

Summary

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

Abstract

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

Introduction

The Radial Monolayer Cell Migration assay was originally developed to measure the infiltrative properties of adherent tumor cells^1-4 on slides coated with extracellular matrix (ECM) proteins^5-7 or with individual ECM components, such as fibronectin or laminin^1,2. The technique involved seeding a single cell suspension of tumor cells in the center of wells using a stainless steel cell sedimentation manifold (CSM). After sedimentation, the tumor cells would adhere to the bottom of the well and the change in the diameter of the initial cell population over time was used to establish a rate of horizontal motility. The Radial Monolayer Cell....

Protocol

1. Slide Preparation

1. Insert Cell Sedimentation Manifold slides into sterilization pouches and seal with autoclave tape. Face the Teflon-coated side of the slide the paper-side of the pouch to avoid plastic deposits on the wells.
2. Autoclave pouches for 15 min at 121 °C.
3. Remove slide from sterilization pouch inside a biosafety cabinet and place in a sterile 150 x 15 mm sterile Petri dish. Up to four slides can fit per dish. Place a 35 x 10 mm Petri dish next to the slides. Add 2-3 ml of sterile H₂O to provide humidification.
4. Pre-coat slides with poly-D-lysine at 100 µg/ml by using enough volume to cover the e....

Results

Viral vectors encoding for fluorescent proteins can be used in addition to, or instead of, fluorescent dye. Viral transduction should be performed in advance of the motility assay. Both adherent and non-adherent cell types can be differentially-labeled. The protocol for transduction will depend on the type of vector employed. Here, we transduced the alloCTL in Figures 3, 5 and 6 with RRV-EMD at least two days in advance of the assay using the protocol described¹²

Discussion

Tumor cells in a single cell suspension were pipetted into the wells of a Teflon-masked slide. The cells were allowed to adhere and then formed monolayers in a humidified 5% CO₂, 37°C incubator (Figure 1A). Established monolayers or ECM proteins derived from the monolayer could be harvested for these assays (Figure 1B). Effector T lymphocytes labeled with vital fluorescent dyes or transduced with vectors coding for EMD were seeded into the center of the well using a CSM. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Teflon-masked microscope slides	Creative Scientific Methods	CSM002
Cell Sedimentation Manifold	Creative Scientific Methods	CSM001
Petri Dish, 150mm	Corning	430597
Petri Dish, 35mm	Corning	430588
Phosphate-Buffered Saline (PBS)	Mediatech	21-040
20 μl Pipetman	Gilson	F123600
200 μl Pipetman	Gilson	F123601
200 μl pipette tips	VWR	89003-060
Distilled, deionized water (sterile)	Mediatech	25-055
Trypsin	Mediatech	25-054-CL
Celltracker Red CMPTX	Invitrogen	C34552
TrypLE Express (optional)	Gibco	12604
Tumor cell culture media (e.g. DMEM)	Mediatech	10-013
AIM-V serum-free media	Invitrogen	12055-083
Fetal Bovine Serum	Omega Scientific	FB-02
Inverted microscope
SPOT Advanced	Diagnostic Instruments
Poly-D-Lysine	Millipore	A-003-E
Fibronectin	BD	354008
31/2 x 51/4 Autoclave Pouches	Crosstex	SCXS2
Trypan Blue	Mediatech	25-900-CI
Cell Proliferation eFluor 670	eBioscience	65-0840-85
ImageJ	NIH