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

The overall goal of the following experiment is to show non-adherent effector T lymphocyte migration over a monolayer of adherent tumor cells. This is achieved by first establishing a monolayer of adherent tumor cells in the wells of poly D lysine coated Teflon masked slides as a second step fluorescently labeled effector, T lymphocytes are sedimented into the center of the monolayer using a cell sedimentation manifold. Next light and fluorescence microscopy are used to visualize the migration of the non-adherent cells over the monolayer.

The results show that the migration and effector functions of non-adherent T lymphocytes in co-culture with an adherent tumor cell monolayer are maintained after the lymphocytes have been transduced with a retroviral vector. The main advantage of this technique over existing methods is that radial migration of non-adherent effector T lymphocytes can be measured in coal culture with adherent tumor cells. This has far reaching implications for patients with primary or metastatic brain tumors because allogeneic cytotoxic T lymphocytes that are modified to express a pro-drug activator gene, such as cytosine deaminase, are being tested as a multimodal approach for eliminating tumor in the brain.

Generally, individuals new to this method will struggle at establishing adherent tumor cell monolayers due to the heterogeneity of tumor cell growth in adherence characteristics. Visual demonstration of this method is critical as the steps for generating an even tumor monolayer and an effective sedimentation of non-adherent L-O-C-T-L require an experienced understanding of the characteristics of both cell types. To begin place up to four sterilized.

10 well Teflon coated slides in a sterile 150 millimeter by 15 millimeter Petri dish. Place a 35 millimeter by 10 millimeter Petri dish containing two to three milliliters of sterile water next to the slides to provide humidity. Next, preco the slides by covering each well with a solution of 100 micrograms per milliliter of either poly D lycine or poly L lysine.

Incubate the slides for one hour at room temperature, then aspirate the solution and rinse the surface of the well twice with PBS with the sample wells prepared. Harvest a confluent flask of adherent tumor cells. Count the number of viable cells by light microscopy, and then suspend the cells at a concentration of five times 10 to the six cells per milliliter.

In buffered growth medium, add five to 10 microliters of the cell suspension to each well, depending on the tumor cell type, bring the volume of the well up to 40 microliters with medium and pipette up and down slowly to evenly distribute the cells. After seeding the wells, allow the tumor cells to settle at room temperature for 30 minutes. Then place the lid on the Petri dish and carefully transfer it to a 37 degree Celsius humidified incubator with 5%carbon dioxide incubate for at least 24 hours.

Change the culture medium on the wells daily by carefully removing some of the medium from the monolayer and replacing it with a larger volume of fresh complete T-cell medium. The cells will typically be confluent within one to three days to prepare cells for sedimentation onto the tumor cell monolayer. Harvest the non-adherent T cells and label them with a cell proliferation dye according to the manufacturer's protocol at least one hour in advance of the assay.

Next place the humidified chamber containing the tumor cell slides in the biological safety cabinet. Wash the wells with PBS by pipetting and then add 45 microliters of complete medium with 10%serum to the wells. Slide a sterilized cell sedimentation manifold over the wells until the hook at the end of the manifold touches the bottom of the slide.

Make sure that the medium is visible in the manifold channels. Then count the non-adherent cells and suspend them at one to two times 10 to the fifth. Cells per microliter in the medium.

Gently disrupt the cells to eliminate any cell clusters that would interfere with cell sedimentation. Slowly pipette one microliter of the cell suspension into the channel of the manifold for each. Well then incubate the apparatus at room temperature for 20 to 30 minutes to allow the cells in the channel to settle on the monolayer.

After the cells have settled, gently lift the manifold straight up without touching the slide. Make sure that the cell pellets have been seated within the circumference of the manifold channel. Using a microscope, the seated cells should adhere slightly to the monolayer so that gently moving the slide will not disperse the pellet.

After confirming that the cells have sedimented, transfer the slide to an inverted microscope equipped with a carbon dioxide and humidity chamber. Use the four x objective for brightfield and multichannel fluorescent imaging of the cells. Acquire images of a given area in brightfield and fluorescent channels, and then add micron bars with image capture software.

Store the slide in a humidified incubator between time points once all of the time points are acquired. Drag and drop the image files into the image editing program and select the add layer option to create an image file with multiple layers. Merge the light and the fluorescent images into one layer.

Use the acquisition software to take pictures of an eight millimeter by eight millimeter area. The software should be set to stitch captured images together automatically using the channel that is best represented across the entire well. If only fluorescence channels are imaged, this should be the channel for imaging the adherent cells.

Alternatively, the images can be stitched using image editing software. This is done by aligning regions of the images that are conserved from one image to another and overlaying the images on top of each other until a complete picture is generated. Stitch the images together using the image software.

Use the combined images to make surface intensity fluorescence maps with Image J software to visualize fluorescent T-cell aggregation or spread over time. Human allo react cytotoxic T lymphocytes known as Allo CTL transduced, with a replication competent retroviral vector encoding an emerald green fluorescent protein expression gene were seated at the center of a monolayer of glioma cells. After four hours, the all OCTL had formed visible aggregates by 24 hours.

Empty patches appeared in the adherent cell monolayer indicating lysis of the tumor cells and some allo CTL had migrated beyond the leading edge. Fluorescently labeled murine Allo CTL were seated at the center of a mono layer of flu fluorescently labeled glioma cells at one hour Allo CTL closely associated with the glioma cells by 48 hours, Allo CTL had migrated away from the center of the mono layer surface intensity. Fluorescence maps were generated from these images with image J software and show substantial migration away from the center after 48 hours.

It is important to change the culture medium daily for the tumor cell monolayer to maintain healthy culture conditions. In addition, once mastered the sedimentation of non-adherent cells should take about 20 minutes Following this procedure. Other methods such as cell path tracing can be used to answer questions about the rate at which allo CTL migrate across the monolayer or the time that they're in contact with tumor cells performing their cytotoxic functions.

This protocol will help researchers in the field of immuno gene therapy explore the migration of gene modified T lymphocytes in co culture with tumor cells. After watching this video, you should have a good understanding of how to prepare non-adherent and adhere cells per co culture and how to use the cell sedimentation manifold to study horizontal radial migration of non-adherent cells over tumor cell monolayer.