A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The generation of iPSC-derived thymic emigrants by a 3D thymic organ culture is the first in vivo method regenerating homogenous population of tumor antigen-specific naive-like T cells. The main advantages are the iTE are more physiological relevant, and this method more is more potent then any other in vivo differential method to produce constantly a specific T cells. iTE is homogeneous upset of CD8 alpha-beta anti-genous specific T cells with a naive T cell-like functional phenotype, including the capacity for proliferation, remodiformation, and tumor suppression in vivo.

Demonstrating the procedure will be Doctor Rafiqul Islam a post-doc in my laboratory. To begin culture OP9/DLL1 cells in OP9 media at 37 degrees Celsius. When the cells reach 80 to 95 percent confluency, wash them once with one X magnesium, calcium, and phenol red free PBS.

Add four milliliters of point zero five percent trypsin and incubate at 37 degrees celsius for five minutes. Then add four milliliters of OP9 media and pipet to disassociate the cell layer and make a single cell suspension. Transfer the cell suspension into a 50 milliliter conical tube through a 100 micrometer cell strainer.

Centrifuge at 300 G and at four degrees Celsius for five minutes. Aspirate the suppurate and re-suspend the cells in 12 milliliters of OP9 media. Then plate two milliliters of the OP9/DLL1 cell suspension into a new 10 centimeter cell culture Petri dish.

Add an additional eight milliliters of OP9 media. Repeat this passage every two to three days. On day zero harvest the induced pluripotent stem cells as a single cell suspension by trypsinization.

Collect the cells and centrifuge at 300 G at four degrees celsius for five minutes. Aspirate the supernatant and re-suspend the IPC's at a density of 100, 000 cells per 10 milliliters of OP9 media. Then plate 100, 000 cells onto a confluent OP9/DLL1 10 centimeter dish.

Continue incubation at 37 degree celsius with five percent carbon dioxide. On day three aspirate the old media and replace it with 10 milliliters of fresh OP9 media. On day six wash each 10 centimeter confluent OP9 dish with 10 milliliters of PBS.

Add three milliliters of point zero five percent trypsin to each dish. And incubate for three to five minutes at room temperature. Add four milliliters of OP9 media and gently pipet to collect the cells.

Pass the cells through a 100 micrometer cell strainer and centrifuge at 300 G and four degree celsius for five minutes. Then discard the supernatant. Re-suspend the cells in 10 milliliters of differentiation media.

Plate the cell suspension onto a new 10 centimeter OP9/DLL1 confluent dish. Continue incubation at 37 degrees celsius with five percent carbon dioxide. On day nine, aspirate the old media and replace it with 10 milliliters of fresh differentiation media.

Continue incubating the cells at 37 degrees celsius and five percent CO2. On day 11 when cardiomyocytes are observed in iPSC colonies use a pipet to mechanically detach non-adherent cells. Filter the cells through a 100 micrometer cell strainer and centrifuge at 300 G and four degree celsius for five minutes.

After this, aspirate the supernatant and re-suspend the cells in 24 milliliters of differentiation media. Plate the iPSC's into a confluent OP9/DLL1 six well plate. Incubate the cells at 37 degrees celsius with five percent carbon dioxide.

On day 15, collect all non-adherent cells and filter them through a 40 micrometer cell strainer. Centrifuge at 300 G and at four degrees celsius for five minutes. Continue passaging the non-adherent cells every three to four days as previously described.

On day seven of DGWO treatment, take out four new 10 centimeter dishes. Fill each dish with 20 milliliters of complete media. Transfer all of the nitrocellulose membranes with thymic lobes into one 10 centimeter dish.

Using forceps, detach the individual lobes from the membrane Allowing them to be submerged in media. Incubate the lobes for one hour at room temperature. Then transfer the thymic lobes to a new 10 centimeter dish with complete media.

And incubate for one hour at room temperature. Repeat this transfer and incubation an additional two times. Using forceps, fix the thymic lobes to the dish while using the other hand to make a 100 to 200 micrometer deep incision in the center and extending half the diameter of the lobe to facilitate T cell progenitor migration into the lobe.

Transfer the thymic lobes to a new 10 centimeter dish filled with complete differentiation media. If using 3D culture plates with lower and upper level grids, fill both grids with steril PPS to prevent the evaporation and drying of the hanging drops. Next transfer 30 microliters of complete media containing one DGWO treated thymic lobe into each well of the 3D culture plate.

Collect the non-adherent T lineage cells from the OP9/DLL1 co-culture. And re-suspend the cells at a density of 2, 000 to 5, 000 T cell lineage cells per 20 microliters of media. Add 20 microliters of the T lineage cell suspension to each thymic lobe in the 3D culture plate.

Incubate overnight at 37 degrees celsius with fiver percent carbon dioxide. The next day, set the P200 pipet to 30 microliters. Pipet the media in each well up and down several times to remove all the cells surrounding the thymic lobes.

Then aspirate the media from each well and replace it of 30 microliters of complete media. Repeat this process, pipeting, removing, and replacing the media five to seven times to remove any extra immature T cells which does not migrate into the lobes. Continue incubating the lobes, making sure to change 25 to 30 microliters of media daily.

On day four or five, use light microscopy to confirm the formation of a halo of iPSC-derived thymic emigrants around the lobes. Collect the iTE's daily by pipeting media without lobe disruption. Change the media every day and continue the collection up to approximately 12 days.

Harvested iTE's are ready to use for molecular analysis or in vivo transplantation experiments. In this study, co-cultured fetal thymuses are sectioned to analyzed whether iPSC derived T cell lineage cells can migrate into the thymic lobes. Un-seeded control lobes have a tissue architecture characterized by an astrocyte-like thymic epithelial web deployed of endogenous CD3 positive cells.

However thymic lobes seeded with iPSC derived immature T cells are repopulated with CD3 positive mononuclear cells indicating migration of iPSC derived immature T cells into the lobes. T cells that migrate into, and mature, within the thymic microenvironment subsequently egress as iTE. Flow cytometric analysis is then used to test the phenotypic characterization of various cells.

Extra thymic T cells show CD4, CD8 double positive T cells and CD8 alpha single positive T cells without expression of the positive selection marker, MHC Class One whereas iTE are a homogenous population of CD8 alpha single positive MHC Class One positive T cell phenotype indicating their successful passage through positive selection prior to egressing from the thymic lobe. iTE have antigen-specificity against cognate peptide. iTE co-cultured with antigen presenting cells preloaded with an irrelevant peptide as nucleoprotein do not show into a cellular production of cytokines.

However iTE co-culture antigen presenting cells pre-pulsed with cognate peptide GP100, produce intracellularly TNF alpha interleukin ii and interferon gamma. Please remember that open line cell culture is a strongly dependent on the ABS slot. Generated iTE can be used in any other assay working with conventional T cells.

For example, T cell clearing assay, cytokine production assay, in vivo tumor regression trials, T cell differentiation study, et cetera. This technology can be applied to many immunological or such project. Including a T cell differentiation, pulse timing T cell maturation, and the generation of antigen-specific T cell from hematocrit progenitor or stem cell.

After watching this video the viewer should understand how to generate induced pluripotent, stem cell derived, tumor antigen specific thymic emigrants using a 3D thymic organ culture system.