Transducing immature thymocytes with retroviral vectors and culturing these cells on OP9 DL4 stromal cells is a challenging technique. This detailed protocol will save researchers time and effort while optimizing these systems. This technique provides a flexible in vitro system for studying the effects of genetic modifications on immature T-cells and it can be useful for studying T-cell development and cancer.

Demonstrating the procedure will be Gisele Rodrigues, a post-doctoral fellow from my laboratory. To begin, seed the retrovirus producer cells 18 to 24 hours before transfection at 70 to 90%confluency in each well of a six-well tissue culture plate containing two milliliters of retrovirus producer cell, or RPC medium. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide.

To transfect the cells, replace the medium with fresh RPC medium one hour before transfection. Prepare lipofection mixtures by diluting four micrograms of DNA containing two micrograms of helper plasmid pCL-Eco and two micrograms of transfer plasmid pMIG in 250 microliters of reduced serum medium and mix gently. Next, mix 10 microliters of transfection reagent and 250 microliters of reduced serum medium and incubate for five minutes at room temperature.

After five minutes of incubation, combine the diluted DNA with the diluted transfection reagent. Mix gently and incubate for 20 to 25 minutes at room temperature. Add the 500 microliters of the DNA and transfection reagent mixture gently to the well containing the retrovirus producer cells by dropping them onto the cells with a circular movement.

Mix gently by rocking the plate back and forth and then incubate the plate in a 37 degree Celsius incubator for 16 to 24 hours. Approximately 16 hours after transfection, replace the old medium with two milliliters of fresh RPC medium and continue incubating the cells at 37 degrees Celsius for 20 to 24 hours. To prepare the thymocyte single-cell suspension, place a harvested thymus from euthanized mice in five milliliters of PBS in a Petri dish.

Using sterile glass slides, place the thymus between the frosted surfaces of the slides and gently rubbed the slides together, rolling the thymus between the two slides. Rinse the glass slides to collect the cells and discard the remnant thymic stromal tissue. Filter five milliliters of the thymic suspension through a 30-or 40-micrometer cell strainer filter and centrifuge the cells at 300 G for 10 minutes.

After removing the supernatant, lyse the red blood cells by adding one milliliter of a ACK lysis buffer per tube for one minute. Add five milliliters of cell depletion buffer to deactivate the ACK lysis buffer, centrifuge the suspension at 300 G for 10 minutes, and resuspend in one to five milliliters of cell depletion buffer for counting. After counting the cells and centrifuging at 300 G for 10 minutes, resuspend the cells at one times 10 to the seventh in 80 microliters of cell depletion buffer.

Add 10 microliters each of CD4 and CD8 microbeads per one times 10 to the seventh cells. Mix well and incubate for 15 minutes in the dark in the refrigerator. Next, prepare a depletion column by rinsing it with two milliliters of depletion buffer and discarding the flow-through.

Wash the incubated cells by adding one to two milliliters of depletion buffer per one times 10 the seven cells. Centrifuge at 300 G for 10 minutes and discard the supernatant. Then, apply the resuspended cell suspension to the column and collect the flow-through of unlabeled cells.

Wash the column twice with one milliliter of buffer and collect the flow-through. Stain the depletion efficiency controls by splitting the 200 microliters of cells collected before depletion into four FACS tubes, unstained, CD4 single-stained, CD8 single-stained, and CD4 and CD8 double-stained. Use the unstained and single-stained samples to set up the flow cytometry parameters.

Use the 1, 000 microliters of cells collected after depletion to stain for CD4 and CD8 and compare with the double-stained sample collected before depletion. To culture the thymocytes, place two to five times 10 to the fifth to one times 10 to the sixth post-depletion thymocytes into a T25 flask of 80 to 90%confluent OP9 DL4 cells in OP9 medium containing cytokines. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide for 24 hours on OP9 DL4 cells.

To harvest the retrovirus, collect the supernatant containing the retroviruses from the transected cells by tilting the six-well plate and then positioning a three-to five-milliliter syringe at the bottom of the plate while pulling the plunger to aspirate the supernatant. Filter the retrovirus supernatant through a 0.45-micrometer syringe filter and collect the filtrate in a 50-milliliter tube. Replace the medium with two milliliters of fresh RPC medium and continue to incubate the cells at 37 degrees Celsius for 20 to 24 hours for the second transduction.

collect the thymocytes from the OP9 DL4 culture by aggressive pipetting to remove the thymocytes and OP9 DL4 cells from the flask surface. Filter the cell suspension through a 40-micrometer cell strainer to remove most OP9 DL4 cells and collect the filtrate in a 50-milliliter tube. Centrifuge the thymocytes and the filtrate at 300 G for five minutes and discard the supernatant.

Resuspend the thymocytes in 0.5 to one milliliter of OP9 medium with cytokines. Add one to two milliliters of RPC medium containing the virus. Then, add hexamethrine bromide at eight micrograms per milliliter of total cell suspension.

Spinoculate the contents by centrifuging the cells at 850 G for one hour at room temperature. Resuspend the cells in six milliliters of OP9 medium with cytokines per flask and add the suspension back onto the OP9 DL4 cell monolayer. Incubate at 37 degree Celsius overnight.

Repeat the steps of retrovirus harvesting using a new well of the retrovirus-containing cell supernatant. Maintain the transduced thymocytes on OP9 DL4 culture for two to five days or freeze as needed. The depletion efficiency was assessed flow cytometrically by labeling the magnetically-unlabeled cell fraction for CD4 and CD8 after immunomagnetic cell separation and analyzing this on a two-dimensional bivariant dot plot.

A good yield of double-negative cells for CD4 and CD8 is 95%or above, as represented here. When using vectors that express screenable markers such as a fluorescence gene, the transection and transduction can be roughly and empirically assessed by fluorescence microscopy. The transduction efficiency was analyzed by harvesting the thymocytes from the OP9 DL4 monolayer.

and looking at the expression of a fluorescence gene by flow cytometry. The efficiency of transduction using an empty retroviral vector with GFP as the reporter gene was 84.2%T-cell differentiation on OP9 DL4 cells was observed four days after transduction. Flow cytometry analysis of cell differentiation induced by the co-culture on OP9 DL4 cells and the transgene expression, where the cells were labeled for CD4, CD8, CD44, and CD 25.

The transduction of double-negative thymocytes with the empty retroviral vector pMIG presented approximately the same proportions of single positives, double positives, double negatives, and its sub-stages double negative one to four as the untransduced thymocytes, indicating that T-cell development was not affected by the transduction process. The most difficult task when attempting to replicate this protocol is making sure that the different cell types are managed simultaneously in a coordinated way. Overexpressing oncogenes in immature thymocytes occasionally can affect normal T-cell development, so the subsequent immunophenotyping by flow cytometry is a helpful way to learn which direction to take.

The oncogenic potential of transduced T-cells differentiated on fetal layer cells can be further investigated by injection into immune-compromised mice.

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