The overall goal of this novel feeder-free system is to provide a method to mass produce high-purity natural killer cells for in vitro as well as in vivo assays. This method can help answer the key questions in innate immunology, including natural killer cell development and cancer immunity. The main advantage of this technique is that this novel feeder-free system can largely increase the purity of the produced natural killer cells.

That significantly facilitates the following assays in vitro and in vivo. The indication of this technique extends to the natural killer immunity because mass production of murine natural killer cells can be produced from the bone marrow of cancer patients for analysis and modification for personal immunotherapy. Generally, individuals new to this method will struggle because the consumed medium should be collected from the OP9 cell undergoing load phase growth.

Facial demonstration of this method is critical as the bone marrow instruction steps are difficult to learn, because the bones need to be sterile with ethanol with optimal time to preserve the viability. To begin this procedure, collect the OP9 cells from a culture flask. Add FBS to stop trypsin digestion.

Collect the OP9 cells, and centrifuge at 465 times gravity for five minutes. After this, re-suspend the cell pellet. Using a hemocytometer, count the cells.

Then, seed two times 10-to-the-six cells in 15 milliliters of alpha-mem medium in a 75 centimeter-square culture flask, and incubate for 48 hours. Once the cells are 80%confluent, wash the cells with 10 millimeters of phosphate-buffered saline and add 20 milliliters of plain alpha-mem medium without antibiotics and fetal bovine serum to the cells. Incubate for 24 hours.

The next day, collect the OP9-conditioned media by removing the cell debris with a filter and preserve at four degrees Celsius. Inject overdose of 100 milligrams per kilogram of pentobarbitone intraperitoneal to euthanize a 12-week-old mouse. After confirming lack of breathing, excise the femur bones with a surgical knife.

Remove the remaining muscles by gently scratching with a blade. Transfer the bones in a 50 milliliter centrifuge tube filled with chilled, sterile alpha-mem medium, and keep in a biosafety cabinet. Then, expel the alpha-mem medium, and wash the bones with 70%ethanol for 30 seconds.

Again, wash the bones with ice-cold sterile phosphate-buffered saline twice to remove any remaining ethanol. After washing, transfer the bones to a mortar containing five milliliters of ice-cold phosphate-buffered saline and fragmentize the bones with a pestle. Swirl the pestle gently to release the bone marrow cells into the phosphate-buffered saline.

Transfer the phosphate-buffered saline containing bone marrow cells in a new 50 milliliter centrifuge tube. Then, centrifuge the tube at 465 times gravity for five minutes at four degrees Celsius. After centrifugation, discard the supernatant.

Extract until the bone turns white in color to get maximum yield. Next, re-suspend the pellet with 18 milliliters of sterile distilled water, and wait for 30 seconds to remove the red blood cells. Then, add two milliliters of 10X ice-cold phosphate-buffered saline to stop the reaction.

Use a 70 micrometer cell strainer to remove the lysed red blood cells. Then, centrifuge the tubes at 465 times gravity for five minutes at four degrees Celsius. Discard the supernatant and re-suspend the pellet in 20 milliliters of ice-cold phosphate-buffered saline.

Centrifuge the cells one more time. Next, transfer the cell pellet in a 100 milliliter sterile petri dish containing 10 milliliters of OP9 conditional medium, supplemented with additional reagents. Transfer the petri dish in an incubator at 37 degrees Celsius with 5%carbon dioxide for two hours.

Remove all unattached cells and seed in a new culture dish containing OP9 conditional medium, and place in an incubator at 37 degrees Celsius for five minutes. On the fourth day, replace the culture medium with OP9 conditional medium, supplemented with 20%fetal bovine serum and 2, 000 units per milliliter of murine interleukin 2. Keep changing the culture medium every three days until the seventh day to obtain the mature natural killer cells.

Follow the product manual to premix the small interfering RNA or non-sense control with the transfection reagent at zero, fourth and seventh day of changing the medium until the experimental endpoint. Add 50 nanomolar of small interfering RNA, or non-sense mixture, to the unattached cells during medium refreshment. Collect the differentiating cells at an appropriate timepoint.

Centrifuge the cells at 465 times gravity for five minutes. Discard the supernatant and wash the cell pellet with ice-cold phosphate-buffered saline. After washing the fixed cells with ice-cold phosphate-buffered saline, re-suspend the cells in 100 microliters of flow cytometry staining buffer containing SY3 conjugated antibodies.

After this, re-suspend the samples in 300 microliters of phosphate-buffered saline for fax analysis. A representative growth curve demonstrating the proliferation rate of the bone marrow cells undergoing differentiation into natural killer cells in feeder-free system. The graph shows a significant increase in the proliferation rate of the cells by Day Seven.

A representative bright field image showing differentiated natural killer cells in a novel feeder-free system. Mature natural killer cells exhibited high nuclear to cytoplasmic ratio with a granular cytoplasmic morphology. A representative bar plot showing the effect of small interfering RNA on the bone marrow cells undergoing differentiation into natural killer cells on Day Six.

The plot shows significant reduction on the E4bp4 mRNA levels and small interfering RNA treated natural killer cells compared to the control group. A representative flow cytometric data demonstrating the effect of silenced E4bp4 on the differentiation of bone marrow derived natural killer cells in a TGF beta-1 Smad3-dependent manner. Data shows knockdown of E4bp4 mRNA vastly decreases in mature natural killer cell production on Day Six in comparison to the non-sense control.

Small interfering RNA mediated decreased production of natural killer cells is shown to be rescued by suppressing TGF beta-1 Smad3 with one micromolar SIS3 Smad3 inhibitor in comparison to the small interfering RNA transfected group. Once mastered, this study can be done in two hours if it's performed properly. Following this procedure allows genetic modifications such as gene lockdown and over-expression can be conducted in order to answer additional questions like the regulation mechanisms for natural killer cell activity as well as the development.

The first I had the idea for this method when I was expanding the natural killer cells in the old system. The presence of the fetal cells decreased the load and efficiency of siRNA to the interest gene. After watching this video, you should have a good understanding of how to produce massive high-quality natural killer cells from this local system in vitro.

Don't forget that working with RNA extraction reagents can be extremely hazardous, and precautions should always be taken while performing this procedure.