正常に伝達する初代ヒト気管支上皮細胞に必要な最適なレンチウイルスの生産および細胞培養条件

The overall goal of this methodology is to achieve highly efficient transduction of primary human bronchial epithelial cells using lentiviruses. This method can help researchers who want to use primary human bronchial epithelial cells rather than cell lines, and might be adaptable to other difficult to transduce cell types. The main advantage of this technique is a high transduction efficiency maintained throughout differentiation into a psuedostratified epithelium.

The protocol should be executed in line with institutional and governmental regulations under enhanced BSL-2 conditions in the USA. After preparing HEK medium in a Collagen I coated dish, according to the text protocol, use 10 milliliters of the medium to plate one million HEK cells onto the coated dish. Incubate the dish at 37 degrees Celsius, and 5%carbon dioxide, until the cells are 50 to 60%confluent.

This will be considered Day 0. On Day 0, in a laminar flow cabinet, using a commercial transfection kit, bring reagents to room temperature. For each 10 centimeter dish of HEK293 cells, in a 15 milliliter centrifuge tube, combine the following:4.5 micrograms of pMD2-VSVG envelope DNA, 7.5 micrograms of pMDLg/pRRE packaging DNA, 3.75 micrograms of pRSV-rev DNA, 10 micrograms of lentiviral expression vector, and 86 microliters of a 2 Molar calcium solution.

Then add sterile water to a final volume of 700 microliters. Still in a laminar flow cabinet, while vortexing, add 700 microliters of 2x Hepes Buffered Saline dropwise, and incubate at room temperature for 30 minutes. Early in the incubation, deposit 5 microliters of the mixture on a small dish.

Under a microscope, with a 10x objective, check the drop by slowly focusing up and down. A fine precipitate should be visible, confirming the calcium phosphate precipitation process. The most critical step is the precipitate.

If none is present, it may indicate a bad plasmid preparation or miscalculation of plasmid or transfection reagents. Proceeding to the next step with our precipitate, may result in low virus preparation and/or pro-transductions. Stop the precipitation after 30 minutes by adding 10 milliliters of HEK medium and pipetting up and down to mix.

Then, remove the medium from the dish of HEK cells and carefully and slowly add the precipitate solution along the edge of the dish. The next day, remove and discard the medium containing the precipitate and replace it with 10 milliliters of HEK medium. Under the microscope, check for fine precipitate.

It should be visible in the dish in empty spaces, between the cells. The following day, using a syringe, collect the medium, and filter it through a 0.45 micrometer filter, into a 15 milliliter centrifuge tube containing 3.8 milliliters of 40%PEG solution. Invert the tube five times to mix, and store at four degrees Celsius for at least 24 hours, until all the virus collections are complete.

On Days 3 and 4, repeat the medium collection and filtering. Then use 10%bleach to decontaminate the dish before discarding. Centrifuge all of the collection tubes at 1, 650 x g and four degrees Celsius for 20 minutes to pellet the virus.

Remove and discard the supernatant, and spin again for five minutes to collect and remove any remaining PEG supernatant. Use 200 microliters of bronchial epithelial growth medium, or BEGM, without amphotericin B, to re-suspend each pellet. Then, pull them together, and prepare 200 microliter aliquots.

Store the aliquots at 80 degrees Celsius. In a Collagen I coated dish, plate one million HBE cells in 10 milliliters of BEGM medium. Incubate at 37 degrees Celsius, and 5%carbon dioxide, replacing the medium every day for a total of four days.

After coating permeable support inserts with Collagen IV, according to the text protocol, to transduce 80 to 90%confluent HBE cells, remove the medium, and add three milliliters of 0.1%trypsin, and one milliMolar EDTA PBS. Incubate at 37 degrees Celsius for five minutes. When the cells have detached, add three milliliters of Soybean Trypsin Inhibitor, suspend the cells, and transfer to a 15 milliliter centrifuge tube.

Then pellet the cells by spinning at 460 x g and room temperature for five minutes. Remove the supernatant, and use three milliliters of BEGM to re-suspend the pellet and proceed with counting. Then, after counting the cells, add more BEGM so that each 12 millimeter permeable support insert contains 200, 000 cells and 400 microliters.

Using a multiplicity of infection, or MOI, factor of four, add the appropriate volume of virus to the cell suspension. Then, add hexidine methylene bromide, at a final concentration of two micrograms per milliliter. Dispense 400 microliters of the cell mixture per 12 millimeter permeable support insert into the apical compartment, and add one milliliter of BEGM alone to the basolateral compartment.

Incubate overnight at 37 degrees Celsius and 5%carbon dioxide. The following day, remove the apical and basolateral medium, and use PBS to wash the cells. Then, use air-liquid interface, or ALI, medium to replace both compartments.

Finally, when the cells reach confluency, remove the apical fluid, and continue to change the medium in the bottom compartment every other day, until the cells reach maturity. Refer to the text protocol for additional details. Shown here are HEK cells at approximately 50 to 60%confluency, at which point, the cells are optimal for transfection.

This figure illustrates the precipitate in a drop in the transfection mixture. A precipitate that looks more like sand grains, rather than agglomerates, will increase the efficiency of the transfection. This image shows an example of primary HBE cells, that are approximately 80%confluent, prior to their trypsinization and transduction.

In this EGFP transduction experiment, using an MOI of four on permeable support inserts, a nearly 100%transduction rate was achieved. Once mastered, the transfection portion of this protocol can be done in 45 minutes. The entire protocol, however, will take at least 10 days.

While attempting this procedure, it is important to remember that this protocol should be executed in line with institutional and governmental regulations on the BSL-2+conditions. Following this procedure, primary human bronchial epithelial cells, rather than cell lines, can easily be manipulated to over-express or silence proteins of specific genes. These primary cells are more representative of in vivo tissues.

After watching this video, you should have a good understanding of how to achieve highly efficient transductions of primary human bronchial epithelial cells using lentiviruses.