This method can help answer key questions in the avian virology field, such as how immunosuppressive viruses interact with B cells. The main advantage of this technique is that cells are more relevant than immortalized cell lines currently used in the lab. Though this method provides insight into how chicken cells respond to IBDV, it can also be applied to ALV and REV.
This method reduces the number of birds in our research, and it's beneficial to the three Rs, which stands for the replacement, reduction, and refinement of the use of animal in research. In a microbiological safety cabinet, wash the BF in 30 milliliters of cold PBS at least three times. Transfer the washed tissue to a Petri dish, and add five milliliters of a 1X collagenase D solution.
Use sterile scissors or a scalpel blade to cut the BF into pieces that are less than five millimeters in diameter. Incubate at 37 degrees Celsius with periodic gentle agitation for 30 minutes. After this, use a sterile Pasteur pipette to repeatedly aspirate the mixture to encourage disintegration of the tissue.
Add another five milliliters of 1X collagenase D solution to the tissue, and incubate it at 37 degrees Celsius with periodic gentle agitation for another 30 minutes. Repeat this process, aspirating the mixture, adding fresh collagenase D solution, and incubating until the tissue is completely digested. Note that there will be small granules that will not dissolve further.
Pass the digested cell suspension through a 100-micron cell strainer into 20 milliliters of 1X HBBS without calcium. Centrifuge at 400 times g for five minutes. Discard the supernatant, resuspend the pellet in 10 milliliters of 1X RPMI with 5%FBS.
Then, overlay 10 milliliters of the cell suspension on top of five milliliters of density gradient media that contains polysucrose and sodium diatrizoate. Make sure that there is a clear interface between the two layers. Centrifuge at 900 times g and four degrees Celsius for 20 minutes.
The cells should form a band at the interface between the cell media and the density gradient media. Next, use a sterile Pasteur pipette to remove the cells and transfer them to a tube containing cold PBS. Wash the cells by centrifuging them at 400 times g for five minutes and then resuspending them in cold PBS.
Repeat this wash three times. First, centrifuge the cell suspension at 400 times g for five minutes. Resuspend the cells in 30 milliliters of 1X complete IMDM.
Take an aliquot of the cell suspension, and add it to a Trypan blue solution. Count the number of viable cells that exclude the Trypan blue, and determine the number of cells and the percentage viability. After this, centrifuge the cell suspension at 400 times g for five minutes.
Resuspend the cells in complete IMDM supplemented with a one-to-20 dilution of chicken CD40 ligand at a density of 10 million cells per milliliter. Culture the cells in either 96-or 24-well plates for 48 to 72 hours. 48 to 72 hours post-isolation, thaw an aliquot of the virus.
Vortex the sample, and store it on ice. Resuspend the primary bursal cells in the IMDM medium. Take a 10-microliter aliquot of the cell suspension, and add it to 10 microliters of a Trypan blue solution.
Then, determine the number of cells and percentage viability. Dilute the virus in 1X complete IMDM to the appropriate multiplicity of infection to make the virus inoculum. Mix this dilution by vortexing.
Centrifuge the cell suspension at 400 times g for five minutes. Next, remove the supernatant, and resuspend the cells in the virus inoculum. Incubate at 37 degrees Celsius for one hour with periodic agitation.
After this, centrifuge the cell suspension at 400 times g for five minutes. Remove the virus inoculum, and wash the cells in 1X complete IMDM media. Culture the cells in either 96-or 24-well plates.
In this study, chicken primary bursal cells are successively cultured ex vivo in the presence of soluble chicken CD40 ligand. Cells cultured in the presence of soluble chicken CD40 ligand are seen to increase fourfold from 902, 000 to 3.63 million per milliliter over a period of six days. Cell viability is also significantly improved in the presence of chicken CD40 ligand.
In representative confocal microscopy images, infected cells are seen to have a green fluorescence around the nucleus, which is consistent with the presence of IBDV in the cytoplasm. This is evident for two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661. RNA is then extracted at five, 18, 24, and 48 hours post-infection and subjected to RT-qPCR with primers specific to a conserved region of the IBDV VP4 gene.
IBDV VP4 expression is seen to increase to 16, 603 copies at 48 hours post-infection with D78 and to 38, 632 copies at 48 hours postinfection with UK661. This data demonstrates that chicken primary bursal cells can support the replication of cell-culture-adapted and very virulent IBDV strains. Following this procedure, other methods like RT-qPCR, microarray, or RNA-Seq can be performed in order to answer additional questions, like how the cells respond to the infection.
We have compared how the cells respond to infection with an attenuated strain and a very virulent strain of IBDV and our now investigating how they respond to other viral infections. The ability to culture these cells allows us to study aspects of virus pathogenesis and immunosuppression without the need to infect birds. This will have a substantial impact on the three Rs, the replacement, refinement, and reduction of the use of animals in research.
