The overall goal of this in vitro organ culture method is to maintain homeostasis and observe the inflammatory response of intervertebral discs in their native anatomy. This method can help answer key questions in intervertebral disc degeneration and low back pain, and it may aid in discovering new drugs and therapies. The main advantage of this method is that it can be done rapidly while maintaining the anatomy of the disc and other tissues.
Generally, individuals new to this method will struggle, because it is hard to maintain sterility, as any contamination my compromise the experimental results. Because of the minute size of mouse intervertebral discs, visual demonstration of the dissection and organ culture steps is critical. Before beginning this protocol, purchase two strains of ten-week-old mice, BALB/c and transgenic animals with the luciferase gene expressed under the control of the nuclear factor kappa B promoter, bred on the BALB/c genetic background.
To begin, bathe the previously euthanized mouse in 70%ethanol for two minutes to ensure its skin and coating is disinfected for the dissection. Then, make a longitudinal cut in the animal's dorsal skin using small dissection scissors to expose the body cavity. Proceed with two longitudinal vertical cuts, starting from the L1 vertebrae to the C8 vertebrae on both sides of the spine.
Use a scalpel, fine forceps, and fine dissection scissors to carefully remove the L1 to C8 spine fragment from the mouse body cavity. Excise any excess soft tissues that surround the spine. Be careful not to scrape or injure the intervertebral disc, or IVD, on the ventral side.
Divide the spinal column into functional spine units, or FSUs, comprising vertebrae disc vertebrae fragments. Then, wash the obtained FSUs in Hank's balanced salt solution supplemented with antibiotics for two minutes. Now that FSUs are washed, assign them randomly to three experimental groups marked as fresh, control, and stab.
Immediately after preparation, freeze the fresh FSUs in liquid nitrogen, and store the samples in a negative 20 degree Celsius freezer. To initiate the organ culture, use ten-milliliter serological pipettes to add two milliliters of DMEM/F-12 culture medium into each well of a 24-well culture plate. Then, transfer each dissected and washed FSU to an individual well of the 24-well plate filled with media.
Incubate the 24-well plate containing the samples in a disinfected incubator at 37 degrees Celsius, 5%carbon dioxide, 20%oxygen, and greater than 90%humidity. After culturing the samples for 24 hours, use a sterile 27-gauge needle to puncture the annulus fibrosus so as to induce mechanical injury in the samples from the stab group. Every 48 hours, change the media by transferring the samples with tweezers to a new 24-well plate filled with media.
On the twenty-first day of culture, transfer all samples into media containing 0.75 milligrams per milliliter nitroblue tetrazolium chloride, and incubate them for an additional 24 hours. After 24 hours of incubation, freeze all the FSUs in liquid nitrogen, and store the samples in a negative 20 degree Celsius freezer for further testing. To assess NF kappa B expression, add ten microliters of a one milligram per milliliter luciferin solution to each well containing FSU isolated from an animal transgenic for NF kappa B luciferase.
Then, incubate the plate in a sterile incubator at 37 degrees Celsius for ten minutes. Under the dissection microscope, use a scalpel and tweezers to remove the bony vertebral bodies from each FSU sample. Be careful not to damage or detach the cartilaginous endplates from the IVD.
Then, using cyanoacrylate glue, attach the isolated IVDs to a one centimeter by one centimeter by 0.2 centimeter aluminum plate. Subsequently, measure the disc height and width with a laser micrometer, and calculate the disc height ratio as described in the text protocol. Next, transfer the samples to the phosphate-buffered saline bath positioned under the compression machine, and preload the disc to 0.02 newtons.
To cyclically compress the disc, apply 20 cycles of a sinusoidal waveform at 1 hertz and conduct three trials for both 1%strain level and 5%strain level. Record the load and displacement data to calculate the average stiffness and loss tangent of the IVD. Presented here are images of the bioluminescent signal corresponding to NF kappa B expression in control and stab samples.
The signal in both control and stab samples indicates that the IVD cultured in vitro remains viable and responsive. Additionally, stab samples reveal increased NF kappa B expression compared to the control on days one, five, 13, and 19. Further mechanical testing conducted for the IVDs cultured in vitro confirms that culture conditions do not influence the properties of the control IVDs, such as disc height ratio, stiffness, or loss tangent.
In addition, the decreased disc height ratio, stiffness, and loss tangent observed for the stab group further confirms viability and responsiveness of the IVD cultured in vitro. Once this technique has been mastered, up to 18 samples can be prepared for culture in an hour. While attempting this procedure, it's important to work slowly and carefully to ensure that sterility is maintained.
Following this procedure, more mechanistic studies can be performed using genetically modified mice to get a better understanding of disc degeneration. This technique can also be used to explore various treatments and therapies for low back pain. After watching this video, you should have a good understanding of how to prepare and culture mouse functional spine units for experimental conditions.
Don't forget that working in a lab can be hazardous, and precautions, including personal protective equipment, should always be taken while performing this procedure.
