Injection of Porcine Adipose Tissue-Derived Stroma Cells via Waterjet Technology

Waterjet injections of active components, such as liquids, nanoparticles, microparticles, encapsulated drugs, or even living cells can be applied much more precise when compared to needle injections in different tissues targeted. Minimally invasive approaches require precise localization of the medication in predetermined layers of the targeted tissue by preselecting injection pressures. Our current focus is the cell therapy for urinary incontinence.

However, this technique has already been extended to the regeneration of infected muscle tissue after severe heart attacks. At present, the technique can be only applied by experienced researchers or licensed physicians in pre-clinical studies. It is not intended to be used in routine laboratory procedures as it is developed for future clinical purposes.

To begin, place the urethra with the connecting bladder dissected from an adult female landrace pig on a sponge which mimics the elasticity of the lower pelvic floor. Then using the bladder, determine the orientation of the urethra. This is possible due to the localization of the ureter and the three ligaments, which fix the bladder in the abdominal and pelvic cavity.

After correctly orienting the urethra, cut it open longitudinally on the dorsal side with the aid of a catheter. After adjusting the cell density of the porcine adipose tissue-derived stromal cells to 2.4 times 10 to the sixth cells per milliliter, label the cells with a green fluorescent membrane permeable live cell stain, then aspirate the cell suspension with the syringe and apply the Williams cytoscopic injection needle. Next, either hold the needle shortly above the two milliliters of growth media in a 15 milliliter centrifugation tube or insert the needle into the open urethra tissue.

In both cases, manually inject 250 microliters of the cell suspension. Cells injected into the cadaveric urethra from an injection dome. Collect the cells injected into the media directly by centrifugation.

For cells injected into the cadaveric urethra, aspirate them out of the injection dome with an 18 gauge needle applied on a syringe. Transfer the aspirated cells into a centrifugation tube and pellet them by centrifugation. Resuspend both cell pellets in four milliliters of growth media.

To determine cell yield and viability, mix 20 of the cell suspension thoroughly with 20 microliters of trypan blue. Then fill 10 microliters of this mixture in each chamber of the hemocytometer. Under a microscope count the cells in all four corner squares.

Count the unstained white cells as viable cells and the blue stained cells as dead cells, then calculate the cell number per milliliter. For injections via the waterjet, adjust the density of the porcine adipose tissue-derived stromal cells to six times 10 to sixth cells per milliliter. Label them as demonstrated earlier and fill the cell suspension into the dosing unit of the needle-free waterjet device.

Then either hold the injection nozzle shortly above the two milliliters of growth media in a 15 milliliter centrifugation tube or shortly above the open urethra tissue. In both cases, inject 100 microliters of the cell suspension. Use high pressure for tissue penetration followed by a low pressure phase for cell injections.

Cells injected into the cadaveric urethra form an injection dome. Collect cells injected into the media directly by centrifugation. For cells injected into the cadaveric urethra, aspirate them out of the injection dome.

Transfer the aspirated cells into a centrifugation tube, and pellet them using centrifugation as demonstrated earlier. After centrifugation, resuspend both pellets in four milliliters of growth media. Seed the cells retrieved after injections in tissue culture dishes at a density of five times 10 to the fifth cells per dish.

After a three hour incubation at 37 degrees Celsius, and right before the AFM measurement, replace the growth media with three milliliters of Leibovitz's L-15 media without L-glutamine. To measure the elasticity of individual cells, visually identify one cell and focus on it. Then place the computer mouse at the middle of the cell and to improve measurements precision, position the AFM tip directly above the cell nucleus.

Next, focus on the cantilever and move it on the computer mouse. Then start the measurement with run and measure at least 50 cells. The set point parameter obtained by calibration of the cantilever is used and one cell is measured three times.

The viability of cells delivered through the Williams needle was higher than injections by the waterjet using the E60-10 settings. Biomechanical assessment of injections in the capture fluid revealed that Williams needle injections displayed no significant difference in the elastic moduli when compared to controls, while the waterjet injections significantly reduced the cellular elastic moduli by 40 to 50%Similarly, in cadaveric urethra tissue samples, Williams needle injections yielded no significant difference in cellular elastic moduli compared to controls. However, a significant reduction of 51%was observed after waterjet injections.

One has to only make sure that the needle injections does not fully penetrate the tissue that's inflicting injury. With waterjet, one has to only place the device in the correct position in the injection area while the rest is automatically done by the device itself. As the waterjet device was developed to match and fit into endoscopic devices, you can use the waterjet applications for any site in the body which can be reached by these cystoscopic or endoscopic technologies to regenerate tissues and do the repair job.