We thank our co-authors from the original publications for their help and support.

The porcine adipose tissue samples were obtained from the Institute for Experimental Surgery at the University of Tuebingen. All procedures were approved by local animal welfare authorities under the animal experiment number CU1/16.
1. Isolation of porcine adipose tissue-derived stromal cells
<ol>
	<li>Use porcine adipose tissue delivered from the Institute for Experimental Surgery in a 50 mL centrifuge tube to the laboratory.</li>
	<li>Transfer the tissue to a sterile Petri...

In the present study, we demonstrated and presented a step-by-step approach for WJ cell delivery procedure and employed a sequela of quantitative investigations to assess the effect of WJ delivery on cellular characteristics: cellular viability and biomechanical features (i.e., EM). Following WJ injection, 85.9% of the harvested cells were viable. In terms of WN injection, 97.2% of the cells retained their viability after injection. Thus, the WJ approach fulfills an absolute requirement for a clinical implementation: mor...

Urinary incontinence (UI) is a widespread disorder with a prevalence of 1.8 - 30.5% in European populations1 and is characterized primarily by malfunctioning of the urethral sphincter. From a clinical perspective, surgical treatment is often offered to patients when conservative therapies or physiotherapy fail to address and alleviate the emerging symptoms.
Cell therapy for the potential regenerative repair of the sphincter complex malfunction has been emerging as an avant-garde approach for the treatment of UI pathology2,3. Its main goals are to replace, repair and restore the biological functionality of the damaged tissue. In animal models for UI, stem cell transplantation has shown promising results in urodynamic outcomes2,4,5. Stem cells arise as optimal cellular candidates as they have the ability to undergo self-renewal and multipotent differentiation, thus, aiding the affected tissue regeneration6. Despite the forthcoming regenerative potential, the practical use of cell therapy remains hindered as minimally invasive delivery of cells still face several challenges concerning the injection precision and coverage of the target. Even though the current approach used for cell delivery is injection through a needle-syringe system7, it usually results in an overall deficit of viable cells, with reported viabilities as low as 1%- 31% post-transplantation8. In addition, cell delivery via needle injection has been also shown to affect the placement, the retention rate, as well as distribution of transplanted cells into the targeted tissue9,10,11. A feasible, novel approach that overcomes the abovementioned limitation is the needle-free cell delivery via water-jet technology.
Waterjet (WJ) technology is emerging as a new approach that enables high throughput delivery of cells by cystoscope under visual control in the urethral sphincter12,13. The WJ enables cell delivery at different pressures (E = effects in bar) ranging from E5 to E8013. In the first phase, (tissue penetration phase) isotonic solution is applied with high pressure (i.e., E60 or E80) in order to loosen the extracellular matrix surrounding the tissue targeted and open small interconnecting micro-lacunae. In the second phase (the injection phase), pressure is lowered within milliseconds (i.e., up to E10) in order to gently deliver the cells into the targeted tissue. Following this two step-phase application, the cells are not subjected to additional pressure against the tissue when ejected but are floating in a low-pressure stream into a liquid-filled cavernous area13. In an ex vivo model setting where stem cells were injected via WJ into cadaveric urethra tissue, viable cells could be afterwards aspirated and retrieved from the tissue and further expanded in vitro13. Though a 2020 study by Weber et al. demonstrated the feasibility and applicability of WJ to deliver footprint-free cardiomyocytes into the myocardium14, it has to be borne in mind the WJ technology is still in a prototype stage.
The following protocol describes how to prepare and label porcine adipose tissue-derived stromal cells (pADSC) and how to deliver them into capture fluid and cadaveric tissue via WJ&#160;technology and Williams cystoscopy needles (WN). Post cellular injection, the cellular vitality and elasticity via atomic force microscopy (AFM) is assessed. Via step-by-step instructions, the protocol gives a clear and concise approach to acquire reliable data. The discussion section presents and describes the major advantages, limitations and future perspectives of the technique. The WJ delivery of cells as well as the sequela post translation analyses reported here are replacing the standard needle injection and provide a solid cell delivery framework for regenerative healing of the target tissue. In our recent studies we provided evidence that WJ delivered cells more precisely and at least at comparable viability when compared to needle injections15,16.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Greiner BioOne</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL BD Luer-LokTM Syringe</td>
			<td>BD Plastik Inc</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m cell sieve</td>
			<td>Greiner BioOne</td>
			<td>542000</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Greiner BioOne</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 tissue culture flask</td>
			<td>Corning Incorporated</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM head</td>
			<td>(CellHesion 200) JPK</td>
			<td>JPK00518</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM processing software</td>
			<td>Bruker</td>
			<td>JPK00518</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM software</td>
			<td>Bruker</td>
			<td>JPK00518</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM system Cell Hesion 200</td>
			<td>Bruker</td>
			<td>JPK00518</td>
			<td></td>
		</tr>
		<tr>
			<td>All-In-One-Al cantilever</td>
			<td>Budget Sensors</td>
			<td>AIO-10</td>
			<td>tip A, Conatct Mode, Shape: Beam 
			Force Constant: 0.2 N/m (0.04 - 0.7 N/m) 
			Resonance Frequency: 15 kHz (10 - 20 kHz) 
			Length: 500 &#181;m (490 - 510 &#181;m) 
			Width: 30 &#181;m (35 - 45 &#181;m) 
			Thickness: 2.7 &#181;m (1.7 - 3.7 &#181;m)</td>
		</tr>
		<tr>
			<td>Amphotericin B solution</td>
			<td>Sigma</td>
			<td>A2942</td>
			<td>250 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Atomic Force Microscope (AFM)</td>
			<td>CellHesion 200, JPK Instruments, Berlin, Germany</td>
			<td>JPK00518</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Microlance 3 18G</td>
			<td>BD</td>
			<td>304622</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>Gibco</td>
			<td>A10008-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cantilever</td>
			<td>&#160;All-In-One-AleTl, Budget Sensors, Sofia, Bulgaria</td>
			<td>AIO-TL-10</td>
			<td>tip A, k &#188; 0.2 N/m</td>
		</tr>
		<tr>
			<td>C-chip disposable hemocytometer</td>
			<td>NanoEnTek</td>
			<td>631-1098</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge: Rotina 420R</td>
			<td>Hettich Zentrifugen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type I, powder</td>
			<td>Gibco</td>
			<td>17100-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium - low glucose</td>
			<td>Sigma</td>
			<td>D5546</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather disposable scalpel (No. 10)</td>
			<td>Feather</td>
			<td>02.001.30.010</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES sodium salt solution (1 M)</td>
			<td>Sigma</td>
			<td>H3662</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted phase contrast microscope (Integrated with AFM)</td>
			<td>AxioObserver D1, Carl Zeiss Microscopy, Jena, Germany</td>
			<td>L201306_03</td>
			<td></td>
		</tr>
		<tr>
			<td>laboratory bags</td>
			<td>Brand</td>
			<td>759705</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 medium without l-glutamine</td>
			<td>Merck</td>
			<td>F1315</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 medium without L-glutamine</td>
			<td>(Merck KGaA, Darmstadt, Germany)</td>
			<td>F1315</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Lonza</td>
			<td>BE 17-605C1</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>LIVE/DEADTM Viability/Cytotoxicity Kit</td>
			<td>Invitrogen by Thermo Fisher Scientific</td>
			<td>L3224</td>
			<td>Calcein AM and EthD-1 are used from this kit.</td>
		</tr>
		<tr>
			<td>Microscope software: Zen 2.6</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope: AxioVertA.1</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nelaton-Catheter female</td>
			<td>Bicakcilar</td>
			<td>19512051</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>10000 U/ml Penicillin 
			10000 &#181;g/ml Streptomycin</td>
		</tr>
		<tr>
			<td>Petri dish heater associated with AFM</td>
			<td>Bruker</td>
			<td>T-05-0117</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish heater associated with AFM</td>
			<td>JPK Instruments AG, Berlin, Germany</td>
			<td>T-05-0117</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Statistical Software: SPSS Statistics 22</td>
			<td>IBM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Petri dish - CellStar</td>
			<td>Greiner BioOne</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dishes</td>
			<td>TPP AG</td>
			<td>TPP93040</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dishes</td>
			<td>TPP Techno Plastic Products AG, Trasadingen, Switzerland</td>
			<td>TPP93040</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4% 
			0.85% NaCl</td>
			<td>Lonza</td>
			<td>17-942E</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma</td>
			<td>T3924</td>
			<td></td>
		</tr>
		<tr>
			<td>Waterjet: ERBEJET2 device</td>
			<td>Erbe Elektromedizin GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Williams Cystoscopic Injection Needle</td>
			<td>Cook Medical</td>
			<td>G14220</td>
			<td>23G, 5.0 Fr, 35 cm</td>
		</tr>
	</tbody>
</table>

injection of porcine adipose tissue-derived stroma cells via waterjet technology

Urinary incontinence (UI) is a highly prevalent condition characterized by the deficiency of the urethral sphincter muscle. Regenerative medicine branches, particularly cell therapy, are novel approaches to improve and restore the urethral sphincter function. Even though injection of active functional cells is routinely performed in clinical settings by needle and syringe, these approaches have significant disadvantages and limitations. In this context, needle-free waterjet (WJ) technology is a feasible and innovative method that can inject viable cells by visual guided cystoscopy in the urethral sphincter. In the present study, we used WJ to deliver porcine adipose tissue-derived stromal cells (pADSCs) into cadaveric urethral tissue and subsequently investigated the effect of WJ delivery on cell yield and viability. We also assessed the biomechanical features (i.e., elasticity) by atomic force microscopy (AFM) measurements. We showed that WJ delivered pADSCs were significantly reduced in their cellular elasticity. The viability was significantly lower compared to controls but is still above 80%.

Following cell delivery via the two approaches, the viability of cells delivered through the WN (97.2 &#177; 2%, n=10, p&#60;0.002) was higher when compared to injections by WJ using the E60-10 settings (85.9 &#177; 0.16%, n=12) (Figure 2). Biomechanical assessment results showed that: WN injections of cells in capture fluid displayed no significant difference with respect to the elastic moduli (EM; 0.992 kPa) when compared to the controls (1.176 kPa; Figure 3A)...

Watch this Scientific Journal Video about Injection of Porcine Adipose Tissue-Derived Stroma Cells via Waterjet Technology at JoVE.com

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

We present a method of cell injection via needle free waterjet technology coupled with a sequela of post-delivery investigations in terms of cellular viability, proliferation, and elasticity measurements.

Urinary incontinence (UI) is a highly prevalent condition characterized by the deficiency of the urethral sphincter muscle. Regenerative medicine ...

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.

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

Abstract