Name: injection of porcine adipose tissue-derived stroma cells via waterjet technology
Uploaded: 2021-11-23T15:00:00
Description: Watch this Scientific Journal Video about Injection of Porcine Adipose Tissue-Derived Stroma Cells via Waterjet Technology at JoVE.com

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 ...

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