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 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%.
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 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.
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
2. Cell cultivation of porcine adipose tissue-derived stromal cells
3. Labelling of cells with calcein-AM
NOTE: Cells that are injected into cadaveric tissues are stained with a green-fluorescent membrane-permeable live-cell stain and a red-fluorescent membrane-impermeant viability indicator to verify that extracted cells are the same as the injected cells and not tissue fragments of the urethra.
4. Prepare urethral tissue samples for injections
5. Injections of cells via a Williams needle in fluids and tissue samples
6. Injections of cells via Waterjet in fluids and tissue samples
7. Biomechanical assessment of cellular elasticity by atomic force microscopy (AFM)
8. Statistical analysis
Following cell delivery via the two approaches, the viability of cells delivered through the WN (97.2 ± 2%, n=10, p<0.002) was higher when compared to injections by WJ using the E60-10 settings (85.9 ± 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), while WJ injections triggered a significant reduction of the cellular EM (0.440 kPa, p<0.001, Figure 3B). A decrease of 40 – 50% of the EM after WJ injections was noted. Even though, WN injections in cadaveric urethra tissue yielded no significant difference in cellular EM (Figure 4A) a significant reduction in EM was noted after WJ injections in tissue samples (0.890 kPa to 0.429 kPa; p<0.00, Figure 4B). Thus, absolute EM values after WJ injection were thereby reduced by 51%. Collectively, the results show that while WJ cell delivery fulfills an absolute requirement for a clinical implementation where more than 80% viable cells post delivery18 , post WJ delivery the cell elastic moduli are affected. A lower cellular EM might facilitate the migration of features of the cells after WJ delivery. In such a wider distribution and range of regenerative capacities in the desired region19.
Figure 1. Anatomy of porcine bladder and urethra and injection sites. A) The ventral side of the porcine bladder and urethra with the three ligaments fixing the bladder in the abdominal and pelvic cavity are shown. Additionally, the ureters that end on the dorsal side of the bladder are shown. B) Representative image of the cadaveric urethra used for WJ and WN injection. Longitudinally, the dorsal opened urethra with injection domes circled in black are shown. Please click here to view a larger version of this figure.
Figure 2. Cellular viability determination injections via WN and WJ. pADSCs injected via WN or WJ were collected after injection and counted via Trypan exclusion to determine the viability. The cellular viability was significantly reduced after WJ injection when compared to cells delivered via WN. *** p<0.001. The data is graphically displayed as mean with standard deviation. Abbreviations: WJ – waterjet, WN – Williams needle. Figure adapted from Danalache et al. 202119. Please click here to view a larger version of this figure.
Figure 3. Comparison of the quantified Young's moduli of WN respectively WJ delivered cells into capture media and their corresponding controls. No notable difference in EM was observed in the boxplots for the control (untreated) cell monolayers and WN delivered cells (A). Contrastingly, a significant decrease in elasticity can be noted between the boxplots of the control cells and the WJ group (B). ns – not significant, p > 0.05, ***p<0.001. Abbreviations: WJ – waterjet, WN – Williams needle. Figure adapted from Danalache et al. 202119. Please click here to view a larger version of this figure.
Figure 4. Comparison of the quantified Young's moduli of WN respectively WJ delivered cells into cadaveric urethra and their corresponding controls. No notable difference was observed between cells delivered via WN cells and their corresponding controls (A). A significant decrease in elasticity was noted between the WJ delivered cells and the control cell monolayer (B). ns – not significant, p > 0.05, ***p<0.001. Abbreviations: WJ – waterjet, WN – Williams needle. Figure adapted from Danalache et al. 202119. Please click here to view a larger version of this figure.
Approach parameter | Value |
Approach IGain | 3.0 Hz |
Approach PGain | 0.0002 |
Approach target height | 10.0 µm |
Approach setpoint | 3.00 V |
Approach baseline | 0.00 V |
Table 1. Approach parameters.
Run parameter | Value |
Set point | 10 nN |
Z Movement/ Extend Speed | Constant speed/ |
5.0 µm/s | |
Contact time | 0.0 s |
Pulling length | 90 µm |
Delay Mode | Constant Force |
Sample rate | 2000 Hz |
Table 2. Run parameters.
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: more than 80% viable cells post delivery18. While a standardized and reproducible protocol is achieved with the WJ approach, the outcome of needle injection delivery is highly dependent on the size and nozzle of the syringe and needle, pressure, flow rate and the physician performing the injection themselves19.
Studies employing WJ cell delivery in living animal models showed that by varying ejection pressure, the penetration depth can be adapted to the targeted tissue and as such, to the desired clinical application13,16. Transurethral cell injections in living animals under visual control reported misplacement or loss of cells in about 50% of animals treated20, while WJ injections reported precise cell injection rates above 90% (Linzenbold et al.16 and unpublished observation). The current golden standard for cell delivery (needle injections) require penetration of the cannula in a targeted tissue. Therefore, needle cell translation causes injury and trauma in all cases. In the urethra, this may actually cause inflammation ad toxification due to germ and toxins found in even in healthy urine. Additionally, the user-operation time in WJ injection is significantly shorter when compared to a needle injection: cells are placed within milliseconds into the intended tissue layer by presetting the pressure levels. In contrast, penetration depth on needle injection in remote areas by endoscopy is dependent on the skills and experience of the surgeon. The reproducibility of WJ injection is also expected to be superior, but presently only pre-clinical data exist15,16,20 and less than 200 animals were investigated. In our recent study we noted that cellular elasticity is reduced by WJ application when compared to needle injections21. This can be attributed to the shear stress of cells in higher velocity during WJ delivery. Moreover, eventual cell loss might be compensated by a higher precision of cell placement and ejection within the region of interest as achieved with WJ guided delivery by visual guided cystoscopy22.
It is well known that mechanical forces direct stem cell behavior, regeneration potential as well as their subsequent viability and functionality post-transplantation 10,23. The advent of atomic AFM provided a powerful tool for quantifying the mechanical properties of single living cells in nano scale resolution in aqueous conditions 24,25,26,27. AFM is a reliable and highly sensitive method that can detect and record stiffnesses ranging from less than 100 Pa to 106 Pa, thus covering a wide range for the majority tissues and cells28. In fact, cell mechanics is emerging as label-free biomarker for evaluating cell state and in both physiological and pathological state29 and cell elasticity is the synergetic and cumulative response of the nucleus – cytoskeleton crosstalk. It is well established that when a cell is subjected to external forces, these forces are transmitted from the plasma membrane via the cytoskeleton to the nucleus, resulting in intra-nuclear deformations and reorganization30,31,32. Therefore the nucleus, long seen as the genomic material and transcription apparatus, is a key player in the cellular mechanotransduction as well32. In fact, the importance of nuclear mechanics and nucleo-cytoskeletal connections, in all cellular functions and mutations, in lamins and linkers of the nucleo-skeleton to the cytoskeleton (LINC) complex – are at the onset foundation of several pathologies 33,34. These forces could also indicate cellular artifacts. Specifically, external generated forces actually propagate along cytoskeletal filaments and are further transmitted to the nuclear lamina across the LINC complex; in response to these forces, the nucleus, in fact, becomes stiffer35,36, thus merging the two closely intertwined and connected processes. This is also the rationale of our approach and our nuclear mechanics measurements. Moreover, a precise placement of the cantilever on top of the nuclear surface also ensures a high degree of reproducibility and reduces variations owing to cell heterogeneity and attachment to the substratum.
Even though cell elasticity is emerging as label-free biomarker for evaluating the cell state and in both physiological and pathological states29, the measured elastic moduli are marked by large variations even in the same cell type37. A method to counteract such variations as suggested by Schillers et al. is the implementation of standardized nanomechanical AFM procedures (SNAP) that ensure a high reproducibility and applicability of elasticity measurements as a reliable quantitative marker to cells in various states38. Also, even though experimental parameters employed in the AFM analyses, such as indentation velocity, indenter shape and size as well as accurate representation of tip geometry in model fitting 39, influence the absolute measured values40,41, these parameters should not impact the results within one study or a measured tendency.
However, bear in mind that AFM indentations are restricted to the analysis of the outer surface of cells and are, thus, incapable of scanning the inside of a cell membrane or particular intracellular structures. Usukura et al. proposed a "unroofing" method that breaks the cellular membrane and removes the cytoplasmic-soluble components42, thus allowing AFM- intracellular investigations. In our study, however, the focus was placed on the assessment of average elastic moduli rather than probing distinct and selective intracellular components.
Collectively, the consistency and reliability of the yielded AFM data strongly depends on the technical experience of the respective operator and could be biased by biological variability38. Accounting for all the sensitive variables that might affect the actual AFM results, the absolute elastic values reported in this study cannot be generalized and are rather specific for our experimental setup.
Overall, our study provides evidence21 as well as a step-by-step protocol for the superiority of WJ injections over needle injections for regenerative cell therapy regimen.
The authors have nothing to disclose.
We thank our co-authors from the original publications for their help and support.
50 mL centrifuge tube | Greiner BioOne | 227261 | |
1 mL BD Luer-LokTM Syringe | BD Plastik Inc | n.a. | |
100 µm cell sieve | Greiner BioOne | 542000 | |
15 mL centrifuge tube | Greiner BioOne | 188271 | |
75 cm2 tissue culture flask | Corning Incorporated | 353136 | |
AFM head | (CellHesion 200) JPK | JPK00518 | |
AFM processing software | Bruker | JPK00518 | |
AFM software | Bruker | JPK00518 | |
AFM system Cell Hesion 200 | Bruker | JPK00518 | |
All-In-One-Al cantilever | Budget Sensors | AIO-10 | 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 µm (490 – 510 µm) Width: 30 µm (35 – 45 µm) Thickness: 2.7 µm (1.7 – 3.7 µm) |
Amphotericin B solution | Sigma | A2942 | 250 µg/ml |
Atomic Force Microscope (AFM) | CellHesion 200, JPK Instruments, Berlin, Germany | JPK00518 | |
BD Microlance 3 18G | BD | 304622 | |
bovine serum albumin | Gibco | A10008-01 | |
Cantilever | All-In-One-AleTl, Budget Sensors, Sofia, Bulgaria | AIO-TL-10 | tip A, k ¼ 0.2 N/m |
C-chip disposable hemocytometer | NanoEnTek | 631-1098 | |
centrifuge: Rotina 420R | Hettich Zentrifugen | ||
Collagenase, Type I, powder | Gibco | 17100-017 | |
Dulbecco’s Modified Eagle’s Medium – low glucose | Sigma | D5546 | |
Feather disposable scalpel (No. 10) | Feather | 02.001.30.010 | |
fetal bovine serum (FBS) | Sigma | F7524 | |
HEPES sodium salt solution (1 M) | Sigma | H3662 | |
Inverted phase contrast microscope (Integrated with AFM) | AxioObserver D1, Carl Zeiss Microscopy, Jena, Germany | L201306_03 | |
laboratory bags | Brand | 759705 | |
Leibovitz's L-15 medium without l-glutamine | Merck | F1315 | |
Leibovitz's L-15 medium without L-glutamine | (Merck KGaA, Darmstadt, Germany) | F1315 | |
L-glutamine | Lonza | BE 17-605C1 | 200 mM |
LIVE/DEADTM Viability/Cytotoxicity Kit | Invitrogen by Thermo Fisher Scientific | L3224 | Calcein AM and EthD-1 are used from this kit. |
Microscope software: Zen 2.6 | Zeiss | ||
Microscope: AxioVertA.1 | Zeiss | ||
Nelaton-Catheter female | Bicakcilar | 19512051 | |
Penicillin-Streptomycin | Gibco | 15140-122 | 10000 U/ml Penicillin 10000 µg/ml Streptomycin |
Petri dish heater associated with AFM | Bruker | T-05-0117 | |
Petri dish heater associated with AFM | JPK Instruments AG, Berlin, Germany | T-05-0117 | |
Phosphate buffered saline (PBS) | Gibco | 10010-015 | |
Statistical Software: SPSS Statistics 22 | IBM | ||
Sterile Petri dish – CellStar | Greiner BioOne | 664160 | |
Tissue culture dishes | TPP AG | TPP93040 | |
Tissue culture dishes | TPP Techno Plastic Products AG, Trasadingen, Switzerland | TPP93040 | |
Trypan Blue 0.4% 0.85% NaCl |
Lonza | 17-942E | |
Trypsin-EDTA solution | Sigma | T3924 | |
Waterjet: ERBEJET2 device | Erbe Elektromedizin GmbH | ||
Williams Cystoscopic Injection Needle | Cook Medical | G14220 | 23G, 5.0 Fr, 35 cm |