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

Summary

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.

Abstract

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%.

Introduction

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 pathology^2,3. 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 outcomes^2,4,5. 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 tissue^9,10,11. 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 sphincter^12,13. 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 injections^15,16.

Protocol

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

1. Use porcine adipose tissue delivered from the Institute for Experimental Surgery in a 50 mL centrifuge tube to the laboratory.
2. Transfer the tissue to a sterile Petri dish under the sterile bench and mince it with two scalpels (No. 10) to small fragments and mush.
   NOTE: Scissors can also be used in order to get small fragments. The smaller the fragments are, the better for the following digestion.
   1. Transfer the small fragments/mush into a 50 mL centrifuge tube and incubate it with 5 mL of homogenizer solution for 30 min at 37 °C on a shaker. The homogenizer solution is PBS with 1% bovine serum albumin (BSA) (stock solution: 1 % (w/v) BSA in PBS) and 0.1% collagenase type I.
      NOTE: Always prepare homogenizer solution freshly. The shaker has a circular shaking motion at a slow speed 25-500 rpm.
3. To stop the incubation, add 10 mL of growth media [Dulbecco's Modified Eagle's Medium - low glucose (DMEM-LG), 2.5% HEPES sodium salt solution (1 M), 10% fetal bovine serum (FBS), 1% L-Glutamin (200 mM), 1% Penicillin-Streptomycin (10000 U/mL Penicillin; 10,000 µg/mL Streptomycin) and 1% Amphotericin B (250 µg/mL)].
   NOTE: Growth media can be prepared beforehand. Antibiotics and antifungal drugs are necessary in the preparation of the growth media for cell culture to protect cells from contaminations.
4. Incubate the centrifuge tubes for 10 min at room temperature.
5. Aspirate the fraction with adipocytes, which is lighter and therefore swimming on top of the liquid, with a 10 mL pipette and discard it.
6. Filter the remaining stromal fraction through a 100 µm cell sieve with a polyethylene terephthalate (PET) membrane free of heavy metals to withhold adipose tissue fragments and to retrieve only the cell suspension.
7. Centrifuge the filtered cell suspension for 7 min at 630 x g at room temperature.
8. Resuspend the cell pellet in 2 mL of PBS to wash the cells once.
9. Centrifuge the cell suspension again for 7 min at 630 x g at room temperature.
10. After centrifugation and repeated resuspension of the cell pellet in 10 mL of growth media per flask, seed cells in 75 cm² cell culture flasks.
    ​NOTE: Depending on the size of the cell pellet after the washing step with PBS, seed cells in one to four flasks.

2. Cell cultivation of porcine adipose tissue-derived stromal cells

1. Harvest and passage cells when they reach 70% confluence.
2. Wash cells with 10 mL of PBS twice and aspirate PBS completely.
   NOTE: This step is necessary to remove remaining growth media, which is complemented by 10% FBS. FBS has several protease inhibitors which can hinder the function of the following trypsinization.
3. Add 3 mL of 0.05%-Trypsin-EDTA per flask to detach the cells.
4. Incubate flasks for 3 min at 37 °C.
5. Check under the microscope if cells are detached.
   NOTE: Sometimes it takes some more time to detach the cells. In this case, incubate flasks for maximum 5 min at 37 °C.
6. To stop the incubation and detaching process, add 3 mL of growth media.
   NOTE: As noted above, growth media is complemented by 10% FBS that contains protease inhibitors.
7. Transfer the detached cells to a centrifugation tube and centrifuge for 7 min at 630 x g at room temperature.
8. Resuspend the cells in 10 mL of growth media and count them with a hemocytometer.
9. Seed cells with an inoculation density of 3 x 10⁵ cells per 75 cm² flask.

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.

1. Wash cells twice with 10 mL of PBS.
2. Add 5 mL of dye solution per 75 cm² flask. The dye solution consists of growth media with 2 µM of the green-fluorescent membrane-permeable live-cell dye and 4 µM red-fluorescent membrane-impermeant viability indicator.
3. Incubate for 30 min at room temperature in the dark.
4. Aspirate and discard the dye solution.
5. Wash cells again twice with 10 mL of PBS.
6. Add growth media and document the green and red fluorescence signal by fluorescence microscopy.
   1. Place the 75 cm² flask on the microscope table, use the 10x objective, select no fluorescence filter and make sure that the transmitted light path is active.
      NOTE: These steps are all performed manually on the microscope.
   2. In parallel to step 3.6.1, open the software program.
   3. Press the Live button under the Locate tab to obtain a live image of the cells in the flask on the table and focus on them with the coarse and fine adjustment drives.
      NOTE: On the right sight the live image will appear once the Live button is activated.
   4. In the sub-tab Microscope Components, select the correct objective (10x).
   5. In the sub-tab Camera, apply a checkmark for Auto Exposure.
   6. Press the button Snap to take the first picture with transmitted light.
   7. Insert the scale bar by using the Graphics tab in the menu bar and selecting Scale Bar.
   8. Save the image by using the Files tab in the menu bar and selecting Save as czi.
   9. For the fluorescence pictures change the filter to the one specific for green or red fluorescence and select the reflected light path.
      NOTE: These changes have to be done manually on the microscope.
   10. Press again the Live button under the Locate tab to obtain a live image of the cells.
   11. Press the button Snap to take the second picture with either green or red fluorescence.
   12. Insert the scale bar by using the Graphics tab in the menu bar and selecting Scale Bar.
   13. Save the image by using the Files tab in the menu bar and selecting Save as czi.
   14. Repeat steps 3.6.9 to 3.6.13 with the other fluorescence channel.

4. Prepare urethral tissue samples for injections

1. Dissect the urethra and the connecting bladder out of the pig.
   NOTE: Urethral tissue samples from fresh cadaveric samples of adult female landrace pigs were used for the experiments.
2. Transport it to the laboratory in bags on wet ice.
   NOTE: The samples should be left on ice until further processing. No additives were added to the samples.
3. Place the urethra with the bladder on a sponge, which mimics the elasticity of the lower pelvic floor.
   1. Use the bladder to determine the orientation (proximal, distal, dorsal, and ventral) of the urethra. This is possible due to the localization of the ureters and the three ligaments which fix the bladder in the abdominal and pelvic cavity. The three ligaments are the two vesicae lateralia ligaments on the sides of the bladder and the vesicae medianum, which arises from the ventral surface of the bladder (Figure 1).
4. Cut open the urethra longitudinally on the dorsal side with aid of a catheter.
5. Inject the cells into the opened urethra as described in the following chapters.

5. Injections of cells via a Williams needle in fluids and tissue samples

1. Harvest cells as described in step 2.
2. In contrast to step 2.9, adjust cell density for injections to 2.4 x 10⁶ cells per mL.
   NOTE: For injections into tissue samples label the cells with a green-fluorescent membrane-permeable live-cell.
3. Aspirate the cell suspension with a syringe and apply the Williams cystoscopic injection needle (WN) to it.
4. Either hold the needle shortly above the 2 mL of growth media in a 15 mL centrifugation tube or insert the needle into the opened urethra tissue. In both cases inject 250 µL of cells manually.
   NOTE: Cells injected into the cadaveric urethra form an injection dome.
5. Collect cells injected into media directly by centrifugation for 7 min at 630 x g at room temperature.
6. For cells injected into the cadaveric urethra, aspirate them out of the injection dome with an 18G needle applied on a syringe.
7. Transfer the injected and aspirated cells into a centrifugation tube and centrifuge for 7 min at 630 x g at room temperature comparably to the cells injected into media.
8. After centrifugation, resuspend the cells in 4 mL of growth media in both cases.
9. Determine cell yield and viability by aid of Trypan blue dye exclusion with a hemocytometer.
   1. Mix 20 µL of cell suspension thoroughly with 20 µL of Trypan blue.
   2. Fill 10 µL of this mixture in each chamber of the hemocytometer.
      NOTE: Both chambers are filled and counted to gain statistical optimization by doubling sampling.
   3. Under a microscope, count cells in all four corner squares.
      NOTE: Count cells shining in white (unstained) as viable cells whereas cells shining blue (stained) are counted as dead cells.
   4. To calculate the cell number per mL, calculate the mean of the two chambers, divide this number by two and multiply it with 10⁴.

6. Injections of cells via Waterjet in fluids and tissue samples

1. Harvest cells as described in step 2.
2. In contrast to steps 2.9 and 5.2, adjust cell density for injections to 6 x 10⁶ cells per mL.
   NOTE: For injections into tissue samples use cells labelled with a green-fluorescent membrane-permeable live-cell.
3. Fill the cell suspension into the dosing unit of the WJ device.
4. Either hold the injection nozzle shortly above the 2 mL of growth media in a 15 mL centrifugation tube or shortly above the opened urethra tissue.
5. In both cases inject 100 µL of cells by the device using a high pressure for tissue penetration followed by a low-pressure phase for cell injections. Use the pressure settings E60-10.
   NOTE: Cells injected into the cadaveric urethra form an injection dome.
6. Collect cells injected into media directly by centrifugation for 7 min at 630 x g at room temperature.
7. For cells injected into the cadaveric urethra, aspirate them out of the injection dome with an 18G needle applied on a syringe.
8. Transfer the injected and aspirated cells into a centrifugation tube and centrifuge for 7 min at 630 x g at room temperature comparably to the cells injected into media.
9. After centrifugation, resuspend cells in 4 mL of growth media in both cases.
10. Determine cell yield and viability by aid of Trypan blue dye exclusion with a hemocytometer as described in detail in 5.10.

7. Biomechanical assessment of cellular elasticity by atomic force microscopy (AFM)

1. Preparation of samples
   NOTE: The retrieved cells following injection are now subject for further analyses by AFM. Additionally, cells that were not injected are used as controls.
   1. Seed cells in tissue culture dishes with a density of 5 x 10⁵ cells per dish.
      NOTE: Seed one tissue culture dish per condition. The conditions are controls, ADSCs injected by WJ or WN into media and ADSCs injected by WJ or WN into cadaveric tissue.
   2. Incubate cells in tissue culture dishes for 3 h at 37 °C.
      NOTE: To avoid variances due to cells in G1 phase and during mitosis, we performed AFM measurements 3 h after the cell seeding step.
   3. Right before the measurement with the AFM replace the growth media with 3 mL of Leibovitz's L-15 media without L-glutamine.
   4. Place the tissue culture dish in the sample holder of the AFM device and turn on the Petri dish heater set at 37 °C.
2. Preparation of AFM and cantilever calibration
   1. Use a glass block that is specified for measurements in liquids and adjust it on the AFM holder.
      NOTE: The upper surface of the glass block must be straight and in parallel to the AFM holder.
   2. Carefully place the cantilever on the surface of the glass block. The tip A must lay over the polished optical plane.
      NOTE: Do not scratch the polished optical surface of the glass block because scratches could lead to interferences in the measurements. The tip A must protrude over the polished optical plane because otherwise the reflection of the laser of the AFM to the photodetector is not possible.
   3. To stabilize the cantilever on the glass block, add a metallic spring with the aid of tweezers.
   4. When the cantilever is fixed on the glass block place the block on the AFM head and lock the integrated locking mechanism.
   5. Mount the AFM head on the AFM device.
      NOTE: The spring must face the left side to be placed correctly. If this is not the case, correct the position of the glass block and adjust the AFM head again.
   6. Initialize the software setup and open the software together with the laser alignment window and the approach parameter settings. Additionally, turn on the stepper motor, the laser light and the CCD-camera.
   7. Identify the cantilever tip A by using the CCD-camera.
   8. Lower the cantilever until it is fully dipped into the medium. Use the stepper motor function to reach this aim.
   9. Once the cantilever is fully covered by medium, the laser is aligned on top of the cantilever by using the adjustment screws. The reflected beam must fall onto the center of the photodetector and the sum of the signals must be 1 V or higher. The lateral and vertical deflections should be close to 0.
      NOTE: If the center is reached can be monitored in the laser alignment function as well as the signal values. If the signal values are not correct, further adjustments are necessary.
   10. Run now the scanner Approach with the approach parameters (Table 1).
   11. Retract the cantilever by 100 µm once it reaches the bottom by pressing the Retract button.
       NOTE: Make sure that no cell is attached at that field were the approach is done because this would falsify the calibration.
   12. Set up the Run parameters according to the following specifications: Setpoint 1 V, Pulling Length 90 µm, Velocity: 5 µm/s, Sample rate: 2000 Hz and as a Delay Mode: Constant Force.
   13. Start the calibration force-distance curve measuring by pressing the button Run.
       NOTE: By clicking the Run button in the software a force-distance curve is obtained.
   14. Select the region of linear fit of the retracted curve in the software on the obtained calibration force-distance curve. Calculate the spring constant measurement by the software.
   15. Calibrate the cantilever following the exact parameters and steps as described by Danalache et al.¹⁷.
3. Measuring the elasticity of individual cells
   1. Visually identify a cell and focus on it. Place the computer mouse at the middle of the cell. To improve the measurements precision, position the AFM tip directly above the cell nucleus.
      NOTE: The computer mouse is used as visual marker to identify the target site of indentation.
   2. Now focus on the cantilever and move it on the computer mouse.
   3. Start the measurement with Run with the parameters given in Table 2.
      NOTE: The set point parameter obtained by calibration of the cantilever are used. Furthermore, one cell is measured three times. Measure at least 50 cells.
4. Data processing
   1. Process the data following exact steps and parameters as previously described by Danalache¹⁷.
   2. Save and export the file.

8. Statistical analysis

1. Open the statistical software.
2. Choose the selection of New Dataset.
   NOTE: Two files are opened. One is the "DataSet" and the other one is the "Output" file.
3. Select the DataSet file and open the Variable View tab.
4. Insert the numeric variables for site injection (capture fluid or cadaveric tissue), category (control, WJ injection, WN injection) and elasticity.
5. Insert the measured elasticity data with their corresponding site injection and category number in the Data View tab.
6. Analyze the data by selecting the menu bar tab Analyze | Descriptive Statistics and choose Exploratory data analysis.
7. As the dependent variable, select Elasticity and factor list select Category.
   NOTE: The results are displayed in the "Output" file including a box plot which is used for the results section.
8. To perform a statistical test, select the Independent Samples within the Nonparametric Test under the menu bar tab Analyze.
9. In the new opened file keep the settings in the tab Objective and Settings.
10. Open the tab Fields and choose Elasticity as Test Fields and Category as Groups.
11. Press Run.
    NOTE: The results are displayed in the "Output" file. For these analyzes a Mann-U-Whitney test is performed.
12. Include the result of the nonparametric test in the box plot of the exploratory data analysis.
13. Save the files by selecting File in the menu bar and choosing Save.

Results

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)...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
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