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, rep....

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 ....

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