Callanan, Anthony

Davis, Niall

English

A urinary bladder bioreactor was constructed to replicate physiological bladder dynamics. A cyclical low-delivery pressure regulator mimicked filling pressures of the human bladder. Cell growth was evaluated by culturing human urothelial cells (UCs) on porcine extracellular matrix scaffolds in the bioreactor and in static growth conditions for 5 consecutive days. UC proliferation was compared with quantitative viability indicators and by fluorescent markers for intracellular esterase activity and plasma membrane integrity. Scaffold integrity was characterized with scanning electron microscopy and 4,6-diamidino-2-phenylindole staining

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     Edinburgh Research Explorer                                      Development of a bladder bioreactor for tissue-engineering inurologyCitation for published version:Davis, N & Callanan, A 2015, 'Development of a bladder bioreactor for tissue-engineering in urology',Methods in Molecular Biology.Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Peer reviewed versionPublished In:Methods in Molecular BiologyGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 11. May. 2020 1  1  2 Development of a bladder bioreactor for tissue-engineering in 3 urology 4  5 Davis NF, Callanan A 6  7 Centre for Applied Biomedical Engineering Research, Department of Mechanical and 8 Aeronautical Engineering and MSSI, University of Limerick Ireland 9  10  11  12  13  14  15  16  17  18 Corresponding author: 19 Dr. Niall Davis 20 Centre for Applied Biomedical Engineering Research 21 University of Limerick  22 Co. Limerick  23 Ireland 24 Email: nialldavis@rcsi.ie 25  2 Keywords 1 Bladder bioreactor 2 Tissue-engineering 3 Regenerative medicine 4 Urothelial cells 5  6 Abstract word count: 7 Manuscript word count: 8 Number of references: 9 Number of figures: 10 Number of tables: 11 12  3 Summary 1 A urinary bladder bioreactor was constructed to replicate physiological bladder 2 dynamics. A cyclical low-delivery pressure regulator mimicked filling pressures of 3 the human bladder. Cell growth was evaluated by culturing human urothelial cells 4 (UCs) on porcine extracellular matrix scaffolds in the bioreactor and in static growth 5 conditions for 5 consecutive days. UC proliferation was compared with quantitative 6 viability indicators and by fluorescent markers for intracellular esterase activity and 7 plasma membrane integrity. Scaffold integrity was characterized with scanning 8 electron microscopy and 4,6-diamidino-2-phenylindole staining. 9 10  4 1.0 Introduction 1 Surgical repair for end stage bladder disease often utilises vascularised, autogenous, 2 mucus-secreting gastrointestinal tissue to either replace the diseased organ or to 3 augment inadequate bladder tissue. Augmentation cystoplasty is a urological 4 procedure that involves the addition of viscoelastic ileal tissue to defective bladder 5 tissue to improve functional bladder capacity. Postoperatively, the compliant smooth 6 muscle of the bowel is often sufficient to restore the basic shape, structure and 7 function of the urinary bladder, however lifelong postoperative complications are 8 common. Morbidities that result from interposition of intestinal tissue may be divided 9 into three broad areas: metabolic, neuromechanical, and technical-surgical. Metabolic 10 complications are the result of altered solute reabsorption by the intestine of the urine 11 that it contains. Neuromechanical aspects involve the configuration of the bowel; 12 which affects storage volume and contraction of the intestine that may lead to 13 difficulties in storage. Finally, technical-surgical complications involve aspects of the 14 procedure that result in surgical morbidity. 15  16 ECMs are decellularised biological scaffolds commonly derived from porcine organs. 17 After their preparation process decellularised ECM scaffolds may be seeded with 18 autologous impermeable urothelial cells (UCs) in vitro. Cell-seeded scaffolds offer 19 important advantages in urological settings when compared to gastrointestinal 20 segments as UCs may provide a protective impermeable epithelial lining prior to in 21 vivo implantation. Limitations associated with cell-seeded scaffolds are harvesting 22 difficulties and low proliferative potential in vitro. Theoretically ‘bladder bioreactors’ 23 may improve urothelial cell proliferation by preconditioning cell-seeded scaffolds in a 24 simulated in vivo physiological environment. The objective of the present study was 25  5 to construct and evaluate a urinary bladder bioreactor for urological tissue-1 engineering purposes. 2 3  6 2.0 Materials  1 2.1 Construction of bioreactor 2 Components were purchased from BOC gases® (United Kingdom and Ireland) unless 3 otherwise indicated. Primary constituents included a sealed pressure chamber with a 4 transparent window, pressurised gas containers and silicone tubing (Centre for 5 Applied Biomedical Engineering and Research [CABER], University of Limerick, 6 Ireland). The principal aim of the bioreactor was to increase hydrostatic pressure on 7 cell seeded ECM scaffolds from 0-10cm H20 via a cyclical low-delivery pressure 8 regulator over preset durations. Urothelial cell viability and proliferative activity on 9 ECM scaffolds were measured and compared for 5 consecutive days in a control and 10 bioreactor experimental group by quantitative viability indicators. 11  12 2.2 Culture and expansion of UCs 13 Cell culture materials were obtained from Innovative Technologies in Biological 14 Systems, Bizkaia, Spain (Innoprot®) unless indicated.  15 1. UCs were initially cultured under standard cell culture conditions in a 16 humidified atmosphere of 95% air and 5% CO2  at 37°C in T-75 vented cap 17 flasks (Sarstedt Ltd, Wexford, Ireland).  18 2. The cell line was grown to confluence in urothelial cell medium (UCM) and 19 supplemented with 1% urothelial cell growth supplement (UCGS) and 1% 20 penicillin/streptomycin solution (P/S solution). For expansion, cells were fed 21 every 48 hours until 85-90% confluency, which was usually achieved after 7-8 22 days.  23 3. Confluent cells were harvested by incubation with 0.0025% trypsin/ 0.5mM 24 EDTA Solution (T/E solution). Trypsin was neutralised with Trypsin 25  7 Neutralisation Solution (TNS) that contained buffered saline solution, 10% 1 foetal bovine serum (FBS) and was calcium and magnesium free. The cells 2 were then centrifuged for 5 minutes (1000rpm) and gathered. Passage 4 cells 3 were used in the study. 4  5 2.3 Measurement of urothelial cell viability  6 Cell viability was assessed and quantified with the Alamar Blue® cell viability 7 reagent (Invitrogen, Ireland) for 5 consecutive days. In the presence of viable cells the 8 dye reduces, turns red and becomes highly fluorescent.  9 1. One tenth of the volume of alamarBlue® reagent was added directly 10 into culture medium based on the protocol illustrated in Table 1. 11 2. Proliferation rates in both experimental groups were measured as a fold 12 increase in the number of viable cells per day. To eliminate the 13 potential for intervention bias between both groups, 2 control groups 14 were created. Control group 1 consisted of increasing cell densities 15 cultured in static growth conditions. Control group 2 consisted of 16 increasing cell densities cultured in the bioreactor under static 17 conditions (i.e. without the influence of cyclical pressure). Calibration 18 curves with linear fit were applied to both control groups (Fig. 1) 19  20 2.4 Scanning electron microscopy (SEM) 21 To assess the structural integrity of UBM scaffolds in the bioreactor SEM was 22 performed before and after 5 days on UBM with and without cells. The samples were 23 sputter coated with a thin layer of gold and palladium. 24  25  8 2.5 4-6-Diamidino-2-phenylindole (DAPI) staining 1 DAPI nucleic acid staining (Invitrogen®, Dublin, Ireland) was performed to confirm 2 the presence of cell nuclei in the bioreactor group after 5 days of growth (see note 1). 3  4 2.6 Measurement of fluorescence activity 5 ‘Viability/cytotoxicity’ fluorescence assays were performed by means of three-6 dimensional confocal microscopy on both experimental groups to compare cell 7 viability after 5 days of culture (LSM 700, Carl Zeiss Microimaging, Gottingen, 8 Germany). Live cells were distinguished by the presence of ubiquitous intracellular 9 esterase activity, determined by the enzymatic conversion of the virtually 10 nonfluorescent cell-permeant calcein-AM to the intensely fluorescent calcein (see 11 note 2).  12  13 EthD-1 enters cells with damaged membranes and undergoes a 40-fold enhancement 14 of fluorescence upon binding to nucleic acids, thereby producing a bright red 15 fluorescence in dead cells (ex/em ~495 nm/~635 nm). EthD-1 is excluded by the 16 intact plasma membrane of live cells. The determination of cell viability depends on 17 these physical and biochemical properties of cells. Cytotoxic events that do not affect 18 these cell properties may not be accurately assessed using this method. Background 19 fluorescence levels are inherently low with this assay technique because the dyes are 20 virtually non-fluorescent before interacting with cells. 21  22  23  24  25  9 3.0 Methods  1 3.1 Bioreactor and physiological intravesical pressures 2 1. A manometer was placed in the sealed pressure chamber for the duration of 3 the study to accurately monitor pressures within the bioreactor throughout the 4 entire experimental time period.  5 2. Cyclical filling pressures of the human bladder were mimicked by connecting 6 a low delivery pressure regulator, with a pressure adjusting screw, to the gas 7 container (95% air and 5% CO2) that increased the fluid pressure on the cell-8 seeded UBM scaffold within the bioreactor from 0-10 cm H20 (Fig. 2).  9 3. Application of cyclical pressure over a preset duration resulted in mechanical 10 strain on the cell-seeded ECM scaffold. Voiding pressures were mimicked by 11 releasing the infused gas from a ‘Y-connector’ silicon outlet source outlet 12 source over a period of approximately 10 seconds every 6-8 hours. 13  14 3.2 Urothelial cell-seeding techniques in control and experimental group 15 1. Specimens of urinary bladder matrix (UBM), a porcine extracellular matrix 16 graft, were cut into circles of 2cm diameter, transferred into 12-well tissue 17 culture plates and weighted with stainless steel rings also 2cm in diameter to 18 inhibit their lifting. Preparation of UBM has previously been described (see 19 note 3). Each stainless steel ring was autoclaved prior to insertion to ensure 20 sterility. UBM scaffolds were then seeded with urothelial cells (2.5- 5.0 x 104 21 cells/cm2 per well).  22 2. Equally matched circles of UBM were inserted into a second modified 6-well 23 plate and transferred into the urinary bladder bioreactor for comparative 24  10 assessment. Luminal surfaces of UBM scaffolds were seeded with 5.0 x 1 104cells/cm2 per well and cultured in 2ml of UCM 2   3 3.3 Measurement of urothelial cell viability  4 1. After adding the alamarBlue® reagent, the solution was incubated for 4 hours 5 at 37°C in a cell culture incubator that is protected from direct light (see note 6 4).  7 2. For greater sensitivity the extent of alamarBlue® dye reduction was quantified 8 by fluorescence spectrophotometry (BioTek, Synergy HT Multi-Mode 9 Microplate Reader). Proliferation rate was measured as a fold increase in the 10 number of viable cells per day 11 3. Fluorescence was read using a fluorescence excitation wavelength of 540-12 570nm (peak excitation is 570nm). Fluorescence emission was read at 580-13 610nm (peak emission was 585nm). Fluorescence methods are more sensitive 14 than absorbance methods and this method was applied to all experiments (see 15 note 5).  16 4. Absorbance of alamarBlue® can also be monitored at 570nm, using 600nm as 17 a reference wavelength value. Average 600nm absorbance values of the cell 18 culture medium alone (background) were subtracted from the 570nm 19 absorbance values of experimental wells. Background subtracted 570nm 20 absorbance versus concentration of the test compound values were then 21 plotted 22 5. Cells were plated in 100μL medium into 96-well tissue culture plates after 23 performing cell number titration in the range of 40 to 10,000 for adherent cells 24 and 2000 to 500,000 for suspension cells. A background control of 100μL of 25  11 medium without cells should be used. Subsequently 10μL of alamarBlue® was 1 added into the medium and the cells incubated at 37°C overnight.  2  3  4 3.4 Scanning electron microscopy (SEM) 5 1. Initially, primary fixation was performed by immersing the sample in 2.5% 6 glutaraldehyde in 0.1M cacodylate buffer, pH 7.4 for 2 hours at room 7 temperature or in a refrigerator at 4°C overnight.  8 2. Samples were washed for 5 minutes on 3 consecutive occasions in 0.1M 9 cacodylate buffer (pH 7.4).  10 3. Secondary fixation was performed by immersing the sample in 1% osmium 11 tetroxide (aqueous) pH 7.4 for 1 hour at room temperature in a lightproof 12 container. The washing process was repeated in 0.1M cacodylate buffer 7.4. 13 4. Dehydration was achieved by the following method (see note 6): 14  1 x 10min. in 25% ethanol,  15  1 x 10min. in 50% ethanol,  16  1 x 10min. in 70% ethanol,  17  1 x 10min. in 85% ethanol,  18  1 x 10min. in 95% ethanol,  19  2 x 10min. in 100% ethanol,  20  1 x 10min. 100% ethanol (EM grade). 21 5. Sputter coating was performed by applying a thin layer of gold and palladium 22 over the sample with an automated sputter coater. This process can take up to 23 10 minutes. 24  25  26  12 3.5 4-6-Diamidino-2-phenylindole (DAPI) staining 1 1. To make a 5 mg/mL DAPI stock solution (14.3 mM for the dihydrochloride or 2 10.9 mM for the dilactate), dissolve the contents of one vial (10 mg) in 2 mL 3 of deionized water (dH2O) or dimethylformamide (DMF). The less water-4 soluble DAPI dihydro-chloride may take some time to completely dissolve in 5 water and sonication may be necessary. 6 2. Counterstaining Protocol 7  Equilibrate the sample briefly with phosphate-buffered saline (PBS). 8  Dilute the DAPI stock solution to 300 nM in PBS. Add approximately 300 μL 9 of this dilute DAPI staining solution to the coverslip preparation, making 10 certain that the cells are completely covered. 11  Incubate for 1–5 minutes. 12  Rinse the sample several times in PBS. Drain excess buffer from the coverslip 13 and mount. 14  View the sample using fluorescence microscopy with appropriate filters. 15  16 3.6 Measurement of fluorescence activity 17 1. Media was aspirated and the culture vessel washed with DPBS. Five millilitres 18 of DPBS were added into a 15ml centrifuge tube followed by 10μl of 19 ethidiumhomodimer-1 (Ethd-1) and 10μl of Calcein to the tube, 20 (concentrations may change depending on the sample size).  21 2. The newly mixed solution was added to the cell sample and placed in a 22 refrigerator at 4°C for 10minutes. After this timeframe, the solution was 23 aspirated and the sample washed with DPBS. A small amount of DBPS was 24 left on the sample to prevent dehydration. 25 26  13 4.0 Notes 1 1 DAPI staining was performed by dissolving 10mg of DAPI into 2mL of 2 deionised water and protecting the solution from light. Subsequently, 300μL 3 of the diluted DAPI staining solution was added to the cell-seeded scaffolds. 4 The samples were then incubated for 5 minutes and evaluated with 5 fluorescence confocal microscopy (LSM 700, Carl Zeiss Microimaging, 6 Gottingen, Germany). 7  8 2. The polyanionic dye calcein is well retained within live cells, producing an 9 intense uniform green fluorescence in live cells (ex/em ~495 nm/~515 nm). 10  11 3. The harvested porcine bladder was rinsed in normal saline to remove the inner 12 luminal urothelial cell lining. The outer abluminal muscular layers were 13 removed by manual delamination. The specimen was bisected along 1 side to 14 form a sheet. Subsequently, the tunica serosa, tunica muscularis externa, 15 tunica submucosa, and the muscularis mucosa were manually removed while 16 preserving an intact basement membrane and lamina propria. The 17 decellularization process was completed by soaking the matrix in buffered 18 saline (pH 7.4), placing it in peracetic acid/4% ethanol for 2 hours and rinsing 19 it in sterile buffered saline. Finally, sterilisation was achieved by exposure to 20 ethylene oxide. 21  22 4. Sensitivity of detection generally increases with longer duration times; 23 therefore samples with fewer cells should use incubation times up to 24 hours. 24  25  14 5. Assay plates can be wrapped in foil, stored at 4°C and read within 1-3 days 1 without affecting the fluorescence or absorbance values. 2  3  4 6. It takes approximately 40 minutes to achieve a critical dry point and the 5 sample can then be mounted onto a metal stub with a double sided carbon 6 tape. 7  8  9  10  11  12 13  15 Figure Legends: 1 Figure 1:  2 A: Calibration curve for human urothelial cells cultured under static growth 3 conditions. B: Calibration curve for human urothelial cells cultured in the bioreactor 4 under static growth conditions.  5 Legend: Applying standard curves eliminated intervention bias between both 6 experimental groups. 7  8 Figure 2: 9 Photograph of bioreactor chamber and ECM scaffold exposed to cyclical 10 physiological urinary bladder pressures. 11  12 Format Volume of Cells & Medium Volume of 10x alamarBlue® to add Cuvette 1mL 100μL 96-well plate 100μL 10μL 284-well plate 40μL 4Μl  13 Table 1: AlamarBlue® protocol for assessing viable cell numbers 14  16  1 Figure 1 2  17  1 Figure 2 2 

Development of a bladder bioreactor for tissue-engineering in urology

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Development of a bladder bioreactor for tissue-engineering in urology

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