(W0930-05-27) Decellularization of Porcine Bladder as a Biocompatible Platform for Evaluating In Vitro Drug Efficacy

Purpose: This research aims to develop a method for decellularizing porcine bladder tissue to isolate the extracellular matrix (ECM) as a substrate for three-dimensional (3D) in vitro assays. Traditional two-dimensional (2D) cell monolayers on polystyrene plates can restrict cell morphology and native differentiation by limiting cell attachment and the resulting stimulus [3]. The proposed approach formulates hydrogel substrates from native ECM proteins, providing cells with a biomimetic seeding platform to more closely simulate in vivo cell differentiation and proliferation.

Methods: Decellularization was performed by treating 0.5 cm² tissue segments with detergent solutions (SDS and Triton X-100/NH₄OH) interspersed with washing cycles. Nuclear content removal was completed using DNase I treatment (in MgCl₂/CaCl₂ solution). The resulting decellularized ECM (dECM) was freeze-dried and stored at -20°C. Decellularization efficacy was confirmed through DNA extraction and quantification, with the extracted sample absorbance measured at 260 nm. The dECM was solubilized through acid-protease digestion for 24 hours. After neutralization with NaOH and buffer adjustment with DPBS, the pre-gel solution was stored at 4°C for short-term use or -20°C for long-term storage [4]. Hydrogels were formed by diluting the pre-gel to concentrations of 4,6, and 8 mg/mL and incubating at 37°C for 40 minutes, with gelation verified through turbidimetric kinetic analysis by measuring the absorbance at a wavelength of 405nm. The biocompatibility of the dECM hydrogel substrate included cytotoxicity assessment using T24 bladder cancer cells and immunogenicity evaluation using RAW 264.7 macrophages [1]. Relative cell viability was determined through LDH release quantification at 24-, 48-, and 72-hours post-seeding. The inflammatory profile was assessed by measuring TNF-α (pro-inflammatory) and IL-10 (anti-inflammatory) cytokine production via sandwich ELISA after 24-hour culture, with RAW cells stimulated with bacterial lipopolysaccharide (LPS) or resiquimod (RSQ) to serve as positive controls for pro-inflammatory or anti-inflammatory states [2]. Quantification of cytokine release in each sample was determined by recombinant protein standard curves. The absorbance of each sample after ELISA processing was measured at 450 nm with 570 nm correction.

Results: The efficacy of the above decellularization protocol was assessed through qualitative observation and DNA quantification. Tissue samples showed an increase in transparency after each initial detergent treatment (Figure 1), and subsequent opacity was observed after each additional treatment step. The DNA content decreased significantly from native tissue (2066.98 ng/mg) to SDS treatment (779.39 ng/mg), which was further reduced after Triton X-100 (251.74 ng/mg), and reached a minimum concentration after DNase I treatment (79.22 ng/mg). This significant reduction in DNA concentration in samples after a complete decellularization protocol confirms the removal of cellular and nuclear content from the tissue samples. Solubilized dECM, after neutralization and incubation at 37 °C, formed hydrogels with concentration-dependent gelation kinetics. All three concentrations exhibited sigmoidal gelation curves, achieving 90% gelation within 30 minutes. Gelation lag time was directly proportional to concentration, with 8 mg/mL showing the longest lag time and 4 mg/mL showing the shortest. T24 cells cultured on dECM hydrogels maintained excellent cell viability across all time points and hydrogel concentrations. Across each time point, relative cell viability at 4 mg/mL was 103.87%, 103.13%, and 102.27% at 24, 48, and 72 hours, respectively. At 6 mg/mL, viability was 105.68%, 103.39%, and 102.33%, respectively. The 8 mg/mL concentration showed viability of 107.12%, 103.39%, and 103.11%, respectively. RAW 264.7 macrophages on hydrogels demonstrated significantly lower inflammatory responses compared to stimulated controls (P < 0.05). LPS-stimulated controls secreted TNF-α at 201.67 pg/mL, while cells on 4, 6, and 8 mg/mL hydrogels produced only 12.34, 12.61, and 15.52 pg/mL, respectively. Similarly, resiquimod-stimulated controls produced IL-10 at 219.69 pg/mL, while cells on hydrogels at 4, 6, and 8 mg/mL concentrations secreted significantly lower levels at 92.42%, 91.29%, and 91.67%, respectively.

Conclusion: Our decellularization method effectively removed cellular components from porcine bladder tissue, as evidenced by the decrease in DNA content across treatment stages. The protocol yielded a decellularized ECM successfully processed into pre-gel solutions with predictable, concentration-dependent gelation kinetics. An in vitro assessment of cytotoxicity and immunogenicity confirmed the biocompatibility of these hydrogels, with T24 bladder cancer cells maintaining excellent viability across all concentrations and time points. The minimal production of pro-inflammatory TNF-α and anti-inflammatory IL-10 cytokines by RAW 264.7 macrophages on the hydrogels indicates low immunogenicity of the substrate material. These findings demonstrate that decellularized porcine bladder ECM hydrogels offer a promising three-dimensional culture platform with the potential to overcome traditional two-dimensional assay limitations by providing a physiologically relevant environment for cell growth to further study drug efficacy and fundamental studies of cell-matrix interactions.

References: [1] A practical guide to hydrogels for cell culture | Nature Methods. (n.d.). Retrieved November 1, 2024,
[2] Ahmad, W., Jantan, I., Kumolosasi, E., Haque, M. A., & Bukhari, S. N. A. (2018). Immunomodulatory effects of Tinospora crispa extract and its major compounds on the immune functions of RAW 264.7 macrophages. International Immunopharmacology, 60, 141–151.
[3] Bodgi, L., Bahmad, H. F., Araji, T., Choboq, J. A., Bou-Gharios, J., Cheaito, K., Zeidan, Y. H., Eid, T., Geara, F., & Abou-Kheir, W. (2019). Assessing Radiosensitivity of Bladder Cancer in vitro: A 2D vs. 3D Approach. Frontiers in Oncology, 9, 153.
[4] Freytes, D. O., Martin, J., Velankar, S. S., Lee, A. S., & Badylak, S. F. (2008). Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix. Biomaterials, 29(11), 1630–1637.
[5] Wolf, M. T., Daly, K. A., Brennan-Pierce, E. P., Johnson, S. A., Carruthers, C. A., D’Amore, A., Nagarkar, S. P., Velankar, S. S., & Badylak, S. F. (2012). A hydrogel derived from decellularized dermal extracellular matrix. Biomaterials, 33(29), 7028–7038.

Acknowledgements: The authors have no conflicts of interest to declare. This work was supported in part by a grant from National Institute of Health (P20GM113117) (H.K.) and (5UL1TR002366-08)(H.K.).


Figure 1. The formation of dECM hydrogels. (A) Progression of lyophilized tissue into pre-gel solution after acid protease digestion and neutralization. Resulting dECM hydrogel after incubation of pre-gel at 37 °C for 40 minutes. (B) Turbidimetric kinetics of dECM hydrogel at varying concentrations of 4, 6, and 8 mg/mL dECM.


Figure 2. Characterizing the biocompatibility of dECM hydrogels. (A) Quantitation of the relative cell viability of T24 cells seeded on the dECM hydrogel at varying concentrations (4, 6, and 8 mg/mL) or directly on the plate (COP) after a 48-hour incubation time. (B) Quantitative representation of the concentration of IL-10 and TNF-α secreted by RAW 264.7 cells seeded on the dECM hydrogel at varying concentrations (4, 6, and 8 mg/mL) or directly on the plate (COP) and stimulated with either LPS or RSQ. Statistical analysis was performed using a one-way ANOVA, where *P < 0.05, **P < 0.005, and *** P < 0.001.