The decellularization of organs has attracted attention as a new functional methodology for regenerative medicine based on tissue engineering. In previous work we developed an L-ECM (Extracellular Matrix) as a substrate-solubilized decellularized liver and demonstrated its effectiveness as a substrate for culturing and transplantation. Importantly, the physical properties of the substrate constitute important factors that control cell behavior. In this study, we aimed to quantify the physical properties of L-ECM and L-ECM gels. L-ECM was prepared as a liver-specific matrix substrate from solubilized decellularized porcine liver. In comparison to type I collagen, L-ECM yielded a lower elasticity and exhibited an abrupt decrease in its elastic modulus at 37 °C. Its elastic modulus increased at increased temperatures, and the storage elastic modulus value never fell below the loss modulus value. An increase in the gel concentration of L-ECM resulted in a decrease in the biodegradation rate and in an increase in mechanical strength. The reported properties of L-ECM gel (10 mg/mL) were equivalent to those of collagen gel (3 mg/mL), which is commonly used in regenerative medicine and gel cultures. Based on reported findings, the physical properties of the novel functional substrate for culturing and regenerative medicine L-ECM were quantified.

Milkfish (Chanos chanos) is an abundant fish commodity in the Philippines that generates a large number of wastes such as skin, scales, viscera, and bones, which, upon disposal, cause environmental pollution. The abundance of these wastes, such as fish skin, rich in bioactive natural products such as collagen, elicits interest in their conversion into high-market-value products. The decellularization of milkfish skin waste can extract its extracellular matrix (ECM), a potential raw material for biomedical applications such as the repair of damaged skin tissues. In particular, this study characterized the developed decellularized ECM with different concentrations (0.1%, 1.0%) of the decellularizing agents (Triton X-100, SDS) and temperature (4 °C, room temperature) using milkfish skin. The decellularized ECM structure was better preserved using Triton X-100, while SDS was more effective in cell component removal, especially at 1% concentration and 4 °C temperature. There were significant effects of varying the temperatures and concentrations on the physical and mechanical properties of the decellularized ECM. Future studies could explore more variables to further establish protocols and more analyses to better characterize the decellularized milkfish skin.

Cells have various applications in biomedical research. Cryopreservation is a cell-preservation technique that provides cells for such applications. After cryopreservation, sensitive cells, such as primary hepatocytes, suffer from low viability due to the physical damage caused by ice crystals, highlighting the need for better methods of cryopreservation to improve cell viability. Given the importance of effectively suppressing ice crystal formation to protect cellular structure, trehalose has attracted attention as cryoprotectant based on its ability to inhibit ice crystal formation; however, trehalose induces osmotic stress. Therefore, to establish a cell-cryopreservation technique, it is necessary to provide an optimal balance between the protective and damaging effects of trehalose. In this study, we evaluated the effects of osmotic stress and ice crystal formation on the viability and function of primary rat hepatocytes at wide range of trehalose concentration. There was no osmotic stress at very low concentrations (2.6 μM) of trehalose, and 2.6 μM trehalose drives the formation of finer ice crystals, which are less damaging to the cell membrane. Furthermore, we found that the number of viable hepatocytes after cryopreservation were 70% higher under the 2.6 μM trehalose-supplemented conditions than under the dimethyl sulfoxide-supplemented conditions. Moreover, non-cryopreserved cells and cells cryopreserved with trehalose showed comparable intracellular dehydrogenase activity. We showed that trehalose at very low concentrations (2.6 μM) improved dramatically viability and liver function of hepatocyte after cryopreservation.

Tilapia, a widely farmed aquaculture fish, produces substantial waste, including viscera that contain extracellular matrix (ECM) utilized as a biomaterial for tissue regeneration applications. Extracting ECM from viscera requires a specific decellularization method, as no standardized protocol exists. This study performed three decellularization methods: sonication, orbital shaking at room temperature, and agitation at 4°C, using SDS and TX100 at concentrations of 0.1% and 0.3%. The effectiveness of each method was assessed through H&E staining, dsDNA quantification, and SEM imaging to verify cellular content removal and ECM structure preservation. Additional analyses, including ATR‐FTIR, SDS‐PAGE, protein quantification, HPLC, and detergent residue tests, were performed to examine functional groups, collagen composition, protein content, amino acid profiles, and detergent residues in the decellularized samples. The results of H&E staining showed a significant reduction in cellular components in all samples, which was confirmed through DNA quantification. Sonication with 0.3% SDS achieved the highest DNA removal rate (96.5 ± 1.1%), while SEM images revealed that agitation at 4°C with 0.3% TX100 better preserved ECM structure. Collagen was present in all samples, as confirmed by ATR‐FTIR analysis, which revealed pronounced spectral peaks in the amide I, II, III, A, and B regions. Samples treated with agitation at 4°C using 0.1% SDS exhibited the highest protein content (875 ± 15 µg/mg), whereas those treated with TX100 had lower detergent residue. Overall, the decellularization methods effectively reduced DNA content while preserving ECM structure and components, highlighting the potential of tilapia viscera as bioscaffolds and offering insights into utilizing fish waste for high‐value products.

Tilapia (Oreochromis niloticus) is a widely cultivated fish in tropical and subtropical regions such as the Philippines, generating substantial waste during processing, including bones that are a valuable source of extracellular matrix (ECM). However, the extraction of ECM from fish bones requires an essential step of demineralization. This study aimed to assess the efficiency of tilapia bone demineralization using 0.5 N HCl at different time durations. By evaluating the residual calcium concentration, reaction kinetics, protein content, and extracellular matrix (ECM) integrity through histological analysis, composition assessment, and thermal analysis, the effectiveness of the process was determined. Results revealed that after 1 h of demineralization, the calcium and protein contents were 1.10 ± 0.12% and 88.7 ± 0.58 μg/mL, respectively. The study found that after 6 h, the calcium content was almost completely removed, but the protein content was only 51.7 ± 1.52 μg/mL compared to 109.0 ± 1.0 μg/mL in native bone tissue. Additionally, the demineralization reaction followed second-order kinetics with an R² value of 0.9964. Histological analysis using H&E staining revealed a gradual disappearance of the basophilic components and the emergence of lacunae, which can be attributed to decellularization and mineral content removal, respectively. As a result, organic components such as collagen remained in the bone samples. ATR-FTIR analysis showed that all demineralized bone samples retained collagen type I markers, including amide I, II, and III, amides A and B, and symmetric and antisymmetric CH₂ bands. These findings provide a route for developing an effective demineralization protocol to extract high-quality ECM from fish bones, which could have important nutraceutical and biomedical applications.

Accurate determination of the amount of glycosaminoglycans (GAGs) in a complex mixture of extracellular matrix (ECM) is important for tissue morphogenesis and homeostasis. The aim of the present study was to investigate an accurate, simple and sensitive alcian blue (AB) method for quantifying heparin in biological samples. A method for analyzing heparin was developed and parameters such as volume, precipitation time, solvent component, and solubility time were evaluated. The AB dye and heparin samples were allowed to react at 4 ℃ for 24 h. The heparin-AB complex was dissolved in 25 N NaOH and 2-Aminoethanol (1:24 v/v). The optical density of the solution was analyzed by UV-Vis spectrometry at 620 nm. The modified AB method was validated in accordance with U.S. Food and Drug Administration guidelines. The limit of detection was found to be 2.95 µg/mL. Intraday and interday precision ranged between 2.14–4.83% and 3.16–7.02% (n = 9), respectively. Overall recovery for three concentration levels varied between 97 ± 3.5%, confirming good accuracy. In addition, this study has discovered the interdisciplinary nature of protein detection using the AB method. The basis for this investigation was that the fibrous protein inhibits heparin-AB complex whereas globular protein does not. Further, we measured the content of sulfated GAGs (sGAGs; expressed as heparin equivalent) in the ECM of decellularized porcine liver. In conclusion, the AB method may be used for the quantitative analysis of heparin in ECM scaffolds for tissue engineering applications.