Liver is the largest organ of the human body; it fulfills hundreds of viable functions, and could be considered as a major crossroad of human physiology. In vitro modelling the different features of this organ has always been considered crucial for investigating both physiological and pathological processes, as well as to support drug development. Structural and functional complexity of the liver, however, represented major boundaries to the realization of accurate models. Recent advantages in biomaterials science, together with the development of new processing techniques allowed to improve the outcomes in this challenge. The availability of tools enabling to grow even different cell types within the same 3D matrix, mimicking their natural microenvironment represents a step forward if compared to classical 2D cell cultures. Alginate is commonly exploited to encapsulate cells in a tailorable 3D environment [1]. Moreover, decellularized liver are commonly used as scaffold to be recellularized, or enzymatically degraded into a viscous solution to encapsulate cells [2]. Decellularized liver-ECM was previously introduced in alginate beads to specifically encapsulate HCCLM3 cells, showing enhanced viability and protein expression if compared to alginate alone [3]. In light of this, this work presents the development of a 3D in vitro model based on liver ECM and alginate to support hepatocytes growth and activity while tuning the rheological properties to meet the ones of human liver. ECM was obtained from porcine (pECM) and bovine (bECM) livers, by a combination of different methods [4]. The decellularization buffer (TRIS-HCl buffer + 1% w/v SDS + 1% v/v Triton X100) was injected in multiple sites of 0.5 cm cubes of liver and then used to submerge the samples while under orbital shacking up to 7 days. Freeze-dried decellularized cubes were grinded into powder of controlled granulometry. Residual immunogenic content after decellularization was quantified through DNeasy Blood and Tissue Kit. Both pECM and bECM protein compounds were characterized through ELISA. Four different alginate hydrogels were produced exploiting internal gelation: alginate 2% w/v, alginate 2% w/v + gelatin 0.8% w/v, alginate 2% w/v + pECM 0.8% w/v, and alginate 2% w/v + bECM 0.8% w/v. Each solution or suspension was obtained in complete medium (EMEM + 10% v/v FBS + 1% v/v P+S + 1% v/v Na pyruvate + 1% v/v L-Gln). Hydrogels were dried in order to determine their water content. Stability was tested in culture conditions (37 °C; 95% humidity; 5% CO2) up to 14 days. Each hydrogel was then subjected to rheological analyses. For cell-loaded hydrogels, HepG2 cell suspension was added to the gel-precursor solution, to obtain a final concentration of 2 x 106 cells/ml; after cross-linking, gels were submerged with complete medium and incubated in culture condition. After 24, 48, and 96 hours, gels were dissolved using Na-citrate, cells were retrieved, seeded, and tested with MTT assay. Gels were stained with live/dead kit, and subsequently observed at the confocal microscope (CLSM) after 24, 48, and 96 hours of culture. Agarose gel electrophoresis confirmed the presence of a negligible amount of residual DNA in both pECM and bECM, thus validating the decellularization procedure. Alginate allowed to tailor the cross-linking; it has been possible to easily tune the reagents amount, thus obtaining rheological characteristics reproducing the ones of physiological liver tissue. Additionally, another set of rheological tests has been applied to evaluate the processability of hydrogels via 3D-bioprinting. The possibility to retrieve the cells from the reversible 3D matrix, even after long culture periods, using a nontoxic Ca2+ ions chelator allowed to quantify cell metabolic activity by MTT assay. An improvement of the viability through time resulted in both gels containing pECM and bECM, if compared to other hydrogels. CLSM analyses with live/dead kit not only confirmed the results of MTT assay, but even allowed to observe 3D clusters, similar to those observed in vivo [5]. This study demonstrated the possibility to synergically combine the chemical features of liver-derived ECM with the structural characteristic of an alginate hydrogel, mimicking the in vivo liver microenvironment. The presence of ECM positively impacted cell viability. Ongoing studies will include the bioprinting of the model, aiming to accelerate and standardize its production.

Three-dimensional ECM-based in vitro model of the liver

G. Guagliano;L. Sardelli;P. Petrini
2021-01-01

Abstract

Liver is the largest organ of the human body; it fulfills hundreds of viable functions, and could be considered as a major crossroad of human physiology. In vitro modelling the different features of this organ has always been considered crucial for investigating both physiological and pathological processes, as well as to support drug development. Structural and functional complexity of the liver, however, represented major boundaries to the realization of accurate models. Recent advantages in biomaterials science, together with the development of new processing techniques allowed to improve the outcomes in this challenge. The availability of tools enabling to grow even different cell types within the same 3D matrix, mimicking their natural microenvironment represents a step forward if compared to classical 2D cell cultures. Alginate is commonly exploited to encapsulate cells in a tailorable 3D environment [1]. Moreover, decellularized liver are commonly used as scaffold to be recellularized, or enzymatically degraded into a viscous solution to encapsulate cells [2]. Decellularized liver-ECM was previously introduced in alginate beads to specifically encapsulate HCCLM3 cells, showing enhanced viability and protein expression if compared to alginate alone [3]. In light of this, this work presents the development of a 3D in vitro model based on liver ECM and alginate to support hepatocytes growth and activity while tuning the rheological properties to meet the ones of human liver. ECM was obtained from porcine (pECM) and bovine (bECM) livers, by a combination of different methods [4]. The decellularization buffer (TRIS-HCl buffer + 1% w/v SDS + 1% v/v Triton X100) was injected in multiple sites of 0.5 cm cubes of liver and then used to submerge the samples while under orbital shacking up to 7 days. Freeze-dried decellularized cubes were grinded into powder of controlled granulometry. Residual immunogenic content after decellularization was quantified through DNeasy Blood and Tissue Kit. Both pECM and bECM protein compounds were characterized through ELISA. Four different alginate hydrogels were produced exploiting internal gelation: alginate 2% w/v, alginate 2% w/v + gelatin 0.8% w/v, alginate 2% w/v + pECM 0.8% w/v, and alginate 2% w/v + bECM 0.8% w/v. Each solution or suspension was obtained in complete medium (EMEM + 10% v/v FBS + 1% v/v P+S + 1% v/v Na pyruvate + 1% v/v L-Gln). Hydrogels were dried in order to determine their water content. Stability was tested in culture conditions (37 °C; 95% humidity; 5% CO2) up to 14 days. Each hydrogel was then subjected to rheological analyses. For cell-loaded hydrogels, HepG2 cell suspension was added to the gel-precursor solution, to obtain a final concentration of 2 x 106 cells/ml; after cross-linking, gels were submerged with complete medium and incubated in culture condition. After 24, 48, and 96 hours, gels were dissolved using Na-citrate, cells were retrieved, seeded, and tested with MTT assay. Gels were stained with live/dead kit, and subsequently observed at the confocal microscope (CLSM) after 24, 48, and 96 hours of culture. Agarose gel electrophoresis confirmed the presence of a negligible amount of residual DNA in both pECM and bECM, thus validating the decellularization procedure. Alginate allowed to tailor the cross-linking; it has been possible to easily tune the reagents amount, thus obtaining rheological characteristics reproducing the ones of physiological liver tissue. Additionally, another set of rheological tests has been applied to evaluate the processability of hydrogels via 3D-bioprinting. The possibility to retrieve the cells from the reversible 3D matrix, even after long culture periods, using a nontoxic Ca2+ ions chelator allowed to quantify cell metabolic activity by MTT assay. An improvement of the viability through time resulted in both gels containing pECM and bECM, if compared to other hydrogels. CLSM analyses with live/dead kit not only confirmed the results of MTT assay, but even allowed to observe 3D clusters, similar to those observed in vivo [5]. This study demonstrated the possibility to synergically combine the chemical features of liver-derived ECM with the structural characteristic of an alginate hydrogel, mimicking the in vivo liver microenvironment. The presence of ECM positively impacted cell viability. Ongoing studies will include the bioprinting of the model, aiming to accelerate and standardize its production.
Liver, extracellular matrix, 3D bioprinting
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11311/1182985
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