Decellularized ECM hydrogels: prior use considerations, applications, and opportunities in tissue engineering and biofabrication.

Tissue development, wound healing, pathogenesis, regeneration, and homeostasis rely upon coordinated and dynamic spatial and temporal remodeling of extracellular matrix (ECM) molecules. ECM reorganization and normal physiological tissue function, require the establishment and maintenance of biological, chemical, and mechanical feedback mechanisms directed by cell-matrix interactions. To replicate the physical and biological environment provided by the ECM in vivo, methods have been developed to decellularize and solubilize tissues which yield organ and tissue-specific bioactive hydrogels. While these biomaterials retain several important traits of the native ECM, the decellularizing process, and subsequent sterilization, and solubilization result in fragmented, cleaved, or partially denatured macromolecules. The final product has decreased viscosity, moduli, and yield strength, when compared to the source tissue, limiting the compatibility of isolated decellularized ECM (dECM) hydrogels with fabrication methods such as extrusion bioprinting. This review describes the physical and bioactive characteristics of dECM hydrogels and their role as biomaterials for biofabrication. In this work, critical variables when selecting the appropriate tissue source and extraction methods are identified. Common manual and automated fabrication techniques compatible with dECM hydrogels are described and compared. Fabrication and post-manufacturing challenges presented by the dECM hydrogels decreased mechanical and structural stability are discussed as well as circumvention strategies. We further highlight and provide examples of the use of dECM hydrogels in tissue engineering and their role in fabricating complex in vitro 3D microenvironments.

Bioprinted cancer-stromalin-vitromodels in a decellularized ECM-based bioink exhibit progressive remodeling and maturation.

Constant matrix remodeling and cellular heterogeneity in cancer are key contributors to its development and can profoundly alter treatment efficacy. Developingin-vitromodels containing relevant features that can recapitulate these aspects of the tumor microenvironment and that are well characterized can circumvent the limitations of conventional 2D cultures and animal models. Automated fabrication methods combined with biomimetic biomaterials have provided the opportunity to create platforms that can potentially incorporate a heterogeneous population of cells in a 3D environment that allows cell-cell and cell-ECM interactions with reproducibility. This study used 3D extrusion bioprinting and a composite bioink containing a reinforced decellularized extracellular matrix (ECM) hydrogel to fabricate a head and neck cancerin-vitromodel. The constituents of this model included fibroblasts and active ECM proteins to represent the stroma, along with HNSCC cells to represent the tumor component. The topographical characterization of the bioink showed a fibrous network with nanometer-sized pores. After cell encapsulation and model fabrication, we observed spheroid development and growth over time with cancer cells in the core and fibroblasts in the periphery. Our model is compatible with matrix metalloproteinase (MMP) quantification techniques and showed significant differences in the presence of MMP-9 and MMP-10 compared to the control groups. This characterized model is proposed as a tool for further translational and drug discovery applications since it provides a biomimetic scenario that allows the study of the tumor microenvironmentin-vitrousing nondestructive longitudinal monitoring over time.

Bioprinted Multicomponent Hydrogel Co-culture Tumor-Immune Model for Assessing and Simulating Tumor-Infiltrated Lymphocyte Migration and Functional Activation.

The immune response against a tumor is characterized by the interplay among components of the immune system and neoplastic cells. Here, we bioprinted a model with two distinct regions containing gastric cancer patient-derived organoids (PDOs) and tumor-infiltrated lymphocytes (TILs). The initial cellular distribution allows for the longitudinal study of TIL migratory patterns concurrently with multiplexed cytokine analysis. The chemical properties of the bioink were designed to present physical barriers that immune T-cells must breech during infiltration and migration toward a tumor with the use of an alginate, gelatin, and basal membrane mix. TIL activity, degranulation, and regulation of proteolytic activity reveal insights into the time-dependent biochemical dynamics. Regulation of the sFas and sFas-ligand present on PDOs and TILs, respectively, and the perforin and granzyme longitudinal secretion confirms TIL activation when encountering PDO formations. TIL migratory profiles were used to create a deterministic reaction-advection diffusion model. The simulation provides insights that decouple passive from active cell migration mechanisms. The mechanisms used by TILs and other adoptive cell therapeutics as they infiltrate the tumor barrier are poorly understood. This study presents a pre-screening strategy for immune cells where motility and activation across ECM environments are crucial indicators of cellular fitness.

Constructing 3D In Vitro Models of Heterocellular Solid Tumors and Stromal Tissues Using Extrusion-Based Bioprinting.

Malignant tumor tissues exhibit inter- and intratumoral heterogeneities, aberrant development, dynamic stromal composition, diverse tissue phenotypes, and cell populations growing within localized mechanical stresses in hypoxic conditions. Experimental tumor models employing engineered systems that isolate and study these complex variables using in vitro techniques are under development as complementary methods to preclinical in vivo models. Here, advances in extrusion bioprinting as an enabling technology to recreate the three-dimensional tumor milieu and its complex heterogeneous characteristics are reviewed. Extrusion bioprinting allows for the deposition of multiple materials, or selected cell types and concentrations, into models based upon physiological features of the tumor. This affords the creation of complex samples with representative extracellular or stromal compositions that replicate the biology of patient tissue. Biomaterial engineering of printable materials that replicate specific features of the tumor microenvironment offer experimental reproducibility, throughput, and physiological relevance compared to animal models. In this review, we describe the potential of extrusion-based bioprinting to recreate the tumor microenvironment within in vitro models.