Assessment and process optimization of high throughput biofabrication of immunocompetent breast cancer model for drug screening applications.

Recent advancements in 3D cancer modeling have significantly enhanced our ability to delve into the intricacies of carcinogenesis. Despite the pharmaceutical industry's substantial investment of both capital and time in the drug screening and development pipeline, a concerning trend persists: drug candidates screened on conventional cancer models exhibit a dismal success rate in clinical trials. One pivotal factor contributing to this discrepancy is the absence of drug testing on pathophysiologically biomimetic 3D cancer models during pre-clinical stages. Unfortunately, current manual methods of 3D cancer modeling, such as spheroids and organoids, suffer from limitations in reproducibility and scalability. In our study, we have meticulously developed 3D bioprinted breast cancer model utilizing decellularized adipose tissue-based hydrogel obtained via a detergent-free decellularization method. Our innovative printing techniques allows for rapid, high-throughput fabrication of 3D cancer models in a 96-well plate format, demonstrating unmatched scalability and reproducibility. Moreover, we have conducted extensive validation, showcasing the efficacy of our platform through drug screening assays involving two potent anti-cancer drugs, 5-Fluorouracil and PRIMA-1Met. Notably, our platform facilitates effortless imaging and gene expression analysis, streamlining the evaluation process. In a bid to enhance the relevance of our cancer model, we have introduced a heterogeneous cell population into the DAT-based bioink. Through meticulous optimization and characterization, we have successfully developed a biomimetic immunocompetent breast cancer model, complete with microenvironmental cues and diverse cell populations. This breakthrough paves the way for rapid multiplex drug screening and the development of personalized cancer models, marking a paradigm shift in cancer research and pharmaceutical development.

Synergistic Coassembly of Folic Acid-Based Supramolecular Polymer with a Covalent Polymer Toward Fabricating Functional Antibacterial Biomaterials.

Supramolecular biomaterials can recapitulate the structural and functional facets of the native extracellular matrix and react to biochemical cues, leveraging the unique attributes of noncovalent interactions, including reversibility and tunability. However, the low mechanical properties of supramolecular biomaterials can restrict their utilization in specific applications. Combining the advantages of supramolecular polymers with covalent polymers can lead to the fabrication of tailor-made biomaterials with enhanced mechanical properties/degradability. Herein, we demonstrate a synergistic coassembled self-healing gel as a multifunctional supramolecular material. As the supramolecular polymer component, we chose folic acid (vitamin B9), an important biomolecule that forms a gel comprising one-dimensional (1D) supramolecular polymers. Integrating polyvinyl alcohol (PVA) into this supramolecular gel alters its ultrastructure and augments its mechanical properties. A drastic improvement of complex modulus (G*) (∼3674 times) was observed in the folic acid-PVA gel with 15% w/v PVA (33215 Pa) compared with the folic acid gel (9.04 Pa). The coassembled hydrogels possessed self-healing and injectable/thixotropic attributes and could be printed into specific three-dimensional (3D) shapes. Synergistically, the supramolecular polymers of folic acid also improve the toughness, durability, and ductility of the PVA films. A nanocomposite of the gels with silver nanoparticles exhibited excellent catalytic efficiency and antibacterial activity. The folic acid-PVA coassembled gels and films also possessed high cytocompatibility, substantiated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live-dead assays. Taken together, the antibacterial and cell-adhesive attributes suggest potential applications of these coassembled biomaterials for tissue engineering and wound healing.

High Throughput Bioprinting Using Decellularized Adipose Tissue-Based Hydrogels for 3D Breast Cancer Modeling.

3D bioprinting allows rapid automated fabrication and can be applied for high throughput generation of biomimetic constructs for in vitro drug screening. Decellularized extracellular matrix (dECM) hydrogel is a popular biomaterial choice for tissue engineering and studying carcinogenesis as a tumor microenvironmental mimetic. This study proposes a method for high throughput bioprinting with decellularized adipose tissue (DAT) based hydrogels for 3D breast cancer modeling. A comparative analysis of decellularization protocol using detergent-based and detergent-free decellularization methods for caprine-origin adipose tissue is performed, and the efficacy of dECM hydrogel for 3D cancer modeling is assessed. Histological, biochemical, morphological, and biological characterization and analysis showcase the cytocompatibility of DAT hydrogel. The rheological property of DAT hydrogel and printing process optimization is assessed to select a bioprinting window to attain 3D breast cancer models. The bioprinted tissues are characterized for cellular viability and tumor cell-matrix interactions. Additionally, an approach for breast cancer modeling is shown by performing rapid high throughput bioprinting in a 96-well plate format, and in vitro drug screening using 5-fluorouracil is performed on 3D bioprinted microtumors. The results of this study suggest that high throughput bioprinting of cancer models can potentially have downstream clinical applications like multi-drug screening platforms and personalized disease models.

Smooth muscle matrix bioink promotes myogenic differentiation of encapsulated adipose-derived stem cells.

Hydrogels derived from decellularized extracellular matrices (dECM) can mimic the biochemical composition of the native tissue. They can also act as a template to culture reseeded cells in vitro. However, detergent-based decellularization methods are known to alter the biochemical compositions, thereby compromising the bioactive potential of dECM. This study proposes a facile detergent-free method to achieve dECM from smooth muscle tissue. We have used the muscle layer of caprine esophageal tissue and decellularized using hypo and hyper-molar sodium chloride solutions alternatingly. Then, a hydrogel was prepared from this decellularized smooth muscle matrix (dSMM) and characterized thoroughly. A comparative analysis of the dSMM prepared with our protocol with the existing detergent-based protocol suggests successful and comparable decellularization with minimal residual DNA content. Interestingly, an 8.78-fold increase in sulfated glycosaminoglycans content and 1.62-fold increased collagen content indicated higher retention of ECM constituents with NaCl-based decellularization strategy. Moreover, the dSMM gel induces differentiation of the encapsulated adipose-derived mesenchymal stem cells toward smooth muscle cells (SMCs) as observed by their expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain, the hallmarks of SMCs. Finally, we optimized the process parameter for productive bioprinting with this dSMM bioink and fabricated 3D muscle constructs. Our results suggest that dSMM has the potential to be used as a bioink to engineer personalized esophageal tissues.

Formulation of Dermal Tissue Matrix Bioink by a Facile Decellularization Method and Process Optimization for 3D Bioprinting toward Translation Research.

Decellularized extracellular matrices (ECMs) are being extensively used for tissue engineering purposes and detergents are predominantly used for this. A facile detergent-free decellularization method is developed for dermal matrix and compared it with the most used detergent-based decellularization methods. An optimized, single-step, cost-effective Hypotonic/Hypertonic (H/H) Sodium Chloride (NaCl) solutions-based method is employed to decellularize goat skin that resulted in much higher yield than other methods. The ECM composition, mechanical property, and cytocompatibility are evaluated and compared with other decellularization methods. Furthermore, this H/H-treated decellularized dermal ECM (ddECM) exhibits a residual DNA content of <50 ng mg-1  of dry tissue. Moreover, 85.64 ± 3.01% of glycosaminoglycans and 65.53 ± 2.9% collagen are retained compared to the native tissue, which is higher than the ddECMs prepared by other methods. The cellular response is superior in ddECM (H/H) than other ddECMs prepared by detergent-based methods. Additionally, a bioink is formulated with the ddECM (H/H), showing good shear thinning and shear recovery properties. Process optimization in terms of print speed, flow rate, and viscosity is done to obtain a bioprinting window for ddECM bioink. The printed constructs with optimized parameters have adequate mechanical and cell adhesive properties and excellent isotropic cellular alignment.