Alginate-based Bioink Research Articles

Incorporating silica nanoparticles with silver patches into alginate-based bioinks for 3D bioprinting

Abstract Alginate dialdehyde-gelatin (ADA-GEL) hydrogels are being studied in bioprinting for their combination of cell adhesion and printability. To incorporate specific functionalities and to improve printability, different additives to ADA-GEL inks are being proposed. Here, a novel type of functional nanoparticles comprising silver patches on silica cores was incorporated into ADA-GEL bioinks. Silver patchy particles (SPPs) were present both on the surface and interior of printed structures. Incorporation of SPPs improved printability of ADA-GEL inks and supported osteosarcoma (MG-63) cells over 7 days of culture. SPPs represent a valuable additive for ADA-GEL hydrogels, being attractive to develop bioinks with advanced functionalities. Graphical abstract

Rational Design of a Triple-Layered Coaxial Extruder System: in silico and in vitro Evaluations Directed Toward Optimizing Cell Viability.

Biofabrication is a rapidly evolving field whose main goal is the manufacturing of three-dimensional (3D) cell-laden constructs that closely mimic tissues and organs. Despite recent advances on materials and techniques directed toward the achievement of this goal, several aspects such as tissue vascularization and prolonged cell functionality are limiting bench-to-bedside translation. Extrusion-based 3D bioprinting has been devised as a promising biofabrication technology to overcome these limitations, due to its versatility and wide availability. Here, we report the development of a triple-layered coaxial nozzle for use in the biomanufacturing of vascular networks and vessels. The design of the coaxial nozzle was first optimized toward guaranteeing high cell viability upon extrusion. This was done with the aid of in silico evaluations and their subsequent experimental validation by investigating the bioprinting of an alginate-based bioink. Results confirmed that the values for pressure distribution predicted by in silico experiments resulted in cell viabilities above 70% and further demonstrated the effect of layer thickness and extrusion pressure on cell viability. Our work paves the way for the rational design of multi-layered coaxial extrusion systems to be used in biofabrication approaches to replicate the very complex structures found in native organs and tissues.

Synergistic effect of Cydonia oblonga and its extracted silver nanoparticles for improving antioxidant and antibacterial activity of 3D printed alginate-based hydrogel as wound dressing

The current research studies the synergistic effect of Cydonia oblonga and its extracted nano bio‑silver as a natural and eco-friendly agent for the improvement of three-dimensional (3D)-printed alginate wound dressings. Therefore, Cydonia oblonga extract was first prepared and silver nanoparticles were extracted from it through a green and simple method. The Cydonia oblonga and its extracted bio-based nanoparticles were then added to 3D printing alginate-based ink. Subsequently, a 3D structural extrusion printer was employed to create the porous hydrogel-based wound dressings. The morphological investigation demonstrated that using the extraction method the bio-based silver nanoparticles were successfully prepared, having an average size of 17.95 ± 4.50 nm. The Cydonia oblonga extract showed comparable antioxidant activity to the commercial antioxidant and an excellent total phenol content. In addition, the results showed the combination of Cydonia oblonga extracts/silver nanoparticles significantly improved the antibacterial performance of alginate-based bioinks. In vivo, and in vitro studies confirmed their biocompatibility and significant efficacy in the treatment of burn wounds.

Light-Responsive and Antibacterial Graphenic Materials as a Holistic Approach to Tissue Engineering.

While the continuous development of advanced bioprinting technologies is under fervent study, enhancing the regenerative potential of hydrogel-based constructs using external stimuli for wound dressing has yet to be tackled. Fibroblasts play a significant role in wound healing and tissue implants at different stages, including extracellular matrix production, collagen synthesis, and wound and tissue remodeling. This study explores the synergistic interplay between photothermal activity and nanomaterial-mediated cell proliferation. The use of different graphene-based materials (GBM) in the development of photoactive bioinks is investigated. In particular, we report the creation of a skin-inspired dressing for wound healing and regenerative medicine. Three distinct GBM, namely, graphene oxide (GO), reduced graphene oxide (rGO), and graphene platelets (GP), were rigorously characterized, and their photothermal capabilities were elucidated. Our investigations revealed that rGO exhibited the highest photothermal efficiency and antibacterial properties when irradiated, even at a concentration as low as 0.05 mg/mL, without compromising human fibroblast viability. Alginate-based bioinks alongside human fibroblasts were employed for the bioprinting with rGO. The scaffold did not affect the survival of fibroblasts for 3 days after bioprinting, as cell viability was not affected. Remarkably, the inclusion of rGO did not compromise the printability of the hydrogel, ensuring the successful fabrication of complex constructs. Furthermore, the presence of rGO in the final scaffold continued to provide the benefits of photothermal antimicrobial therapy without detrimentally affecting fibroblast growth. This outcome underscores the potential of rGO-enhanced hydrogels in tissue engineering and regenerative medicine applications. Our findings hold promise for developing game-changer strategies in 4D bioprinting to create smart and functional tissue constructs with high fibroblast proliferation and promising therapeutic capabilities in drug delivery and bactericidal skin-inspired dressings.

Characterization of two different alginate-based bioinks and the influence of melanoma growth within

Extrusion-based bioprinting is an established method in biofabrication. Suitable bioinks have fundamentally different compositions and characteristics, which should be examined, in order to find a perfect model system. Here, we investigate the effect of two alginate-based, yet unalike 3D-printed bioinks, pre-crosslinked alginate-dialdehyde gelatin (ADA-GEL) and a mixture of alginate, hyaluronic acid, and gelatin (Alg/HA/Gel), on the melanoma cell line Mel Im and vice versa in terms of stiffness, shrinkage, cellular behavior and colony formation over 15 days. Rheological stiffness measurements revealed two soft gels with similar storage moduli. The cells did not have a significant impact on the overall stiffness, whereas ADA-GEL (2.5/2.5%) was significantly stiffer than Alg/HA/Gel (0.5/0.1/3%). Regarding the shrinkage of printed constructs, cells had a significant influence, especially in ADA-GEL, which has covalent bonds between the oxidized alginate and gelatin. Multi-photon microscopy exhibited proliferation, cell spreading and migration in ADA-GEL with cell–cell and cell–matrix interaction, dissimilarly to Alg/HA/Gel, in which cells formed spherical, encapsulated colonies. Scanning electron microscopy and histology showed degradation and multi-layered growth on ADA-GEL and fewer examples of escaped cells on Alg/HA/Gel. Both gels serve as proliferation bioink for melanoma with more necrosis in deeper Alg/HA/Gel colonies and differences in spreading and matrix interaction. These findings show the importance of proper characterization of the bioinks for different applications.

Fabrication of a Novel 3D Extrusion Bioink Containing Processed Human Articular Cartilage Matrix for Cartilage Tissue Engineering.

Cartilage damage presents a significant clinical challenge due to its intrinsic avascular nature which limits self-repair. Addressing this, our study focuses on an alginate-based bioink, integrating human articular cartilage, for cartilage tissue engineering. This novel bioink was formulated by encapsulating C20A4 human articular chondrocytes in sodium alginate, polyvinyl alcohol, gum arabic, and cartilage extracellular matrix powder sourced from allograft femoral condyle shavings. Using a 3D bioprinter, constructs were biofabricated and cross-linked, followed by culture in standard medium. Evaluations were conducted on cellular viability and gene expression at various stages. Results indicated that the printed constructs maintained a porous structure conducive to cell growth. Cellular viability was 87% post printing, which decreased to 76% after seven days, and significantly recovered to 86% by day 14. There was also a notable upregulation of chondrogenic genes, COL2A1 (p = 0.008) and SOX9 (p = 0.021), suggesting an enhancement in cartilage formation. This study concludes that the innovative bioink shows promise for cartilage regeneration, demonstrating substantial viability and gene expression conducive to repair and suggesting its potential for future therapeutic applications in cartilage repair.

Cell viability prediction and optimization in extrusion-based bioprinting via neural network-based Bayesian optimization models

The fields of regenerative medicine and cancer modeling have witnessed tremendous growth in the application of 3D bioprinting. Maintaining high cell viability throughout the bioprinting process is crucial for the success of this technology, as it directly affects the accuracy of the 3D bioprinted models, the validity of experimental results, and the discovery of new therapeutic approaches. Therefore, optimizing bioprinting conditions, which include numerous variables influencing cell viability during and after the procedure, is of utmost importance to achieve desirable results. So far, these optimizations have been accomplished primarily through trial and error and repeating multiple time-consuming and costly experiments. To address this challenge, we initiated the process by creating a dataset of these parameters for gelatin and alginate-based bioinks and the corresponding cell viability by integrating data obtained in our laboratory and those derived from the literature. Then, we developed machine learning models to predict cell viability based on different bioprinting variables. The trained neural network yielded regression R2 value of 0.71 and classification accuracy of 0.86. Compared to models that have been developed so far, the performance of our models is superior and shows great prediction results. The study further introduces a novel optimization strategy that employs the Bayesian optimization model in combination with the developed regression neural network to determine the optimal combination of the selected bioprinting parameters to maximize cell viability and eliminate trial-and-error experiments. Finally, we experimentally validated the optimization model’s performance.

3D-bioprinted alginate-based bioink scaffolds with β-tricalcium phosphate for bone regeneration applications

Background/purpose3D-printed bone tissue engineering is becoming recognized as a key approach in dentistry for creating customized bone regeneration treatments fitting patients bone defects requirements. 3D bioprinting offers an innovative method to fabricate detailed 3D structures, closely emulating the native bone micro-environment and better bone regeneration. This study aimed to develop an 3D-bioprintable scaffold using a combination of alginate and β-tricalcium phosphate (β-TCP) with the Cellink® BioX printer, aiming to advance the field of tissue engineering. Materials and methodsThe physical and biological properties of the resulting 3D-printed scaffolds were evaluated at 10 %, 12 %, and 15 % alginate combined with 10 % β-TCP. The scaffolds were characterized through printability, swelling behavior, degradability, and element analysis. The biological assessment included cell viability, alkaline phosphatase (ALP) activity. Results10 % alginate/β-TCP 3D printed at 25 °C scaffold demonstrated the optimal condition for printability, swelling capability, and degradability of cell growth and nutrient diffusion. Addition of β-TCP particles significantly improved the 3D printed material viscosity over only alginate (P < 0.05). 10 % alginate/β-TCP enhanced MG-63 cell's proliferation (P < 0.05) and alkaline phosphatase activity (P < 0.001). ConclusionThis study demonstrated in vitro that 10 % alginate/β-TCP bioink characteristic for fabricating 3D acellular bioprinted scaffolds was the best approach. 10 % alginate/β-TCP bioink 3D-printed scaffold exhibited superior physical properties and promoted enhanced cell viability and alkaline phosphatase activity, showing great potential for personalized bone regeneration treatments.

Engineering peptide-modified alginate-based bioinks with cell-adhesive properties for biofabrication.

Alginate (ALG) and its oxidised form alginate-dialdehyde (ADA) are highly attractive materials for hydrogels used in 3D bioprinting as well as drop-on-demand (DoD) approaches. However, both polymers need to be modified using cell-adhesive peptide sequences, to obtain bioinks exhibiting promising cell-material interactions. Our study explores the modification of ALG- and ADA-based bioinks with the adhesive peptides YIGSR (derived from laminin), RRETEWA (derived from fibronectin) and IKVAV (derived from laminin) for 3D bioprinting. Two coupling methods, carbodiimide and Schiff base reactions, were employed to modify the polymers with peptides. Analytical techniques, including FTIR and NMR were used to assess the chemical composition, revealing challenges in confirming the presence of peptides. The modified bioinks exhibited decreased stability, viscosity, and stiffness, particularly-ADA-based bioinks in contrast to ALG. Sterile hydrogel capsules or droplets were produced using a manual manufacturing process and DoD printing. NIH/3T3 cell spreading analysis showed enhanced cell spreading in carbodiimide-modified ADA, Schiff base-modified ADA, and PEG-Mal-modified ADA. The carbodiimide coupling of peptides worked for ADA, however the same was not observed for ALG. Finally, a novel mixture of all used peptides was evaluated regarding synergistic effects on cell spreading which was found to be effective, showing higher aspect ratios compared to the single peptide coupled hydrogels in all cases. The study suggests potential applications of these modified bioinks in 3D bioprinting approaches and highlights the importance of peptide selection as well as their combination for improved cell-material interactions.

Biopatterning of 3D Cellular Model by Contactless Magnetic Manipulation for Cardiotoxicity Screening.

Impact statement Contactless manipulation and cell patterning techniques provide rapid and cost-effective three-dimensional (3D) cell culture model formation for tissue engineering applications. The present study introduces a new methodology that comprised alginate-based bioink to pattern cells via contactless magnetic manipulation to fabricate 3D cardiac structures. The developed cardiac model was evaluated in terms of Doxorubicin-induced cardiotoxicity and biopatterned 3D cardiac structures were found more resistant to drug exposure compared to two-dimensional control.

Coaxial bioprinting of cellulose nanocrystal-reinforced core-sheath strands for alginate hydrogel construct fabrication

Alginate is among the most popular bioinks for 3D bioprinting because it is biocompatible, biodegradable, and is easily crosslinked using divalent cations. Here, we investigate the addition of cellulose nanocrystals (CNCs) to an alginate-based bioink to strengthen bioprinted structures. Using coaxial 3D bioprinting, alginate strands were constructed with a core-sheath structure reinforced by CNCs within the core. The addition of CNCs resulted in an increase in the storage modulus. Formulations containing CNCs resulted in more stable strands with uniformity factors and printability numbers close to 1. Based on the uniformity factor, the formulations containing 1.5 wt% CNCs deviated the least from a theoretically flawless strand. The alginate strands displayed microporous internal structures that were evident by scanning electron microscopy of the strand cross-sections. The addition of CNCs to the core compartment doubled both the modulus and the tensile strength, and the material was able to resist a strain above 50% before failure, thus demonstrating that the addition of the CNCs to the core of the strand leads to resilient printed structures.

Nanocrystalline Cellulose as a Versatile Engineering Material for Extrusion-Based Bioprinting.

Naturally derived polysaccharide-based hydrogels, such as alginate, are frequently used in the design of bioinks for 3D bioprinting. Traditionally, the formulation of such bioinks requires the use of pre-reticulated materials with low viscosities, which favour cell viability but can negatively influence the resolution and shape fidelity of the printed constructs. In this work, we propose the use of cellulose nanocrystals (CNCs) as a rheological modifier to improve the printability of alginate-based bioinks whilst ensuring a high viability of encapsulated cells. Through rheological analysis, we demonstrate that the addition of CNCs (1% and 2% (w/v)) to alginate hydrogels (1% (w/v)) improves shear-thinning behaviour and mechanical stability, resulting in the high-fidelity printing of constructs with superior resolution. Importantly, LIVE/DEAD results confirm that the presence of CNCs does not seem to affect the health of immortalised chondrocytes (TC28a2) that remain viable over a period of seven days post-encapsulation. Taken together, our results indicate a favourable effect of the CNCs on the rheological and biocompatibility properties of alginate hydrogels, opening up new perspectives for the application of CNCs in the formulation of bioinks for extrusion-based bioprinting.

Rheology-informed hierarchical machine learning model for the prediction of printing resolution in extrusion-based bioprinting

In this study, a rheology-informed hierarchical machine learning (RIHML) model was developed to improve the prediction accuracy of the printing resolution of constructs fabricated by extrusion-based bioprinting. Specifically, the RIHML model, as well as conventional models such as the concentration-dependent model and printing parameter-dependent model, was trained and tested using a small dataset of bioink properties and printing parameters. Interestingly, the results showed that the RIHML model exhibited the lowest error percentage in predicting the printing resolution for different printing parameters such as nozzle velocities and pressures, as well as for different concentrations of the bioink constituents. Besides, the RIHML model could predict the printing resolution with reasonably low errors even when using a new material added to the alginate-based bioink, which is a challenging task for conventional models. Overall, the results indicate that the RIHML model can be a useful tool to predict the printing resolution of extrusion-based bioprinting, and it is versatile and expandable compared to conventional models since the RIHML model can easily generalize and embrace new data.

Multimodal and dynamic cross-linking of modular thiolated alginate-based bioinks

Engineered extracellular matrix-mimicking hydrogels can facilitate 3D cell culture and fabrication of tissue-like constructs and biologically relevant disease models. Processing of cell-laden hydrogels using additive manufacturing techniques further allows for the development of tissue-mimetic structures with higher spatial complexity. Whereas a wide range of printable hydrogels is available, they tend to lack biological functionality and cell compatibility. Here we show an enzymatically mediated thiol-based cross-linking strategy for the design of modular and cytocompatible hydrogel-based bioinks for 3D bioprinting of dynamic multi-material architectures. Alginate is functionalized with cysteines modified with an enzyme-labile thiol protection group. Deprotection using penicillin G acylase (PGA) generates free thiols on-demand, enabling hydrogel cross-linking using thiol-reactive cross-linkers and intermolecular disulfides while avoiding undesired and uncontrolled thiol oxidation. Remaining free thiols can be used for post-printing hydrogel functionalization and lamination of multilayer structures. Moreover, the addition of PGA to a thermo-reversible hydrogel support bath enables the bioprinting of cell-laden 3D structures with high cell viability and excellent shape fidelity. The possibilities to enzymatically generate free thiols during bioprinting facilitate cross-linking and tuning of bioink properties using cytocompatible chemistries and allow for the printing of complex and dynamic cell-laden structures.

Internally crosslinked alginate-based bioinks for the fabrication of in vitro hepatic tissue models

Bioprinting is a key technique to fabricate cell-laden volumetric constructs with controlled geometry. It can be used not only to replicate the architecture of a target organ but also to produce shapes that allow for the mimicry, in vitro, of specific desired features. Among the various materials suitable to be processed with this technique, sodium alginate is currently considered one of the most appealing because of its versatility. To date, the most widespread strategies to print alginate-based bioinks exploit external gelation as a primary process, by directly extruding the hydrogel-precursor solution into a crosslinking bath or within a sacrificial crosslinking hydrogel, where the gelation takes place. In this work, we describe the print optimization and the processing of Hep3Gel: an internally crosslinked alginate and ECM-based bioink for the production of volumetric hepatic tissue models. We adopted an unconventional strategy, by moving from the reproduction of the geometry and the architecture of liver tissue to the use of bioprinting to fabricate structures that can promote a high degree of oxygenation, as is the case with hepatic tissue. To this end, the design of structures was optimized by employing computational methods. The printability of the bioink was then studied and optimized through a combination of different a priori and a posteriori analyses. We produced 14-layered constructs, thus highlighting the possibility to exploit internal gelation alone to directly print self-standing structures with finely controlled viscoelastic properties. Constructs loaded with HepG2 cells were successfully printed and cultured in static conditions for up to 12 d, underlining the suitability of Hep3Gel to support mid/long-term cultures.

In vitro construction of liver organoids with biomimetic lobule structure by a multicellular 3D bioprinting strategy

Liver disease is one of the serious threats to human life and health. Three-dimensional (3D) liver models, which simulate the structure and function of natural liver tissue in vitro, have become a common demand in medical, scientific and pharmaceutical fields nowadays. However, the complex cellular composition and multi-scale spatial arrangement of liver tissue make it extremely challenging to construct liver models in vitro. According to HepaRG preference and printing strategy, the formulation of bioink system with opposite charge is optimized. The sodium alginate-based bioink 1 and dipeptide-based bioink 2 are used to ensure structural integrity and provide flexible designability, respectively. The HepaRG/HUVECs/LX-2-laden liver organoids with biomimetic lobule structure are fabricated by a multicellular 3D droplet-based bioprinting strategy, to mimic the cell heterogeneity, spatial structure and extracellular matrix (ECM) features. The liver organoids can maintain structural integrity and multicellular distribution within the printed lobule-like structure after 7 days of culture. Compared with the 2D monolayer culture, the constructed 3D organoids show high cell viability, ALB secretion and urea synthesis levels. This study provides a droplet-based and layer-by-layer 3D bioprinting strategy for in vitro construction of liver organoids with biomimetic lobule structure, giving meaningful insights in the fields of new drugs, disease modelling, and tissue regeneration.

Marine Collagen-Based Bioink for 3D Bioprinting of a Bilayered Skin Model.

Marine organisms (i.e., fish, jellyfish, sponges or seaweeds) represent an abundant and eco-friendly source of collagen. Marine collagen, compared to mammalian collagen, can be easily extracted, is water-soluble, avoids transmissible diseases and owns anti-microbial activities. Recent studies have reported marine collagen as a suitable biomaterial for skin tissue regeneration. The aim of this work was to investigate, for the first time, marine collagen from basa fish skin for the development of a bioink for extrusion 3D bioprinting of a bilayered skin model. The bioinks were obtained by mixing semi-crosslinked alginate with 10 and 20 mg/mL of collagen. The bioinks were characterised by evaluating the printability in terms of homogeneity, spreading ratio, shape fidelity and rheological properties. Morphology, degradation rate, swelling properties and antibacterial activity were also evaluated. The alginate-based bioink containing 20 mg/mL of marine collagen was selected for 3D bioprinting of skin-like constructs with human fibroblasts and keratinocytes. The bioprinted constructs showed a homogeneous distribution of viable and proliferating cells at days 1, 7 and 14 of culture evaluated by qualitative (live/dead) and qualitative (XTT) assays, and histological (H&E) and gene expression analysis. In conclusion, marine collagen can be successfully used to formulate a bioink for 3D bioprinting. In particular, the obtained bioink can be printed in 3D structures and is able to support fibroblasts and keratinocytes viability and proliferation.

How to Determine a Suitable Alginate for Biofabrication Approaches using an Extensive Alginate Library?

Alginate-based hydrogels are a promising class of biomaterials due to their usability, biocompatibility, and high water-binding capacity which is the reason for their broad use in biofabrication. One challenge of these biomaterials is, however, the lack of cell adhesion motifs. This drawback can be overcome by oxidizing alginate to alginate dialdehyde (ADA) and by subsequent cross-linking with gelatin (GEL) to fabricate ADA-GEL hydrogels, which offer improved cell-material interactions. The present work investigates four pharmaceutical grade alginates of different algae sources and their respective oxidized forms regarding their molecular weight and M/G ratio using 1H NMR spectroscopy and gel permeation chromatography. In addition, three different methods for determining the degree of oxidation (% DO) of ADA, including iodometric, spectroscopic, and titration methods, are applied and compared. Furthermore, the aforementioned properties are correlated with the resulting viscosity, degradation behavior, and cell-material interactions to predict the material behavior in vitro and thus choose a suitable alginate for an intended application in biofabrication. In the framework of the present work, easy and practicable detection methods for the investigations of alginate-based bioinks were summarized and shown. In this regard, the success of oxidation of alginate was confirmed by the three aforementioned methods and was further proven by solid-state 13C NMR, for the first time in the literature, that only guluronic acid (G) was attacked during the oxidation, leading to the formation of hemiacetals. Furthermore, it was shown that ADA-GEL hydrogels of alginates with longer G-blocks are more suitable for long-term experiments due to their stability over an incubation period of 21 days, while ADA-GEL hydrogels of alginates with longer mannuronic acid (M)-blocks are more suitable for short-term applications such as sacrificial inks due to their extensive swelling and subsequent loss of shape. Finally, it was proven that the M/G ratio did not show any influence on the biocompatibility or printability of the investigated alginate-based hydrogels. The physicochemical findings provide an alginate library for tailored application in biofabrication.

Abstract P6-01-38: Predictive efficacy biomarker for chemotherapy agents against triple-negative breast cancer bioprinted organoid tumors (BOTs) using solid tumor biopsy-on-a-chip

Abstract Background: Despite $90 billion in preclinical research and clinical trials every year, 90% of cancer clinical trials are unsuccessful. Thus, there is considerable interest in predicting clinical efficacy of preclinical formulations early in discovery using patient derived ex vivo platforms. However, extracting adequate tissue for such models can be difficult depending on tumor type and site. Dominant mechanisms for expanding patient tissue include xenografts and organoids. However, the former imposes a large time window to establish while the latter is constrained in space by sizescales. Here, we present a novel approach for expanding TNBC tissue, specifically for use in ex vivo models, that addresses these constrains with 3D bioprinted organoid tumors (BOTs). Objective: The aim of this study is to generate TNBC BOTs that mimic core biopsy tissue for use in ex vivo precision and personalized predictive biomarkers for chemotherapies. Methods: BOTs were generated using alginate-based bioink prepared with MDA-MB-231 TNBC cells. Briefly, specially prepared fresh TNBC bioink was deposited layer-by-layer using a Cellink BIO X6 bioprinter in geometrical configurations to mimic 14- to 18- gauge tumor biopsies. These were chemically cross-linked and cured in stages to allow cells and matrix to self-assemble with limited degrees of freedom. Fully cured BOTs were loaded in our ex vivo solid tumor biopsy-on-a-chip and treated with chemotherapy agents to evaluate sensitivity and resistance, with outcomes determined using immunofluorescent live and dead cell staining methods. Results: A 3-minute crosslinking time with calcium chloride provided a stable and functional sodium alginate medium for treating cellular BOTs. Two layers of TNBC bioink was determined to be the optimal size and shape for compatibility with our ex vivo biopsy-on-a-chip predictive efficacy biomarker platform. TNBC cells were verified to be evenly distributed within the cured sodium alginate matrix using live cell nuclear stains. Successful diffusion of multiple agents to spatially distinct regions of the bioprinted tissue was verified with fluorescently labeled small molecules and nucleic acid stains up to 200 µM deep. Impact: Patient derived BOT core mimics and other configurations could be used in ex vivo breast cancer chemotherapy screening models to obtain sensitivity and resistance profiles as predictive functional biomarkers both on the bedside for personalized treatment strategy development and on the bench to uncover new therapeutic targets. Due to their potential to replicate biophysical and biochemical characteristics of a tumor and its microenvironment, BOT based precision and personalized medicine platforms can provide more accurate drug efficacy readout compared to in vitro cancer models. Citation Format: Seth Bollenbecker, Zeelu Patel, Zeenia Punjani, Areesha Charania, Heli Patel, Alyssa Abbott, Kaitlyn Kunkle, Mary Kathryn Sewell-Loftin, Gregory Grossman, Karim Budhwani. Predictive efficacy biomarker for chemotherapy agents against triple-negative breast cancer bioprinted organoid tumors (BOTs) using solid tumor biopsy-on-a-chip [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-38.

Cellular adhesion and chondrogenic differentiation inside an alginate-based bioink in response to tailorable artificial matrices and tannic acid treatment

Many established bioinks fulfill important requirements regarding fabrication standards and cytocompatibility. Current research focuses on development of functionalized bioinks with an improved support of tissue-specific cell differentiation. Many approaches primarily depend on decellularized extracellular matrices or blood components. In this study, we investigated the combination of a highly viscous alginate-methylcellulose (algMC) bioink with collagen-based artificial extracellular matrix (aECM) as a finely controllable and tailorable system composed of collagen type I (col) with and without chondroitin sulfate (CS) or sulfated hyaluronan (sHA). As an additional stabilizer, the polyphenol tannic acid (TA) was integrated into the inks. The assessment of rheological properties and printability as well as hydrogel microstructure revealed no adverse effect of the integrated components on the inks. Viability, adhesion, and proliferation of bioprinted immortalized human mesenchymal stem cells (hTERT-MSC) was improved indicating enhanced interaction with the designed microenvironment. Furthermore, chondrogenic matrix production (collagen type II and sulfated glycosaminoglycans) by primary human chondrocytes (hChon) was enhanced by aECM. Supplementing the inks with TA was required for these positive effects but caused cytotoxicity as soon as TA concentrations exceeded a certain amount. Thus, combining tailorable aECM with algMC and balanced TA addition proved to be a promising approach for promoting adhesion of immortalized stem cells and differentiation of chondrocytes in bioprinted scaffolds.