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1. Cryopreservation of 3D Bioprinted Scaffolds with Temperature-Controlled-Cryoprinting

   https://doi.org/10.3390/gels9060502  

   Warburton, Linnea; Rubinsky, Boris (June 2023, Gels)

   Temperature-Controlled-Cryoprinting (TCC) is a new 3D bioprinting technology that allows for the fabrication and cryopreservation of complex and large cell-laden scaffolds. During TCC, bioink is deposited on a freezing plate that descends further into a cooling bath, keeping the temperature at the nozzle constant. To demonstrate the effectiveness of TCC, we used it to fabricate and cryopreserve cell-laden 3D alginate-based scaffolds with high cell viability and no size limitations. Our results show that Vero cells in a 3D TCC bioprinted scaffold can survive cryopreservation with a viability of 71%, and cell viability does not decrease as higher layers are printed. In contrast, previous methods had either low cell viability or decreasing efficacy for tall or thick scaffolds. We used an optimal temperature profile for freezing during 3D printing using the two-step interrupted cryopreservation method and evaluated drops in cell viability during the various stages of TCC. Our findings suggest that TCC has significant potential for advancing 3D cell culture and tissue engineering. 

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2. Temperature-Controlled 3D Cryoprinting Inks Made of Mixtures of Alginate and Agar

   https://doi.org/10.3390/gels9090689  

   Lou, Leo; Rubinsky, Boris (September 2023, Gels)

   Temperature-controlled 3D cryoprinting (TCC) is an emerging tissue engineering technology aimed at overcoming limitations of conventional 3D printing for large organs: (a) size constraints due to low print rigidity and (b) the preservation of living cells during printing and subsequent tissue storage. TCC addresses these challenges by freezing each printed voxel with controlled cooling rates during deposition. This generates a rigid structure upon printing and ensures cell cryopreservation as an integral part of the process. Previous studies used alginate-based ink, which has limitations: (a) low diffusivity of the CaCl2 crosslinker during TCC’s crosslinking process and (b) typical loss of print fidelity with alginate ink. This study explores the use of an ink made of agar and alginate to overcome TCC protocol limitations. When an agar/alginate voxel is deposited, agar first gels at above-freezing temperatures, capturing the desired structure without compromising fidelity, while alginate remains uncrosslinked. During subsequent freezing, both frozen agar and alginate maintain the structure. However, agar gel loses its gel form and water-retaining ability. In TCC, alginate crosslinking occurs by immersing the frozen structure in a warm crosslinking bath. This enables CaCl2 diffusion into the crosslinked alginate congruent with the melting process. Melted agar domains, with reduced water-binding ability, enhance crosslinker diffusivity, reducing TCC procedure duration. Additionally, agar overcomes the typical fidelity loss associated with alginate ink printing. 

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3. Rheological Study of Low-Viscous Hydrogels for 3d Bio-Printing Processes

   Tuladhar, Slesha; Nelson, Cartwright; Habib, Ahasan. (July 2021, Proceedings of the 2021 IISE Annual Virtual Conference)

   The promising success of 3D printing technique with synthetic polymers like nylon, ABS, PLA and epoxy motivates the researchers to put efforts into fabricating constructs with biocompatible natural polymers. The efforts have been broadened into various fields such as bioengineering, manufacturing, and regenerative medicine. Additive biomanufacturing commonly known as 3D bioprinting shows a lot of potential in tissue engineering with those natural polymers. Some challenges such as achieving printability, maintaining geometry in post printing stage, comforting encapsulated cells, and ensuring high proliferation are to be resolved to turn this process into a successful trial. Appropriate design of experiments with a detail rheological investigation can identify useful mechanical properties which is directly related to shape fidelity of 3D bio-printed scaffolds. As candidate natural polymers, Alginate-low viscous Carboxymethyl Cellulose (CMC) was used restricting the solid content 8% (w/v). Various rheological tests, such as the Steady Rate Sweep Test, Thixotropic (3ITT), Amplitude, and Frequency test were performed. The result indicated that rheological properties are CMC dependent. Printability and shape fidelity were analyzed of the filaments and scaffolds fabricated with all the combinations. The rheological results were co-related with the printability and shape fidelity result. 

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4. Translational biomaterials of four-dimensional bioprinting for tissue regeneration

   https://doi.org/10.1088/1758-5090/acfdd0  

   Faber, Leah; Yau, Anne; Chen, Yupeng (October 2023, Biofabrication)

   Abstract Bioprinting is an additive manufacturing technique that combines living cells, biomaterials, and biological molecules to develop biologically functional constructs. Three-dimensional (3D) bioprinting is commonly used as anin vitromodeling system and is a more accurate representation ofin vivoconditions in comparison to two-dimensional cell culture. Although 3D bioprinting has been utilized in various tissue engineering and clinical applications, it only takes into consideration the initial state of the printed scaffold or object. Four-dimensional (4D) bioprinting has emerged in recent years to incorporate the additional dimension of time within the printed 3D scaffolds. During the 4D bioprinting process, an external stimulus is exposed to the printed construct, which ultimately changes its shape or functionality. By studying how the structures and the embedded cells respond to various stimuli, researchers can gain a deeper understanding of the functionality of native tissues. This review paper will focus on the biomaterial breakthroughs in the newly advancing field of 4D bioprinting and their applications in tissue engineering and regeneration. In addition, the use of smart biomaterials and 4D printing mechanisms for tissue engineering applications is discussed to demonstrate potential insights for novel 4D bioprinting applications. To address the current challenges with this technology, we will conclude with future perspectives involving the incorporation of biological scaffolds and self-assembling nanomaterials in bioprinted tissue constructs. 

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5. Compensating the cell-induced light scattering effect in light-based bioprinting using deep learning

   https://doi.org/10.1088/1758-5090/ac3b92  

   Guan, Jiaao; You, Shangting; Xiang, Yi; Schimelman, Jacob; Alido, Jeffrey; Ma, Xinyue; Tang, Min; Chen, Shaochen (December 2021, Biofabrication)

   Abstract Digital light processing (DLP)-based three-dimensional (3D) printing technology has the advantages of speed and precision comparing with other 3D printing technologies like extrusion-based 3D printing. Therefore, it is a promising biomaterial fabrication technique for tissue engineering and regenerative medicine. When printing cell-laden biomaterials, one challenge of DLP-based bioprinting is the light scattering effect of the cells in the bioink, and therefore induce unpredictable effects on the photopolymerization process. In consequence, the DLP-based bioprinting requires extra trial-and-error efforts for parameters optimization for each specific printable structure to compensate the scattering effects induced by cells, which is often difficult and time-consuming for a machine operator. Such trial-and-error style optimization for each different structure is also very wasteful for those expensive biomaterials and cell lines. Here, we use machine learning to learn from a few trial sample printings and automatically provide printer the optimal parameters to compensate the cell-induced scattering effects. We employ a deep learning method with a learning-based data augmentation which only requires a small amount of training data. After learning from the data, the algorithm can automatically generate the printer parameters to compensate the scattering effects. Our method shows strong improvement in the intra-layer printing resolution for bioprinting, which can be further extended to solve the light scattering problems in multilayer 3D bioprinting processes. 

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