Advancing Tissue Engineering: High-Throughput Bioprinting of Spheroids

Welcome to our tenth newsletter! This edition highlights a very interesting study that was recently published in Nature Communications, that introduces HITS-Bio.

The “HITS- Bio”

HITS-Bio (High-throughput Integrated Tissue Fabrication System for Bioprinting) marks a major leap in rapid bioprinting of spheroids for scalable tissue fabrication. This technique allows for the precise positioning of spheroids (and even organoids) in a high-throughput process that operates at an unprecedented speed about ten folds faster than the existing techniques with a high cell viability rate of over > 90%.

The innovation completely lies in its use of a Digitally controlled nozzle array (DCNA), which enables the simultaneous patterning and spatial arrangement of multiple spheroids with exceptional precision.

DCNA, or Digitally-Controlled Nozzle Array, is a critical component of the HITS-Bio (High-throughput Integrated Tissue Fabrication System for Bioprinting) platform.

DCNA consists of a multi-array system with multiple nozzles that can be independently controlled. This allows for the simultaneous aspiration, positioning, and deposition of multiple spheroids, significantly reducing the time required for tissue fabrication compared to traditional single-nozzle methods. The controlled aspiration pressure allows to pick up spheroids from a culture medium and deposit them on a substrate. The number of nozzles in the DCNA can be customized, making it adaptable for various scales of bioprinting. The array’s movements are digitally controlled to ensure accurate placement of spheroids at specific positions.

Real-time imaging systems, such as integrated cameras, verify the exact placement of spheroids during bioprinting.

Custom Bioinks for Enhanced Functionality:

Researchers also focussed on customizing bioinks specifically for this platform, acting as cement-like substrates to retain spheroids in desired patterns while promoting extracellular matrix (ECM) formation.

They developed BONink and CARink with tailored properties for bone and cartilage tissue engineering respectively, ensuring compatibility , shear thinning, self healing properties, high cell viability and structural integrity.

BONink(Bone Ink):
BONink composed of Gelatin methacryloyl (GelMA): Provides tunable mechanical properties and biocompatibility.
Nanohydroxyapatite (HA): Enhances osteoconductivity, which is critical for bone regeneration.
β-glycerophosphate disodium salt hydrate (β-GP): Enables thermal gelation, improving printability and stability.
Hyaluronic acid (HyA): Promotes cell adhesion and proliferation.
Fibrinogen: Facilitates cell-matrix interactions.
Lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP): A photoinitiator for light-induced crosslinking.

CARink (Cartilage Ink): CARink composed of GelMA (20% concentration for enhanced mechanical properties). Additives similar to BONink but without components specific to bone regeneration like β-GP and nanohydroxyapatite.

To demonstrate the practical potential of this technology, researchers focused on regenerating calvarial bone by repairing critical-size defects using intraoperative bioprinting (IOB) with osteogenically-committed bone spheroids. This approach allows tissues to be fabricated on-demand during surgery, drastically reducing the time needed for the procedure.

The process utilized combinatorial micro-RNA (miR) technology to promote osteogenic differentiation in the spheroids, while HITS-Bio enabled smooth, on-demand aspiration and bioprinting of these miR-transfected spheroids.

BONink was used in the study for calvarial bone regeneration in a rat model. The bioink acted as a supportive substrate for osteogenically committed spheroids, enabling them to form new bone tissue. It demonstrated effective bone healing, with nearly complete defect closure and significant mineral deposition.

CARink was used to fabricate large-scale cartilage constructs, such as a 1 cm³ cartilage tissue composed of 576 chondrogenically committed spheroids, each construct was assembled in under 40 minutes, showcasing a level of speed and efficiency far surpassing that of current bioprinting technologies. The constructs exhibited high ECM deposition and mechanical properties resembling native cartilage.

While HITS-Bio shows great potential, there are certainly areas of limitations and improvements:

First, the DCNA system can experience clogging, typically caused by cell debris accumulating in the spheroid chamber, which may slow down the bioprinting process. Second, proper alignment of all nozzles on a uniform plane is essential for accurate spheroid patterning. Misalignment could cause spheroids to penetrate too deeply or remain elevated, leading to imprecise bioprinting or potential damage due to insufficient surface contact. This can be prevented by ensuring that the defect area for example on the rat is level with the DCNA plane before starting the process. This can be achieved by adjusting the DCNA’s roll, pitch, and yaw angles or by aligning the rat’s head parallel to the DCNA surface.

Third, like other pressure-driven systems, HITS-Bio is susceptible to spheroid damage from aspiration forces during bioprinting. To address this, they optimized nozzle size and pressure, and ensured all nozzles were aligned on a uniform plane. This reduced stress on the spheroids, maintaining their structural integrity. This also helped them to identify the elastic moduli range suitable for bioprinting, finding that spheroids with an elastic modulus greater than 50 Pa could be successfully bioprinted, while those below 40 Pa were not viable.

While the current DCNA setup uses a 4x4 nozzle array, a reconfiguration to a 10x10 nozzle array could further accelerate the bioprinting process, eliminating periodicity constraints and improving tissue fabrication efficiency for a broader range of applications.

The inter-nozzle capillary interactions can impact spheroid picking accuracy, especially with closely spaced nozzles. However this was prevented by coating the nozzles with silicon and adjusting their spacing, as well as potentially incorporating advanced fluid dynamics management. These optimizations reduced liquid elevation between nozzles, improving spheroid manipulation precision. Additionally, the DCNA setup was calibrated to correlate with spheroid size, ensuring optimal performance. Larger spheroids required increased nozzle spacing to prevent interference, while smaller spheroids benefited from tighter spacing and smaller nozzles to maintain precision.

Finally, during spheroid aspiration, maintaining a minimum pressure of ~3 mmHg throughout the DCNA prevented media leakage when spheroids were placed with closed channels. Media leakage could cause spheroids to shift or hinder proper placement. Hemostasis before the intraoperative bioprinting process is also essential to prevent bleeding, which could affect spheroid positioning.

Conclusion:

In conclusion, this study presents HITS-Bio, a high-throughput bioprinting platform that enables scalable tissue fabrication by precisely positioning spheroids at unprecedented speeds using the DCNA platform. HITS-Bio significantly outperforms existing techniques, as demonstrated by the rapid bioprinting of various tissues, including bone and cartilage. Notably, intraoperative bioprinting with miR-enhanced spheroids derived from hADSCs showed potential for repairing calvarial defects in rats. The platform also achieved efficient fabrication of cartilage constructs, completing each in under 40 minutes—far surpassing current methods. Future advancements, such as adding more nozzles, enabling bioprinting on complex surfaces, and increasing automation, could further enhance its scalability and versatility in tissue biofabrication.

As we look forward to such advancements, we are excited to share the highlights of our Bioprinting Winter School India 2024. We can't help but reflect on the amazing journey so far. Starting on December 12, this program brought together a diverse cohort of passionate learners from various academic and professional backgrounds.

The past weeks have been nothing short of transformative. From diving into the foundational principles of bioprinting, biomaterials, and bioink applications to exploring cutting-edge techniques like Digital Light Processing (DLP), Embedded Bioprinting, and Coaxial-Triaxial technologies, every session has been an eye-opener. The knowledge shared by our globally renowned speakers has not only expanded our horizons but also connected theory with real-world applications in ways that left us inspired.

The camaraderie, team support, and lively discussions fostered an environment where ideas thrived, confidence grew, and participants bonded through challenges, laughter, and stepping out of their comfort zones.

And there’s so much more to look forward to! In January 2025, Week 3 kicks off with hands-on training on the advanced Trivima Bioprinters, providing an up-close look at the technologies shaping the future of bioprinting.

This journey is just the beginning. We can’t wait to see the groundbreaking research and innovations this cohort will achieve in the coming years. Here’s to pushing boundaries and creating a future we can all be proud of!

Stay tuned as we continue to explore and innovate together!

Reference - https://www.nature.com/articles/s41467-024-54504-7