Current 3D printing of tissue is restricted by the use of biomaterials that do not recapitulate the native properties of the extracellular matrix (ECM). These restrictions have thus far prevented optimization of composition and structure of the in vivo tissue microenvironment. The artificial nature of currently used biomaterials affects cellular phenotype and function of the bioprinted tissues, and results in inaccurate modeling of disease and drug metabolism significantly. Collagen type I is the major structural component in the ECM, and is widely used as a 3D hydrogel, but is less applicable for 3D bioprinting due to low viscosity and slow polymerization. We have hypothesized that a combination of hyaluronic acid with collagen I yields a bioink with the properties required for extrusion bioprinting, while supporting native cell–matrix interactions and preservation of the native microenvironment properties. To test this hypothesis, we tested the viscoelastic properties of three bioink formulations –2:1, 3:1, and 4:1 collagen type I to hyaluronic acid, and examined cellular behavior in order to determine an optimal formulation that allows for bioprinting while supporting biological activity. We then employed this formulation to bioprint 3D liver tissue constructs containing primary human hepatocytes and liver stellate cells and tested the effects of acetaminophen, a common liver toxicant. Our results have shown that the combination of methacrylated collagen type I and thiolated hyaluronic acid yield a simple, printable bioink that allows for modulation that was directly related to stromal cell elongation. Further, the bioink adequately allowed for implementation as a support hydrogel for hepatocytes which were able to remain viable over two weeks and responded to drug treatment appropriately.

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The current drug development pipeline takes approximately fifteen years and $2.6 billion to get a new drug to market. Typically, drugs are tested on two-dimensional (2D) cell cultures and animal models to estimate their efficacy before reaching human trials. However, these models are often not representative of the human body. The 2D culture changes the morphology and physiology of cells, and animal models often have a vastly different anatomy and physiology than humans. The use of bioengineered human cell-based organoids may increase the probability of success during human trials by providing human-specific preclinical data. They could also be deployed for personalized medicine diagnostics to optimize therapies in diseases such as cancer. However, one limitation in employing organoids in drug screening has been the difficulty in creating large numbers of homogeneous organoids in form factors compatible with high-throughput screening (e.g., 96-and 384-well plates). Bioprinting can be used to scale up deposition of such organoids and tissue constructs. Unfortunately, it has been challenging to 3D print hydrogel bioinks into small-sized wells due to well-bioink interactions that can result in bioinks spreading out and wetting the well surface instead of maintaining a spherical form. Here, we demonstrate an immersion printing technique to bioprint tissue organoids in 96-well plates to increase the throughput of 3D drug screening. A hydrogel bioink comprised of hyaluronic acid and collagen is bioprinted into a viscous gelatin bath, which blocks the bioink from interacting with the well walls and provides support to maintain a spherical form. This method was validated using several cancerous cell lines, and then applied to patient-derived glioblastoma (GBM) and sarcoma biospecimens for drug screening.