BioPrint meets the AI age: development of artificial intelligence-based ADMET models for the drug-discovery platform SAFIRE.

Background: To prioritize compounds with a higher likelihood of success, artificial intelligence models can be used to predict absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of molecules quickly and efficiently. Methods: Models were trained with BioPrint database proprietary data along with public datasets to predict various ADMET end points for the SAFIRE platform. Results: SAFIRE models performed at or above 75% accuracy and 0.4 Matthew's correlation coefficient with validation sets. Training with both proprietary and public data improved model performance and expanded the chemical space on which the models were trained. The platform features scoring functionality to guide user decision-making. Conclusion: High-quality datasets along with chemical space considerations yielded ADMET models performing favorably with utility in the drug discovery process.

3D bio-printed hydrogel inks promoting lung cancer cell growth in a lab-on-chip culturing platform.

The results of a lab-on-chip (LOC) platform fabrication equipped with a hydrogel matrix is reported. A 3D printing technique was used to provide a hybrid, "sandwiched" type structure, including two microfluidic substrates of different origins. Special attention was paid to achieving uniformly bio-printed microfluidic hydrogel layers of a unique composition. Six different hydrogel inks were proposed containing sodium alginate, agar, chitosan, gelatin, methylcellulose, deionized water, or 0.9% NaCl, varying in proportions. All of them exhibited appropriate mechanical properties showing, e.g., the value of elasticity modulus as similar to that of biological tissues, such as skin. Utilizing our biocompatible, entirely 3D bio-printed structure, for the first time, a multi-drug-resistant lung cancer cell line (H69AR) was cultured on-chip. Biological validation of the device was performed qualitatively and quantitatively utilizing LIVE/DEAD assays and Presto blue staining. Although all bio-inks exhibited acceptable cell viability, the best results were obtained for the hydrogel composition including 3% sodium alginate + 7% gelatin + 90% NaCl (0.9%), reaching approximately 127.2% after 24 h and 105.4% after 48 h compared to the control group (100%). Further research in this area will focus on the microfluidic culture of the chosen cancer cell line (H69AR) and the development of novel drug delivery strategies towards appropriate in vivo models for chemotherapy and polychemotherapy treatment.

Balance between the cell viability and death in 3D.

Cell death is a phenomenon, frequently perceived as an absolute event for cell, tissue and the organ. However, the rising popularity and complexity of such 3D multicellular 'tissue building blocks' as heterocellular spheroids, organoids, and 'assembloids' prompts to revise the definition and quantification of cell viability and death. It raises several questions on the overall viability of all the cells within 3D volume and on choosing the appropriate, continuous, and non-destructive viability assay enabling for a single-cell analysis. In this review, we look at cell viability and cell death modalities with attention to the intrinsic features of such 3D models as spheroids, organoids, and bioprints. Furthermore, we look at emerging and promising methodologies, which can help define and understand the balance between cell viability and death in dynamic and complex 3D environments. We conclude that the recent innovations in biofabrication, biosensor probe development, and fluorescence microscopy can help answer these questions.

Scaffold-free 3D cell culture of primary skin fibroblasts induces profound changes of the matrisome.

The human skin has a highly developed extracellular matrix (ECM) that is vital for proper skin functioning, its 3D architecture playing a pivotal role in support and guidance of resident and invading cells. To establish relevant in vitro models mimicking the complex design observed in vivo, scaffold-based and scaffold-free 3D cell culture systems have been developed. Here we show that scaffold-free systems are well suited for the analysis of ECM protein regulation. Using quantitative mass spectrometry-based proteomics in combination with magnetic 3D bioprinting we characterize changes in the proteome of skin fibroblasts and squamous cell carcinoma cells. Transferring cells from 2D to 3D without any additional scaffold induces a profound upregulation of matrisome proteins indicating the generation of a complex, tissue-like ECM.

A Reproducible Bioprinted 3D Tumor Model Serves as a Preselection Tool for CAR T Cell Therapy Optimization.

Chimeric antigen receptor (CAR) T cell performance against solid tumors in mouse models and clinical trials is often less effective than predicted by CAR construct selection in two-dimensional (2D) cocultures. Three-dimensional (3D) solid tumor architecture is likely to be crucial for CAR T cell efficacy. We used a three-dimensional (3D) bioprinting approach for large-scale generation of highly reproducible 3D human tumor models for the test case, neuroblastoma, and compared these to 2D cocultures for evaluation of CAR T cells targeting the L1 cell adhesion molecule, L1CAM. CAR T cells infiltrated the model, and both CAR T and tumor cells were viable for long-term experiments and could be isolated as single-cell suspensions for whole-cell assays quantifying CAR T cell activation, effector function and tumor cell cytotoxicity. L1CAM-specific CAR T cell activation by neuroblastoma cells was stronger in the 3D model than in 2D cocultures, but neuroblastoma cell lysis was lower. The bioprinted 3D neuroblastoma model is highly reproducible and allows detection and quantification of CAR T cell tumor infiltration, representing a superior in vitro analysis tool for preclinical CAR T cell characterization likely to better select CAR T cells for in vivo performance than 2D cocultures.

3D-bioprinted gradient-structured scaffold generates anisotropic cartilage with vascularization by pore-size-dependent activation of HIF1α/FAK signaling axis.

Articular cartilage injury is one of the most common diseases in orthopedics, which seriously affects patients' life quality, the development of a biomimetic scaffold that mimics the multi-layered gradient structure of native cartilage is a new cartilage repair strategy. It has been shown that scaffold topography affects cell attachment, proliferation, and differentiation; the underlying molecular mechanism of cell-scaffold interaction is still unclear. In the present study, we construct an anisotropic gradient-structured cartilage scaffold by three-dimensional (3D) bioprinting, in which bone marrow stromal cell (BMSC)-laden anisotropic hydrogels micropatterns were used for heterogeneous chondrogenic differentiation and physically gradient synthetic poly (ε-caprolactone) (PCL) to impart mechanical strength. In vitro and in vivo, we demonstrated that gradient-structured cartilage scaffold displayed better cartilage repair effect. The heterogeneous cartilage tissue maturation and blood vessel ingrowth were mediated by a pore-size-dependent mechanism and HIF1α/FAK axis activation. In summary, our results provided a theoretical basis for employing 3D bioprinting gradient-structured constructs for anisotropic cartilage regeneration and revealed HIF1α/FAK axis as a crucial regulator for cell-material interactions, so as to provide a new perspective for cartilage regeneration and repair.

3D-bioprinting ready-to-implant anisotropic menisci recapitulate healthy meniscus phenotype and prevent secondary joint degeneration.

Objectives: Disruption of anisotropic phenotypes of the meniscus would contribute to OA progression. Exploring phenotype changes of the anisotropic meniscus in joint degeneration would help understand the biologic interaction between the meniscus and OA, and further facilitate the therapeutic strategies of meniscus injury-related joint degeneration. Meanwhile, engineering biomimetic meniscal tissue mimicking the anisotropy of the healthy meniscus remains a challenge. Methods & Results: Meniscal disruption of phenotype anisotropy (PBV growth, cellular phenotype and ECM depositions) was confirmed in OA patient samples. To recapitulate healthy meniscus phenotypes, 3D-bioprinted anisotropic TCM meniscus constructs with PBV growth and regional differential cell and ECM depositions were generated. Transplanted 3D-bioprinted meniscus into rabbit knees recapitulated phenotypes of native healthy meniscus and conferred long-term protection against secondary joint degeneration. Conclusion: 3D-bioprinted TCM meniscus not only restored the anisotropy of native healthy meniscus with PBV infiltration and better shape retention, but better maintained joint function and prevented secondary joint degeneration, which provided a new strategy for the clinical treatment of meniscus injury-related joint degenerative diseases.

3D-engineered GelMA conduit filled with ECM promotes regeneration of peripheral nerve.

Autologous transplantation remains the golden standard for peripheral nerve repair. However, many drawbacks, such as the risk of reoperation or nerve injury remain associated with this method. To date, commercially available artificial nerve conduits comprise hollow tubes. By providing physical guiding and biological cues, tissue engineered conduits are promising for bridging peripheral nerve defects. The present study focuses on the preparation of artificial composite nerve conduits by 3D bio-printing. 3D-printed molds with a tubular cavity were filled with an Engelbreth-Holm-Swarm (EHS) Hydrogel mimicking the extracellular matrix (ECM) basement membrane. Chemically cross-linked gelatin methacryloyl (GelMA) was used to form the conduit backbone, while EHS Hydrogels improved nerve fiber growth while shortening repair time. Statistical significant difference had been found between the blank conduit and the composite conduit group on compound muscle action potential after 4 months. On the other hand, results between the composite conduit group and the autograft group were of no statistical differences. All results above showed that the composite conduit filled with EHS Hydrogel can promote the repair of peripheral nerve and may become a promising way to treat peripheral nerve defects.

A Review of Recent Advances in 3D Bioprinting With an Eye on Future Regenerative Therapies in Veterinary Medicine.

3D bioprinting is a rapidly evolving industry that has been utilized for a variety of biomedical applications. It differs from traditional 3D printing in that it utilizes bioinks comprised of cells and other biomaterials to allow for the generation of complex functional tissues. Bioprinting involves computational modeling, bioink preparation, bioink deposition, and subsequent maturation of printed products; it is an intricate process where bioink composition, bioprinting approach, and bioprinter type must be considered during construct development. This technology has already found success in human studies, where a variety of functional tissues have been generated for both in vitro and in vivo applications. Although the main driving force behind innovation in 3D bioprinting has been utility in human medicine, recent efforts investigating its veterinary application have begun to emerge. To date, 3D bioprinting has been utilized to create bone, cardiovascular, cartilage, corneal and neural constructs in animal species. Furthermore, the use of animal-derived cells and various animal models in human research have provided additional information regarding its capacity for veterinary translation. While these studies have produced some promising results, technological limitations as well as ethical and regulatory challenges have impeded clinical acceptance. This article reviews the current understanding of 3D bioprinting technology and its recent advancements with a focus on recent successes and future translation in veterinary medicine.

Ex Vivo Functionality of 3D Bioprinted Corneal Endothelium Engineered with Ribonuclease 5-Overexpressing Human Corneal Endothelial Cells.

Human corneal endothelial cells (HCECs) are scarcely proliferative in vivo. The cultured HCECs engineered to overexpress ribonuclease (RNase) 5 (R5-HCECs) are prepared after transient transfection with RNase 5 plasmid vector. As candidate targets of R5-HCECs for enhancement of cellular proliferation and survival of R5-HCECs, programmed cell death protein 4 is inhibited, and cyclin D1 and cyclin E1 are activated. The cultured R5-HCECs and control HCECs on lyophilized amniotic membrane (AM) are deposited as a carrier by extrusion-based 3D bioprinting to prepare transplantable RNase 5 vector-transfected HCECs-laden AM graft (R5-Graft) and the control HCECs-laden AM graft (Ct-Graft), respectively. The ready-to-use R5-Graft shows clearer basolateral expression of Na+ -K+ ATPase pump and higher cell confluency than Ct-Graft. From 2 weeks after graft transplantation, both R5-Graft and Ct-Graft start restoring clarity of the rabbit corneas, and their central corneal edema are much less than those in the control group at 3 and 4 weeks. The ex vivo expression of corneal endothelial phenotypical markers is clear in R5-Grafs rather than in Ct-Grafts at 4 weeks. In conclusion, the fabricated corneal endothelium with cultured HCECs easily survives and functions as corneal endothelium in vivo. Furthermore, the use of the cultured HCECs engineered to overexpress RNase 5 (R5-HCECs) may be an option to obtain higher graft cellularity and to enhance the function of transplanted grafts.

Reverse Reconstruction and Bioprinting of Bacterial Cellulose-Based Functional Total Intervertebral Disc for Therapeutic Implantation.

The degradation of intervertebral discs (IVD), a typical hierarchical structured tissue, causes serious neck and back pain. The current methods cannot fully reconstitute the unique structure and function of native IVD. In this study, by reverse reconstruction of the structure of native IVD and bioprinting bacterial cellulose (BC) nanofibers with a high-throughput optimized micropattern screening microchip, a total IVD is created that contained type II collagen-based nucleus pulposus (NP) and hierarchically organized and micropatterned BC-based annulus fibrosus (AF), mimicking native IVD tissue. The artificial NP contains rat NP cells, whereas the AF contains concentrically arranged BC layers with aligned micropatterns and attached AF cells in +/-30° alternate directions between adjacent layers. Long-term (3 months) implantation experiments on rats demonstrate the excellent structural (shape maintenance, hydration, tissue integration) and functional (mechanical support and flexibility) performance of the artificial IVD. This study provides a novel strategy for creating highly sophisticated artificial tissues.

Comparing Measures of Promiscuity and Exploring Their Relationship to Toxicity.

Recent research has focused on algorithms to derive numerical measures of selectivity based on panels of in vitro pharmacology assays so that one molecule's activity profile may be compared easily with that of another. However, the questions concerning which method or algorithm is best to use, the optimal number of assays required to give an accurate measure of selectivity and the correlation of these measures to in vivo toxicity have remained largely unexplored. In this manuscript we describe a systematic approach to compare and contrast different calculation methods for promiscuity and determine the optimal number and constitution of a panel of assays to measure the selectivity/promiscuity of compounds across all targets. We then go on to examine their relationship to toxicity using a Pfizer proprietary compound set that has both selectivity profiles and exploratory toxicology study results. From this study we conclude that all five methods studied are useful in estimating compound selectivity; that a small panel of between 15 to 30 binding assays can be used as a surrogate for a broader panel enabling higher throughput with lower costs and this panel will most likely have the highest prediction power when correlating this measure to in vivo effects.