Jacob A. Gage

Three-Dimensional In Vitro Co-Culture Model of Breast Tumor using Magnetic Levitation

In this study, we investigate a novel in vitro model to mimic heterogeneous breast tumors without the use of a scaffold while allowing for cell-cell and tumor-fibroblast interactions. Previous studies have shown that magnetic levitation system under conventional culturing conditions results in the formation of three-dimensional (3D) structures, closely resembling in vivo tissues (fat tissue, vasculature, etc.). Three-dimensional heterogeneous tumor models for breast cancer were designed to effectively model the influences of the tumor microenvironment on drug efficiency. Various breast cancer cells were co-cultured with fibroblasts and then magnetically levitated. Size and cell density of the resulting tumors were measured. The model was phenotypically compared to in vivo tumors and examined for the presence of ECM proteins. Lastly, the effects of tumor stroma in the 3D in vitro model on drug transport and efficiency were assessed. Our data suggest that the proposed 3D in vitro breast tumor is advantageous due to the ability to: (1) form large-sized (millimeter in diameter) breast tumor models within 24 h; (2) control tumor cell composition and density; (3) accurately mimic the in vivo tumor microenvironment; and (4) test drug efficiency in an in vitro model that is comparable to in vivo tumors.

Three-dimensional cell culturing by magnetic levitation

Recently, biomedical research has moved toward cell culture in three dimensions to better recapitulate native cellular environments. This protocol describes one method for 3D culture, the magnetic levitation method (MLM), in which cells bind with a magnetic nanoparticle assembly overnight to render them magnetic. When resuspended in medium, an external magnetic field levitates and concentrates cells at the air-liquid interface, where they aggregate to form larger 3D cultures. The resulting cultures are dense, can synthesize extracellular matrix (ECM) and can be analyzed similarly to the other culture systems using techniques such as immunohistochemical analysis (IHC), western blotting and other biochemical assays. This protocol details the MLM and other associated techniques (cell culture, imaging and IHC) adapted for the MLM. The MLM requires 45 min of working time over 2 d to create 3D cultures that can be cultured in the long term (>7 d).

A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.

A spheroid toxicity assay using magnetic 3D bioprinting and real-time mobile device-based imaging

An ongoing challenge in biomedical research is the search for simple, yet robust assays using 3D cell cultures for toxicity screening. This study addresses that challenge with a novel spheroid assay, wherein spheroids, formed by magnetic 3D bioprinting, contract immediately as cells rearrange and compact the spheroid in relation to viability and cytoskeletal organization. Thus, spheroid size can be used as a simple metric for toxicity. The goal of this study was to validate spheroid contraction as a cytotoxic endpoint using 3T3 fibroblasts in response to 5 toxic compounds (all-trans retinoic acid, dexamethasone, doxorubicin, 5′-fluorouracil, forskolin), sodium dodecyl sulfate (+control), and penicillin-G (−control). Real-time imaging was performed with a mobile device to increase throughput and efficiency. All compounds but penicillin-G significantly slowed contraction in a dose-dependent manner (Z’ = 0.88). Cells in 3D were more resistant to toxicity than cells in 2D, whose toxicity was measured by the MTT assay. Fluorescent staining and gene expression profiling of spheroids confirmed these findings. The results of this study validate spheroid contraction within this assay as an easy, biologically relevant endpoint for high-throughput compound screening in representative 3D environments.

A high-throughput in vitro ring assay for vasoactivity using magnetic 3D bioprinting

Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology, and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives.

Magnetically Bioprinted Human Myometrial 3D Cell Rings as A Model for Uterine Contractility

Deregulation in uterine contractility can cause common pathological disorders of the female reproductive system, including preterm labor, infertility, inappropriate implantation, and irregular menstrual cycle. A better understanding of human myometrium contractility is essential to designing and testing interventions for these important clinical problems. Robust studies on the physiology of human uterine contractions require in vitro models, utilizing a human source. Importantly, uterine contractility is a three-dimensionally (3D)-coordinated phenomenon and should be studied in a 3D environment. Here, we propose and assess for the first time a 3D in vitro model for the evaluation of human uterine contractility. Magnetic 3D bioprinting is applied to pattern human myometrium cells into rings, which are then monitored for contractility over time and as a function of various clinically relevant agents. Commercially available and patient-derived myometrium cells were magnetically bioprinted into rings in 384-well formats for throughput uterine contractility analysis. The bioprinted uterine rings from various cell origins and patients show different patterns of contractility and respond differently to clinically relevant uterine contractility inhibitors, indomethacin and nifedipine. We believe that the novel system will serve as a useful tool to evaluate the physiology of human parturition while enabling high-throughput testing of multiple agents and conditions.

Somatic mutation detection from liquid biopsy-derived cellular aggregates formed by magnetic 3D bioprinting.

291 Background: A challenge in the analysis of circulating tumor cells (CTC) is their scarcity, and the inability to expand them for further analysis. To overcome this obstacle, we used magnetic 3D bioprinting to form CTC spheroids that could grow. The principle of magnetic 3D bioprinting is the magnetization of cells with nanoparticles and their subsequent printing into spheroids. For this project, CTCs can be aggregated into close contact to interact and grow in culture. In this study, we demonstrated the ability to aggregate CTCs and perform next generation sequencing (NGS) to detect somatic mutations from renal and prostate cancers. Methods: Blood samples from prostate and kidney cancer patients were enriched for CTCs (Isoflux, Fluxion), from a starting blood volume of 7.5-14 mL. CTCs were isolated immunomagnetically for EpCAM+ EGFR+ cells, then enumerated for CK+ CD45-. The cells were then removed of microbeads, then magnetized by incubation with NanoShuttle (NS, Nano3D), a magnetic nanoparticle asse...

Abstract 4251: Development of spheroids derived from tumor biopsies and patient-derived xenografts using magnetic 3D bioprinting

Precision medicine holds the promise of designing patient-specific therapies to improve therapeutic efficiency. However, the scarcity of tumor and biopsy tissue is a limiting factor in the development of diagnostic assays. Cells isolated from these tissues could be used to overcome these issues, while serving as the basis for assays to diagnose and guide treatment. It is critical that the in vitro culture of these cells be performed in three-dimensional (3D) environments that can better replicate the native tumor microenvironment. However, currently available 3D cell culture platforms, like Matrigel, suffer from technical limitations in reproducibility and handling that make the development of such assays difficult. Towards that end, this study isolates cells from human prostate cancer (PC) and renal cell carcinoma (RCC) tumor biopsies and patient-derived xenografts (PDX) and prints them into spheroids using magnetic 3D bioprinting. The core principle of magnetic 3D bioprinting is the magnetization of cells and their aggregation using mild magnetic forces. Once aggregated, these cells form spheroids that mimic native tumor environments in extracellular matrix and cell-cell and cell-ECM interactions. This technique can be used to actively magnetize cells and generate spheroids from a scarce cell source, while overcoming the limitations of other 3D cell culture platforms. In this study, we demonstrated our ability to print spheroids from cells isolated from human tumor biopsies and PDX. Isolation techniques ranging from simple mincing and filtration to enzymatic digestion were employed. Next, these cells were magnetized by incubation with a biocompatible magnetic nanoparticle assembly, NanoShuttle. Once magnetized, these cells were printed into spheroids of varying sizes, from 1,000-20,000 cells, in 384-well plates. These cells were cultured for days, after which viability was measured using CellTiter-Glo. Our preliminary studies demonstrated our ability to isolate cells and print them into spheroids. Isolation was best with either mincing and filtration alone or collagenase II (400 U/mL) digestion for 1 h. These cells were then successfully magnetized and printed into spheroids, which remained viable after 72 h. Spheroids of 10,000-20,000 cells were the most successful, and further optimization is needed to reduce the size needed for viable spheroids to take full advantage of scarce resources such as tumor biopsies. We also demonstrated the ability to assay compound toxicity, showing a dose-dependent toxicity on spheroids derived from PDX tumors. In all, we demonstrated our ability to isolate cells from human tumor biopsies and PDX models and print them into spheroids with high throughput. These preliminary results will serve as a platform for the further development of precision medicine assays to optimize PC and RCC treatment. Citation Format: Hubert Tseng, Jacob A. Gage, Pujan K. Desai, Reynolds Brobey, Sheri Skinner, Mehdi Dehghani, Kevin P. Rosenblatt, Wenliang Li, Robert J. Amato, Glauco R. Souza. Development of spheroids derived from tumor biopsies and patient-derived xenografts using magnetic 3D bioprinting. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4251.

Three-dimensional cell culture used as a model for PDT dosimetry

a commercial needle free injection system. The Protoporphyrin IX (PPIX) production was monitored using widefield fluorescence imaging at skin surface until 3 h of treatment. The visual damage after 48h of PDT was evaluated and the biopsies were removed from skin to perform the histological analyses. The superficial damage extension and the threshold dose were calculated. The results found to threshold dose is the smallest for application using needle free injection followed by ALA cream and light. These results prove the efficiency of the needle free injection to induce skin damage in the lowest light dose. The damage induce only by light can be suggested as a thermal damage. However, this damage is lower when compared toother conditions after PDT. It is possible to assume that the IPL can be useful to perform topical PDT using ALA. However, the thermal damage needs to be controlled carefully.

Abstract LB-B08: Somatic mutation detection from liquid biopsy-derived cellular aggregates formed by magnetic 3D bioprinting

Background - A challenge in the analysis of circulating tumor cells (CTCs) is their scarcity and the inability to expand them for further analysis. To overcome this obstacle, we used magnetic 3D bioprinting to form CTC spheroids that could grow. The principle of magnetic 3D bioprinting is the magnetization of cells with nanoparticles and their subsequent printing into spheroids. For this project, CTCs were aggregated into close contact to facilitate interactions and growth in culture. We then demonstrated the ability to perform next generation sequencing (NGS) of the spheroids to detect somatic mutations from renal and prostate cancers. Methods - Blood samples from prostate and kidney cancer patients were enriched for CTCs (Isoflux, Fluxion Biosciences), from a starting blood volume of 7.5-14 mL. CTCs were isolated immunomagnetically for EpCAM+ EGFR+ cells, then enumerated for CK+ CD45-. The cells were then magnetized by incubation with NanoShuttle (NS, Nano3D Biosciences) and printed into spheroids in 384-well plates. After 4 d of growth, the cells were lysed and DNA was amplified by whole genome amplification (WGA) with the NGA kit (Fluxion Biosciences) and quantified via qPCR. Targeted libraries were sequenced using the PGM (ThermoFisher) sequencing instrument; data was analyzed using a customized variant calling/filtering pipeline based on standard Ion Reporter alignment tools and VarSeqTM for variant filtering and functional interpretation. Results - CTCs were successfully aggregated using magnetic 3D bioprinting and grew over 4 d. For both prostate and renal cancers, we then demonstrated the detection of somatic variants within a majority of the samples. Using the commercial Oncomine® test kits (ThermoFisher), we found a median of 5 COSMIC variants (32 total) per sample using cell cultures. Conclusions - We successfully developed a method to aggregate CTCs using magnetic 3D bioprinting, expanded them, and then identified somatic mutations using NGS. This procedure may form the basis of a liquid biopsy-derived molecular testing platform for monitoring urological tumor progression and planning treatment strategies. Citation Format: Hubert Tseng, Robert J. Amato, Reynolds Brobey, Cristian Ionescu-Zanetti, Jeff Jensen, Jacob Gage, Pujan Desai, Angela Liao, Mehdi Dehghani, Kevin Rosenblatt, Glauco Souza. Somatic mutation detection from liquid biopsy-derived cellular aggregates formed by magnetic 3D bioprinting. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B08.