Recapitulation of the tumor microenvironment is critical for probing mechanisms involved in cancer, and for evaluating the tumor-killing potential of chemotherapeutic agents, targeted therapies and immunotherapies. Microfluidic devices have emerged as valuable tools for both mechanistic studies and for preclinical evaluation of therapeutic agents, due to their ability to precisely control drug concentrations and gradients of oxygen and other species in a scalable and potentially high throughput manner. Most existing in vitro microfluidic cancer models are comprised of cultured cancer cells embedded in a physiologically relevant matrix, collocated with vascular-like structures. However, the recent emergence of immune checkpoint inhibitors (ICI) as a powerful therapeutic modality against many cancers has created a need for preclinical in vitro models that accommodate interactions between tumors and immune cells, particularly for assessment of unprocessed tumor fragments harvested directly from patient biopsies. Here we report on a microfluidic model, termed EVIDENT (ex vivo immuno-oncology dynamic environment for tumor biopsies), that accommodates up to 12 separate tumor biopsy fragments interacting with flowing tumor-infiltrating lymphocytes (TILs) in a dynamic microenvironment. Flow control is achieved with a single pump in a simple and scalable configuration, and the entire system is constructed using low-sorption materials, addressing two principal concerns with existing microfluidic cancer models. The system sustains tumor fragments for multiple days, and permits real-time, high-resolution imaging of the interaction between autologous TILs and tumor fragments, enabling mapping of TIL-mediated tumor killing and testing of various ICI treatments versus tumor response. Custom image analytic algorithms based on machine learning reported here provide automated and quantitative assessment of experimental results. Initial studies indicate that the system is capable of quantifying temporal levels of TIL infiltration and tumor death, and that the EVIDENT model mimics the known in vivo tumor response to anti-PD-1 ICI treatment of flowing TILs relative to isotype control treatments for syngeneic mouse MC38 tumors.
The development and approval of engineered cellular therapies are revolutionizing approaches to treatment of diseases. However, these life-saving therapies require extensive use of inefficient bioprocessing equipment and specialized reagents that can drive up the price of treatment. Integration of new genetic material into the target cells, such as viral transduction, is one of the most costly and labor-intensive steps in the production of cellular therapies. Approaches to reducing the costs associated with gene delivery have been developed using microfluidic devices to increase overall efficiency. However, these microfluidic approaches either require large quantities of virus or pre-concentration of cells with high-titer viral particles. Here, we describe the development of a microfluidic transduction device (MTD) that combines microfluidic spatial confinement with advective flow through a membrane to efficiently colocalize target cells and virus particles. We demonstrate that the MTD can improve the efficiency of lentiviral transduction for both T-cell and hematopoietic stem-cell (HSC) targets by greater than two fold relative to static controls. Furthermore, transduction saturation in the MTD is reached with only half the virus required to reach saturation under static conditions. Moreover, we show that MTD transduction does not adversely affect cell viability or expansion potential.
Microfluidic acoustophoresis is a label-free technique that isolates a purified product from a complex mixture of cells. This technique is well-studied but thus far has lacked the throughput and device manufacturability needed for many medical and industrial uses. Scale-up of acoustofluidic devices can be more challenging than in other microfluidic systems because the channel walls are integral to the resonant behavior and coupling to neighboring channels can inhibit performance. Additionally, the increased device area needed for parallel channels becomes less practical in the silicon or glass materials usually used for acoustofluidic devices. Here, we report an acoustic separator with 12 parallel channels made entirely from polystyrene that achieves blood cell separation at a flow rate greater than 1 ml/min. We discuss the design and optimization of the device and the electrical drive parameters and compare the separation performance using channels of two different designs. To demonstrate the utility of the device, we test its ability to purify lymphocytes from apheresis product, a process that is critical to new immunotherapies used to treat blood cancers. We process a leukapheresis sample with a volume greater than 100 ml in less than 2 h in a single pass without interruption, achieving greater than 90% purity of lymphocytes, without any prepurification steps. These advances suggest that acoustophoresis could in the future aid in cell therapy bioprocessing and that further scale-up is possible.
The immune checkpoint blockade represents a revolution in cancer therapy, with the potential to increase survival for many patients for whom current treatments are not effective. However, response rates to current immune checkpoint inhibitors vary widely between patients and different types of cancer, and the mechanisms underlying these varied responses are poorly understood. Insights into the antitumor activities of checkpoint inhibitors are often obtained using syngeneic mouse models, which provide an in vivo preclinical basis for predicting efficacy in human clinical trials. Efforts to establish in vitro syngeneic mouse equivalents, which could increase throughput and permit real-time evaluation of lymphocyte infiltration and tumor killing, have been hampered by difficulties in recapitulating the tumor microenvironment in laboratory systems. Here, we describe a multiplex in vitro system that overcomes many of the deficiencies seen in current static histocultures, which we applied to the evaluation of checkpoint blockade in tumors derived from syngeneic mouse models. Our system enables both precision-controlled perfusion across biopsied tumor fragments and the introduction of checkpoint-inhibited tumor-infiltrating lymphocytes in a single experiment. Through real-time high-resolution confocal imaging and analytics, we demonstrated excellent correlations between in vivo syngeneic mouse and in vitro tumor biopsy responses to checkpoint inhibitors, suggesting the use of this platform for higher throughput evaluation of checkpoint efficacy as a tool for drug development.
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