(507c) Fabrication and Maturation of Cancer Spheroids (Micro-Tumors) in a 3D-Printed Continuous Stirred Tank Mini-Reactor (CSTmR)

Cancer continues to be a leading cause of mortality in modern societies, so improved and more reliable in vitro cancer models are needed to expedite fundamental cancer research and anti-cancer drug development. A vast body of evidence has demonstrated that 3D culture systems recapitulate the response of tumors to anticancer drugs in a more faithful manner than conventional 2D culture systems. Cancer spheroids (cancer-cell aggregates) are arguably the simplest and most commonly used 3D model for conducting cancer research, and diverse methods are used to fabricate them. Most are based on cumbersome, artisanal protocols that yield heterogenous populations of spheroids in terms of size and shape.

Here, we describe a simple, robust method for the fabrication and maturation of breast cancer (MCF7) spheroids based on the use of a 3D-printed continuous stirred tank mini-reactor (CSTmR). A portable incubator was fabricated in house and used to create homogeneous, well-controlled environments for the culture of cancerous 3D micro-tissues. Our 2-mL CSTmR features an off-center agitation system that enables homogeneous chaotic laminar mixing at low speeds (10–200 RPM). Gentile agitation of MCF7 cell suspensions (0.5X10⁶ cells/mL) results in the formation of small tumor spheroids (~200 µm in diameter) after two days of batch culture. These microtissues are continuously fed with culture media in the CSTmR for an extended time (weeks). We studied the effect of various residence times, rates of agitation, and inlet glucose concentrations on the growth and rate of maturation of the spheroids. In addition, we characterized the morphology (i.e., shape and size), the 3D architecture (i.e., cell viability as a function of the radial distance from the spheroid center), and the expression of relevant tumor biomarkers (i.e., ER, PR, Ki67, CDH1, EPCAM, ICAM, p53, BCL2, HIFa, LDHA, CAIX, VEGF A) in spheroids in diverse operational conditions and maturation times. The spheroids progressively increased in size during the first 5 to 6 days of culture to reach a steady diameter (between 400 and 1000 µm) as a function of residence time and glucose availability. For example, at low residence times and high glucose concentrations, the spheroids reached a diameter of ~900 ± 1000 µm.

This CSTR culture strategy enables the fabrication of spheroids under a wide range of conditions (in terms of substrate availability, dynamic forces and residence times) and may offer great potential for studying the effects of diverse effectors in tumor progression. For example, we envision the use of this agitated system as a tumor-on-chip platform to expedite testing the efficacy and safety of novel anti-cancer drugs and to enable personalized medicine applications.