Date Published: May 31, 2019
Publisher: Public Library of Science
Author(s): Aneetta E. Kuriakose, Wenjing Hu, Kytai T. Nguyen, Jyothi U. Menon, Nils Cordes.
Scaffold-based cancer cell culture techniques have been gaining prominence especially in the last two decades. These techniques can potentially overcome some of the limitations of current three-dimensional cell culture methods, such as uneven cell distribution, inadequate nutrient diffusion, and uncontrollable size of cell aggregates. Porous scaffolds can provide a convenient support for cell attachment, proliferation and migration, and also allows diffusion of oxygen, nutrients and waste. In this paper, a comparative study was done on porous poly (lactic-co-glycolic acid) (PLGA) microparticles prepared using three porogens—gelatin, sodium bicarbonate (SBC) or novel poly N-isopropylacrylamide [PNIPAAm] particles, as substrates for lung cancer cell culture. These fibronectin-coated, stable particles (19–42 μm) supported A549 cell attachment at an optimal cell seeding density of 250,000 cells/ mg of particles. PLGA-SBC porous particles had comparatively larger, more interconnected pores, and favored greater cell proliferation up to 9 days than their counterparts. This indicates that pore diameters and interconnectivity have direct implications on scaffold-based cell culture compared to substrates with minimally interconnected pores (PLGA-gelatin) or pores of uniform sizes (PLGA-PMPs). Therefore, PLGA-SBC-based tumor models were chosen for preliminary drug screening studies. The greater drug resistance observed in the lung cancer cells grown on porous particles compared to conventional cell monolayers agrees with previous literature, and indicates that the PLGA-SBC porous microparticle substrates are promising for in vitro tumor or tissue development.
The practice of tissue and cell culture has been in existence as early as 1885 when Wilhelm Roux demonstrated that the medullary plate of a chick embryo can be maintained on glass plates with warm saline solution [1, 2]. Since then, cells have been traditionally cultured in vitro on two-dimensional (2D) polystyrene or glass surfaces. 2D cell culture models are still in use in pharmacology today for drug screening and cytocompatibility studies. However, these conventional 2D systems differ from in vivo tissues in cell surface receptor expression, extracellular matrix synthesis, cell density, and metabolic functions . They are also unable to develop hypoxia or mimic the cell arrangement seen in different parts of the tissues and tumors . Further, studies have shown that tumor cell monolayers grown on tissue culture plates develop a non-natural morphology, which could be a major factor affecting their responses to drugs . According to recent reports, the promising effects of therapeutic agents in in vitro 2D cell culture systems have not translated into successful results in animals, and in humans. Only about 5% of the chemotherapeutic agents that showed promising preclinical activity have demonstrated significant therapeutic efficacy in phase III clinical trials . Therefore, there is a vital need for an in vitro cell culture model that mimics in vivo tissues more closely, for cancer drug screening and personalized medicine applications.
All chemicals, unless mentioned specifically, were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. No human participants or vertebrate animals were used in this research. Immortalized A549 lung adenocarcinoma cell line was purchased from ATCC (CCL-185) for in vitro cell culture experiments. The cell culture protocols were approved by the Institutional Review Board (IRB) at the University of Texas at Arlington.
In this work, three types of porous PLGA MPs were prepared using different porogens, i.e. gelatin, SBC or the novel PMPs, to choose the optimal MP formulation with surface characteristics most suitable for lung cancer cell attachment and growth. Porous polymeric microparticle substrates have not been studied before for lung cancer cell culture in vitro. The most important finding of this work is that PLGA-SBC porous particles which had larger, more interconnected pores facilitated longer-term cell proliferation and viability than the other porous models indicating that pore diameter and porosity have direct implications on cell culture and viability on porous microparticle substrates. The lung cancer cells cultured on PLGA-SBC microparticles showed higher drug resistance compared to the 2D model at the same concentrations of cancer drugs, indicating that cells tend to respond differently to treatment depending on their arrangement. This is consistent with previously published reports comparing cell monolayers with cells cultured in a 3D format [5, 58]. Our results indicate the need for further investigation into PLGA-SBC porous particles-based tumor models and validating them in vivo to find the most promising model for pharmacological studies that will closely mimic reactions seen in vivo. We have also introduced a novel method of forming porous MPs using PMPs. The pores of these MPs can be tailored by varying the diameters of the PMPs used.
In summary, biodegradable porous PLGA MPs prepared using three different porogens—gelatin, SBC and PMPs were synthesized and compared to determine the most promising formulation to be used as a substrate for in vitro lung tumor models. An innovative method of preparing porous MPs with uniform pores, using PMPs was also presented. Although all the three types of particles were stable and biodegradable, PLGA-SBC-based porous particles had relatively larger pores and better interconnectivity, and favored cell attachment, growth and viability more, and were thus chosen for further studies. Preliminary drug screening studies conducted using PLGA-SBC particles to determine the therapeutic efficacy of various lung cancer drugs demonstrated that the 3D tumor model responded differently to drugs of the same concentration, compared to the 2D cell monolayers. These results need to be confirmed with in vivo results to determine the most appropriate and comparatively more accurate model for future drug screening applications.