3D Models of Cellular Spheroids As a Universal Tool for Studying the Cytotoxic Properties of Anticancer Compounds In Vitro

Abstract

The aim of this work is to develop a 3D cell culture model based on cell spheroids for predicting the functional activity of various compounds in vivo. Agarose gel molds were made using 3D printing. The solidified agarose gel is a matrix consisting of nine low-adhesive U-shaped microwells of 2.3 3.3 mm for 3D cell spheroid formation and growth. This matrix is placed into a single well of a 12-well plate. The effectiveness of the cell culture method was demonstrated using human ovarian carcinoma SKOVip-kat cells stably expressing the red fluorescent protein Katushka in the cytoplasm and overexpressing the membrane-associated tumor marker HER2. The SKOVip-kat cell spheroids were visualized by fluorescence microscopy. The cell concentration required for the formation of same-shape and same-size spheroids with tight intercellular contacts was optimized. To verify the developed model, the cytotoxicity of the targeted immunotoxin anti-HER2 consisting of the anti-HER2 scaffold DARP 9_29 and a fragment of the Pseudomonas aeroginosa exotoxin, DARP-LoPE, was studied in 2D and 3D SKOVip-kat cell cultures. The existence of a difference in the cytotoxic properties of DARP-LoPE between the 2D and 3D cultures has been demonstrated: the IC50 value in the 3D culture is an order of magnitude higher than that in the monolayer culture. The present work describes a universal tool for 3D cultivation of mammalian cells based on reusable agarose gel molds that allows for reproducible formation of multicellular spheroids with tight contacts for molecular and cell biology studies.

Genetically Encoded Self-Assembling Protein Nanoparticles for the Targeted Delivery In Vitro and In Vivo

“…As natural carriers, self-assembled protein nanoparticles exhibit unique properties for drug delivery in vivo—the therapeutic capabilities can be incorporated into the protein structure, e.g., by the creation of self-assembling proteins fused with truncated genetically encoded fragments of bacterial toxins (such as, e.g., fragments of Pseudomonas aeruginosa exotoxin A [ 119 , 235 , 236 ]) or cytotoxic properties can be incorporated into the nanoparticle structure via loading with a chemotherapeutic drug, such as doxorubicin or paclitaxel. The drug loading process is an additional serious obstacle in the creation of novel medical nanoformulations.…”

“…There are many commercially available IgG antibodies to various antigens. One of the best-known antibodies is trastuzumab, which recognizes the receptor HER2 which is overexpressed in some breast cancer cells [ 119 , 120 ]. Due to their ability to target malignant cells or pathogenic agents, immunoglobulins could be a well-suitable platform for the design of targeted nanoparticles in case the protein’s targeting abilities remain intact during NP formation.…”

Tracing 2D Growth of Pancreatic Tumoroids Using the Combination of Image Processing Techniques and Mini-Opto Tomography Imaging System

“…Also, agarose is an optically transparent substance that works well for microscopic spheroid viewing. 12 For these reasons, in our study, a pancreatic cancer tumoroid was created using the MIA PaCa-2 cell line, which is widely used in vitro studies of pancreatic cancer, 13 , 14 and agarose gel-based matrix.…”

Photothermal Therapy with HER2-Targeted Silver Nanoparticles Leading to Cancer Remission

“…Monoclonal IgG antibodies and their derivatives were traditionally used for diagnostics and targeted drug delivery; however, they have a whole range of undesirable side effects [ 21 , 22 , 23 ]. Synthetic nonimmunoglobulin-recognizing scaffold proteins, e.g., affibodies, DARPins, or ADAPTs, appear to be much more effective tools for targeting nanostructures to cancer cells than traditional IgGs due to the ease of large-scale biotechnological production in bacteria, high stability in severe conditions, and the absence of immunogenicity in vivo [ 19 , 21 , 22 , 24 , 25 , 26 , 27 , 28 ]. In particular, one of the most promising scaffolds is the affibody Z HER2:342 , which highly selectively recognizes the clinically relevant oncomarker HER2 and is already undergoing clinical trials [ 29 ].…”