Rapid Generation of In Vitro Multicellular Spheroids for the Study of Monoclonal Antibody Therapy

Abstract

Tumor microenvironments present significant barriers to penetration by antibodies and immunoconjugates and are difficult to study in vitro. Cells cultured as monolayers typically exhibit less resistance to therapy than those grown in vivo. Therefore, it is important to develop an alternative research model that better represents in vivo tumors. We have developed a protocol to produce multicellular spheroids, a simple and more relevant model of in vivo tumors that allows for further investigations of the microenvironmental effects on drug penetration and tumor cell killing. The protocol is used to produce in vitro three-dimensional tumor spheroids from established human cancer cell lines and primary cancer cells isolated from patients without the use of any extracellular components. To study the ability of tumor-targeting immunoconjugates to penetrate these tumor spheroids in vitro, we have used an immunotoxin targeting mesothelin, a surface protein expressed in malignant mesotheliomas. This method for producing consistent, reproducible 3D spheroids may allow for improved testing of novel monoclonal antibodies and other agents for their ability to penetrate solid tumors for cancer therapy.

Spheroids Formation on Non‐Adhesive Surfaces by Liquid Overlay Technique: Considerations and Practical Approaches

“…• Solutions preparation is very time consuming • Plate-coating procedure is very time consuming [19,[73][74][75][76][77] www.advancedsciencenews.com www.biotechnology-journal.com dissolve the polymer, at 37 C. [73] Furthermore, Phung et al denoted that the complete evaporation of the ethanol from the poly-HEMA coated plates at RT (15)(16)(17)(18)(19)(20) C) takes 1-2 weeks. [77] To surpass these drawbacks, Lawrenson et al suggested to raise the temperature of the poly-HEMA solution (up to 65 C), in order to facilitate the polymer dissolution and to dry the coating inside a humidity-free incubator at 37 C, for 48 h. Such procedure allows the creation of a uniform, solvent free plate coating.…”

“…[77] To surpass these drawbacks, Lawrenson et al suggested to raise the temperature of the poly-HEMA solution (up to 65 C), in order to facilitate the polymer dissolution and to dry the coating inside a humidity-free incubator at 37 C, for 48 h. Such procedure allows the creation of a uniform, solvent free plate coating. [19] Recently, in addition to the previous referred non-adhesive biomaterials, HA applicability to cover surfaces for spheroids formation has also been investigated by Lai and Tu [78] and by our lab.…”

“…In this context, the addition of supplements (e.g., viscosityincreasing additives, ECM related components, and others) to the [37,[73][74][75][76] It takes several hours to dissolve poly-HEMA • Put the poly-HEMA solution in a water bath at 65 C [73,77] Poly-HEMA coating drying is slow (1 to 2 weeks at RT)…”

“…• Poly-HEMA drying time can be reduced to 48 h using an incubator at 37 C [77] The coating is not uniform • Increase the volume of the coating solution • Do not allow agarose to cool down below 60 C before the coating process • Do not reheat the solutions [15,38,61] The coating has some cracks • Do not store the cell culture platforms during long periods of time • For long storage periods, it may be helpful to cover the coating with cell culture medium [15,38] Plate-coating procedure is time consuming [3,37] Spheroids formation, growth and culture Some cells (e.g., MDA-MB-231) cannot grow into spheroids by LOT…”

In vitro skin three-dimensional models and their applications

“…However, depending on the application, small spheroids can be generated to avoid a necrotic core [19]. Spheroids are mostly made by the hanging drop method [20,21] or by the use of non-adherent U-bottom shaped plates [22]. Furthermore, some cells like melanocytes need the addition of a coating such as chitosan to form spheroids [23].…”