3-D Microwell Array System for Culturing Virus Infected Tumor Cells

Abstract

Cancer cells have been increasingly grown in pharmaceutical research to understand tumorigenesis and develop new therapeutic drugs. Currently, cells are typically grown using two-dimensional (2-D) cell culture approaches, where the native tumor microenvironment is difficult to recapitulate. Thus, one of the main obstacles in oncology is the lack of proper infection models that recount main features present in tumors. In recent years, microtechnology-based platforms have been employed to generate three-dimensional (3-D) models that better mimic the native microenvironment in cell culture. Here, we present an innovative approach to culture Kaposi’s sarcoma-associated herpesvirus (KSHV) infected human B cells in 3-D using a microwell array system. The results demonstrate that the KSHV-infected B cells can be grown up to 15 days in a 3-D culture. Compared with 2-D, cells grown in 3-D had increased numbers of KSHV latency-associated nuclear antigen (LANA) dots, as detected by immunofluorescence microscopy, indicating a higher viral genome copy number. Cells in 3-D also demonstrated a higher rate of lytic reactivation. The 3-D microwell array system has the potential to improve 3-D cell oncology models and allow for better-controlled studies for drug discovery.

An endothelial cell line infected by Kaposi’s sarcoma–associated herpes virus (KSHV) allows the investigation of Kaposi’s sarcoma and the validation of novel viral inhibitors in vitro and in vivo

“…In contrast, maintenance of the virus in 3D cell culture at least partially depended on lytic reactivation, as indicated by reduction of viral copy number in presence of PhA. This observation corresponds to recent studies indicating that 3D, but not 2D cell culture conditions supports lytic reactivation of the virus[10,29] and indicates that the 3D cell culture allows validation of the compounds targeting not only latent maintenance, but also lytic replication of the KSHV.…”

“…Several studies indicate that 3D cell culture is beneficial for viral gene expression in lymphatic endothelial as well as in B cells and KSHV-induced cell transformation is more pronounced under these conditions [10,29]. Therefore, we tested if viral maintenance is better reflected by 3D culture conditions.…”

miRNA-36 inhibits KSHV, EBV, HSV-2 infection of cells via stifling expression of interferon induced transmembrane protein 1 (IFITM1)

“…IFITM1 suppression by miR-36 may have direct and indirect effects of KSHV pathobiology: (i) directly limit KSHV infection of cells; (ii) block cell proliferation/division 55 and thereby promote KSHV latency 56 ; and (iii) reduce virus-induced inflammation. Such effects of IFITM1 on the biology of KSHV will be better understood by employing the three-dimensional (3-D) cell culture models as they mimic certain aspects of the tissue environment 57 , 58 . For all this time, studies on virus entry have always focused primarily on the roles of virus encoded glycoproteins and their cognate host cell receptors.…”

Rapid fabrication of functionalised poly(dimethylsiloxane) microwells for cell aggregate formation

“…As well as the hangingdrop technique 12 and non-adherent surface culture, 13 microwell culture has proven to be particularly efficient for aggregate formation while being compatible with standard biological characterisation assays. [14][15][16][17][18][19] Several techniques have been investigated for manufacturing microwells for the formation of MIN6 cell aggregates, which are used as a model to study islet biology. Frequently, microwell arrays are cast from low-fouling materials such as agarose, poly(ethylene glycol) or poly(dimethylsiloxane) (PDMS) using moulds manufactured through intensive micromachining, laser ablation or lithography techniques.…”