An inverse small molecule screen to design a chemically defined medium supporting long-term growth of Drosophila cell lines

Abstract

Drosophila cell culture is used as a model system with multiple applications including the identification of new therapeutic targets in screens, the study of conserved signal transduction pathway mechanisms, and as an expression system for recombinant proteins. However, in vitro methods for Drosophila cell and organ culture are relatively undeveloped. To characterize the minimal requirements for long-term maintenance of Drosophila cell lines, we developed an inverse screening strategy to identify small molecules and synergies stimulating proliferation in a chemically defined medium. In this chemical-genetics approach, a compound-protein interaction database is used to systematically score genetic targets on a screen-wide scale to extract further information about cell growth. In the pilot screen, we focused on two well-characterized cell lines, Clone 8 (Cl.8) and Schneider 2 (S2). Validated factors were investigated for their ability to maintain cell growth over multiple passages in the chemically defined medium (CDM). The polyamine spermidine proved to be the critical component that enables the CDM to support the long-term maintenance of Cl.8 cells. Spermidine supplementation upregulates DNA synthesis for Cl.8 and S2 cells and increases MAPK signaling for Cl.8 cells. The CDM also supports the long-term growth of Kc167 cells. Our target scoring approach validated the importance of polyamines, with enrichment for multiple polyamine ontologies found for both cell lines. Future iterations of the screen will enable the identification of compound combinations optimized for specific applications—maintenance and generation of new cell lines or the production and purification of recombinant proteins— thus increasing the versatility of Drosophila cell culture as both a genetic and biochemical model system. Our cumulative target scoring approach improves on traditional chemical-genetics methods and is extensible to biological processes in other species.

A regulated environment for micro-organs defines essential conditions for intercellular Ca2+waves

“…3). ZB media was engineered to support long-term proliferation and passaging of the wing-disc derived Cl.8 cell line (23). These results demonstrate that specific chemical stimulation by FEX is specifically required for the generation of both spontaneous and mechanically stimulated ICWs.…”

In vivo relevance of intercellular calcium signaling inDrosophilawing development

“…Surprisingly, we observed Ca 2+ oscillations in Clone 8 without fly extract ( Fig. 2G, Movie S11) in a chemically defined media (Burnette et al, 2014). These oscillations occurred on the scale of roughly one per minute ( Fig.…”

Intercellular calcium signaling is regulated by morphogens duringDrosophilawing development

“…2). We used ex vivo culture in the chemically-defined ZB media to reduce the effect of batch variability (Burnette et al, 2014). Although the accompanying paper shows that ZB media with 2.5% fly extract best recapitulates in vivo Ca 2+ dynamics (DEVELOP 2017/159251), we found that 15% fly extract was ideal to yield oscillatory signals of sufficient strength for reliable quantification.…”

“…1d, d', Extended Data Movie 1). However, in vivo imaging provides limited information due to larval motion, mechanical compression, and an inability to specifically perturb the organ with drugs [25][26][27] .…”

“…Organ culture media ZB media + 15% FEX contains 79.4% (v/v) ZB media, 0.6% insulin, 15% ZB-based fly extract and 5% penicillin/streptomycin 25 . ZB media was developed as a chemically defined media to support Drosophila cell culture 26 , and was used as the basal media for most experiments. ZBbased fly extract (FEX) is undefined serum extracted from ground flies using ZB media as the base.…”