Artificial cells eavesdropping on HepG2 cells

Abstract

Cellular communication is a fundamental feature to ensure the survival of cellular assemblies, such as multicellular tissue, via coordinated adaption to changes in their surroundings. Consequently, the development of integrated semi-synthetic systems consisting of artificial cells (ACs) and mammalian cells requires feedback-based interactions. Here, we illustrate that ACs can eavesdrop on HepG2 cells focusing on the activity of cytochrome P450 1A2 (CYP1A2), an enzyme from the cytochrome P450 enzyme family. Specifically, d-cysteine is sent as a signal from the ACs via the triggered reduction of disulfide bonds. Simultaneously, HepG2 cells enzymatically convert 2-cyano-6-methoxybenzothiazole into 2-cyano-6-hydroxybenzothiazole that is released in the extracellular space. d-Cysteine and 2-cyano-6-hydroxybenzothiazole react to form d-luciferin. The ACs respond to this signal by converting d-luciferin into luminescence due to the presence of encapsulated luciferase in the ACs. As a result, the ACs can eavesdrop on the mammalian cells to evaluate the level of hepatic CYP1A2 function.

Keywords: artificial cell; cell mimicry; cellular communication; cytochrome P450.

Scheme 1.

Reciprocal eavesdropping of ACs on HepG2 cells. (a) Schematic of the reciprocal eavesdropping. Alginate-based ACs are conjugated with d-cysteine that can be cleaved upon treatment with GR or DTT to reduce the disulfide bond between alginate and d-cysteine (b). CYP1A2 in HepG2 cells converts the luminometric compound CMB into CHB and (c) d-cysteine and CHB react to produce d-luciferin. d-Luciferin is converted by encapsulated luciferase to give a luminescence signal depending on the CYP1A2 expression level in HepG2 cells.

Figure 1.

Characterization of alginate beads conjugated with d-cysteine (AC^Cys). (a) Bright field microscopy images of AC⁰ and AC^Cys. Scale bar is 100 µm. (b) d-Cysteine is conjugated to alginate in AC^Cys and DTT or GR is used to reduce the disulfide bond to release d-cysteine. (c) Quantification of d-cysteine after treating AC⁰ or AC^Cys with GR or DTT. 50 mg AC^Cys yielded approximately 60 µM d-cysteine after 1 h incubation with 2.5 mM DTT (n = 3). *p < 0.05, determined using a one-way ANOVA followed by a Tukey's multiple comparison post hoc test.

Figure 2.

Eavesdropping in microslides. (a) CYP1A2 expression is induced in HepG2 cells using indirubin. CYP1A2 converts CMB to CHB through dealkylation. CHB reacts with d-cysteine provided by the AC^Cys to yield d-luciferin, which is converted by luciferase to produce luminescence. (b) Determined CHB production of HepG2 cells based on the detected amount of d-luciferin (n = 3). *p < 0.05, determined using a one-way ANOVA followed by a Tukey's multiple comparison post hoc test.

Figure 3.

Eavesdropping in semi-synthetic aggregates. (a) Representative CLSM images of aggregates consisting of HepG2 cells and AC⁰ grown for 5 days. Calcein-AM is used to visualize the live cells (green) and the AC⁰s are coated with PLL^R (red). Scale bar is 100 µm. (b) Cartoon showing the eavesdropping of AC^Cys on the HepG2 cells in the aggregates. (c) d-Luciferin made from CHB produced by HepG2 cells and d-cysteine cleaved from AC^Cys (n = 4). *p < 0.05, determined using a one-way ANOVA followed by a Tukey's multiple comparison post hoc test.

Figure 4.

(a) (i) Reciprocal eavesdropping of ${\mathrm{AC}}_{\mathrm{Luc}}^{\mathrm{Cys}}$ on HepG2 cells. (ii) Schematic showing the experimental set-up. (b) d-Luciferin production as a result of CHB made by HepG2 cells and cleavage of d-cysteine from ACs. *p < 0.05, determined using a one-way ANOVA followed by a Tukey's multiple comparison post hoc test.