Artificial cells made of molecular components and lipid membrane are emerging platforms to characterize living systems properties. Cell-free transcription-translation (TXTL) offers advantages for the bottom-up synthesis of cellular reactors. Yet, scaling up their design within well-defined geometries remains challenging. We present a microfluidic device hosting TXTL reactions of a reporter gene in thousands of microwells separated from an external buffer by a phospholipid membrane. In the presence of nutrients in the buffer, microreactors are stable beyond 24 h and yield a few mg/mL of proteins. Nutrients in the external solution feed the TXTL reaction at the picoliter scale via passive transport across the phospholipid membrane of each microfluidic well, despite the absence of pores. Replacing nutrients with an inert polymer and fatty acids at an isotonic concentration reduces microreactors efficiency, and a significant fraction yields no protein. This emphasizes the crucial role of the membrane for designing cell-free TXTL microreactors as efficient artificial cells.
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