In situ hybridization of synthetic nucleic acids within living cell environments has proved to be a powerful strategy to selectively detect and/or interact with biological targets, with applications ranging from biosensing to therapeutic actions. In a very different context, this ability of nucleic acids to self-assemble through complementary base pairing has been exploited by researchers to build elaborate nanostructures in a user-friendly and highly programmable manner, leading to the development of structural DNA nanotechnology. Within the different methods, DNA origami self-assembly has emerged as one of the most widely studied and applied technologies. Based on the guided folding of a circular single-stranded DNA scaffold through the addition of a designed cocktail of hundreds of short oligonucleotides acting as staples, DNA origamis can be produced at a high yield, with virtually any desired two-(2D) or three-dimensional (3D) morphology. With typical size around 100 nm, these structures offer precise site-specific functionalization at subnanometric resolution, making them powerful platforms for nanoscale organization of matter with exceptional degree of programmability and versatility. Among various application fields, DNA origamis have been particularly recognized as valuable tools to interact with living organisms, leading to emerging applications including virus recognition and capture, mechanobiology, synthetic immunology, or drug delivery. However, their conventional preparation is slow, taking hours to days, and relies on a constraining thermal annealing process (heating to around 80 °C followed by a slow cooling ramp over several hours to days) that is incompatible with living cells. Additionally, the self-assembly of DNA origamis typically requires magnesium concentrations ranging from 10 mM to 25 mM, far exceeding physiological levels and posing further challenges for direct applications in biological environments. As a result, and as of today, origamis have always been first fabricated before being added to cells for further studies. There is thus a critical scientific and methodological gap: while in situ hybridization enables nucleic acids to interact with biological targets, it currently lacks the capacity for elaborate structural organization, whereas DNA origami assembly offers exquisite programmability but requires ex situ preparation under conditions unsuitable for live cells. To bridge this gap, we thus looked for a method allowing in situ assembly of synthetic nucleic acid cocktails into user-defined DNA origamis directly in the presence of live cells. We recently reported that DNA origamis can be isothermally self-assembled in a magnesiumfree, monovalent salt buffer (e.g., 100 mM or 150 mM NaCl) at room temperature, but the assembly remained long (typically 24 h) and buffer conditions were not compatible with cell culture and manipulation. In this work, our strategy was thus to look for existing cell culture buffers containing a sufficiently high amount of monovalent salts (typically around 150 mM), along with essential divalent cations (e.g., Mg 2+ , Ca 2+ ) required for cell culture but at a concentration low enough, to ensure isothermal assembly conditions at a temperature favourable to live cell growth. In fact, several commercially available and widely used buffers satisfy these conditions. We discovered that directly mixing DNA origami scaffold and staples in such buffers resulted in the isothermal self-assembly of well-formed DNA origamis at 37 °C in a few minutes only. This is not only one to two orders of magnitude faster than existing methods whether they require thermal annealing or not, but also constitutes the first origami assembly conditions fully compatible with live cell maintenance, culture and growth. We thus applied and characterized this method for the in situ programmable isothermal self-assembly of various 2D and 3D origami morphologies directly in the presence of live cells, ranging from conventional 2D culture to more complex 3D tissue-like systems (here, cerebral organoids). Ultra-fast DNA origami self-assembly in cell culture media at 37 °C.