Ensuring flawless formation of DNA origamis is a challenge as hundreds of like-charged DNA strands have to associate while ensuring the formation of around 10 000 to 20 000 hydrogen bonds through specific base complementarity. A typical approach to achieve this involves a thermal annealing process in the presence of a high concentration of stabilizing dications, typically magnesium (usually at a concentration ranging from 10 mM to 25 mM, depending on the object). Isothermal assembly at room temperature has been demonstrated as an alternative by replacing dications with a higher concentration of monovalent salts (typically 100 mM or 150 mM), ensuring both sufficient stability and reconfigurability for nearly flawless DNA assembly. However, these conditions remain incompatible with cell culture and growth. With the aim to achieve isothermal assembly of DNA origamis in the presence of live cells at 37 °C, we first selected the widely used Dulbecco's Modified Eagle Medium (DMEM) for its high concentration in monovalent cations (around 160 mM, Table ) and millimolar amounts of dications (mainly Ca 2+ , Mg 2+ , Table ). We simply mixed a DNA origami-coding molecular program (scaffold and staple cocktail) in DMEM and incubated it at 37 °C for in situ selfassembly (Fig. ). With a program coding for triangle origamis, atomic force microscopy (AFM) revealed successful origami formation after a few hours of incubation (Fig. left, Fig. . The experiment was repeated in the presence of 10 vol% of foetal bovin serum (FBS), a commonly required supplement for cell culture. Although the presence of proteins rendered less accurate the imaging of the DNA origamis, the distinction of well-formed triangles (Fig. right, Fig. bottom) indicated proper origami folding in these conditions as well. Isothermal self-assembly at 37 °C also led to well-formed DNA origamis in i) RPMI (Roswell Park Memorial Institute medium), another commonly used cell culture medium; ii) Essential 8 TM , a medium for induced pluripotent stem cells (iPSCs) culture; and iii) PBS (Phosphate Buffered Saline), a usual buffer for biological research (Fig. ). All these buffers share a concentration of monovalent cations between 140 and 160 mM and divalent ones below 3 mM (Table ), ensuring robust isothermal self-assembly at 37 °C. To investigate the kinetics of this isothermal self-assembly, we used AFM to analyse the morphology evolution after adding the molecular program into FBS-free DMEM as a function of incubation time at 37 °C. Using conventional concentrations of scaffold (1 nM) and staples (40 nM each), we observed the formation of ill-shaped assemblies within 5 min, which progressively evolved into distinguishable triangles after 15 min and into particularly well folded structures after 1 h (Fig. ). Decreasing the staple concentration to a 10-fold staple excess slowed the assembly but still resulted in complete folding within 1 h (Fig. top). Strikingly, increasing the scaffold concentration to 10 nM while maintaining a 40-fold staple excess led to the formation of well-folded triangles after 5 min only (Fig. bottom), which represents a considerably shorter time than the hours to days needed for known methods with 5 or without 29 thermal annealing. With a fixed scaffold concentration (10 nM), we assembled molecular programs coding for different morphologies in DMEM and let the self-assembly occur at 37 °C for 15 min (Fig. ). Using M13 scaffold, 2D structures including compact (rectangles) and circular (smileys) ones formed well (Fig. left). We also explored the challenge of a more complex design, consisting of a hollow 3D structure forming a toroid shape with a single layer of adjacent double helices. Using a p7560 scaffold and a 10fold excess of staples, a high number of toroidal structures were detected by both AFM and TEM (Fig. right, Figs. S3-S4), with a diameter 78 ± 3 nm (mean ± SD, n = 18) consistent with the design (Fig. ). All these results show that mixing all DNA strands coding for DNA origami in a monovalent cation-rich mammalian cell culture medium, characteristic of most commonly used media, results in ultra-fast and isothermal self-assembly into user-programmed 2D or 3D morphology at 37 °C. The successful formation of origamis across various culture media led us to explore their in situ selfassembly in the presence of different living cell systems including 2D cell culture and organoid formation. In situ origami assembly with live cells in 2D culture.