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This paper describes a method of generating three-dimensional (3D) cell-laden microstructures by applying the principle of origami folding technique and cell traction force (CTF). We harness the CTF as a biological driving force to fold the microstructures. Cells stretch and adhere across multiple microplates. Upon detaching the microplates from a substrate, CTF causes the plates to lift and fold according to a prescribed pattern. This self-folding technique using cells is highly biocompatible and does not involve special material requirements for the microplates and hinges to induce folding. We successfully produced various 3D cell-laden microstructures by just changing the geometry of the patterned 2D plates. We also achieved mass-production of the 3D cell-laden microstructures without causing damage to the cells. We believe that our methods will be useful for biotechnology applications that require analysis of cells in 3D configurations and for self-assembly of cell-based micro-medical devices.
In this research, we harness living cells as the self-folding driving forces to create diverse range of 3D cell-laden microstructures: this technique is named cell origami. Cells naturally exert a contractile force [24], known as the cell traction force (CTF), that is generated by actomyosin interactions and actin polymerization, and pulls toward the center of the cell body (Figure 1A). The CTF plays a vital role in many biological processes including cell migration, proliferation, and differentiation. Here, we use the CTF to fold 2D microstructures by patterning cells across a pair of microplates and detaching the microplates from the glass substrate (Figure 1B). Cell origami is highly biocompatible and does not require any special materials for the microplates and hinges to induce folding. In addition, we can produce various 3D cell-laden microstructures by just changing the geometrical design of the patterned 2D plates (Figure 1C).
(A) The cells adhere and stretch across two microplates, and CTFs are generated toward the center of the cell body. Green and blue colors show actin and nucleus, respectively. (B and C) Schematic image of the cell origami: (B) the cells are cultured on micro-fabricated parylene microplates. The plates are self-folded by CTF. (C) Various 3D cell-laden microstructures can be produced by changing the geometry of the plates. (D) Schematic of the parylene microplates without a flexible joint. The cells are seeded onto the microplates coated with FN. Unwanted cells do not adhere on the glass substrate because of MPC polymer coating. (E) A fluorescent image merged with phase contrast image of NIH/3T3 cells patterned only on the microplates. The cells are bridged across the microplates. (F) Schematic of the parylene microplates with a flexible joint to achieve precise 3D configurations after folding. (G) A SEM image of the microplates with the flexible joint. Scale bars, 50 µm.
We examined the basic mechanism and design criteria of our cell origami by culturing cells on a set of two microplates that are put side by side to form a single folded microstructure. We applied two types of cell origami: microplates with and without a flexible joint (Figures 1D and F, Figures S1 and S2). The detail of the microplate preparation steps is described in the Materials and Methods section. In both cases, selective patterning of the cells on the microplates was achieved by coating the glass substrate areas, where the microplates do not exist, with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer; this polymer inhibits protein adsorption and cell adhesion [25]. As a microplate, we choose a micro-patterned parylene (poly(p-xylylene) polymer) film coated with fibronectin (FN). It is known that the effect of the material properties of the parylene coated with FN on CTF, including cell stretching and cell spreading, is comparable to that of standard tissue culture substrates such as polystyrene [26]. Thus, parylene coated with FN can be a suitable material for culturing cells. Between the microplates and the glass substrate, there is a sacrificial gelatin layer in order to allow detachment of the microplates from the substrate. The critical conditions for using the CTF as the self-folding driving force include: (i) the concentration of cells; (ii) the distance between the two microplates; and (iii) the adherence of the sacrificial gelatin layer under the microplates.
After the cells were patterned, they extended their filopodia and bridged across pairs of the microplates. The spacing between the plates is a critical criterion that determines whether the cells can bridge them in order to fold the microplates by the CTF (Figure 3A). We found that about 80% of the cells could bridge microplates with spacing less than 7 µm (Figure 3B). Most of the cells, however, could not bridge when the spacing was more than 15 µm. Therefore, the spacing between the plates should be less than 7 µm to produce the cell origami.
An important parameter for producing desired 3D cell-laden microstructures is the folding angle, θ, between the folded microplate and the glass substrate (Figure 4A). The folding continues until the microplates are blocked by the cells. Thus, the folding angle can approximately be determined by the number of the cells on the microplates. When two or less cells bridged across two microplates without a flexible joint, the plate folded almost completely (folding angle >1605) (Figure 4B). With an increase in the number of cells on the microplates, the microplates were blocked by the multiple cells and could not be folded further, thus the folding angle decreased.
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