Producing 3D Cell-Laden Microstructures using Origami Folding Technique

Kaori Kuribayashi-Shigetomi1, Hiroaki Onoe2,3, and Shoji Takeuchi2,3

1 Grad School of Inform Sci and Tech, Hokkaido University, Sapporo, Japan 2 IIS, The University of Tokyo, Tokyo, Japan, 3 Takeuchi Biohybrid Innovation Project, ERATO, Tokyo, Japan

Abstract

Three-dimensional (3D) cell culture is widely employed for fundamental research in cell biology, drug discovery, tissue engineering, and regenerative medicine. Here we harness the cell traction force (CTF) to produce 3D cell-laden 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. This cell-based origami is expected to be useful in biotechnological applications that require analysis of cells in 3D configurations.

Keywords –Origami, Cell Traction Force, Microstructures

1 Introduction Nature utilizes controlled folding schemes to

produce intricate architectures, ranging from proteins to plants. Origami engineering harnesses novel pathways, inspired by the Japanese art of paper folding, for technological applications, including the fabrication of nanoscale DNA-based objects [1], solar panels for space deployment [2,3], and flexible medical stents [4].

In the area of microfabrication, origami folding strategies have also proved to be promising approaches for producing 3D microstructures [5] since they are simple and time-effective compared to other 3D microfabrication techniques such as stereolithography and laser micromachining. In particular, the origami folding techniques have recently been explored to produce various 3D cell-laden microstructures including micro-sized containers and scaffolds for artificial tissues. The folding of these microstructures is typically performed by surface tension, stress-induced forces, and shrinkage of the hingeswith external triggers such as temperature and electrical/chemical signals. However, such driving forces require functional materials and thermo-sensitive polymers that involve complicated preparation processes. In addition, the compatibility of the external triggers to living cells must be considered in these folding mechanisms.

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, 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 (Fig. 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 (Fig. 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 (Fig. 1C).

Fig 1. (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.

Fig. 2. (A) Schematic of 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 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer coating. (B) Schematic of the parylene microplates with a flexible joint to achieve precise 3D configurations after folding.

Table 1 Concentration of gelatin for folding

microplates with and without a flexible joint.

2 Materials and Methods

2.1 Fabrication of Microplates There are two folding approaches (Fig. 2). In the

first approach (Fig. 2A), we used 0.05–0.1% gelatin as the sacrificial layer under the microplates that was produced by a standard Micro Electro Mechanical Systems (MEMS) lithography technique, and the edges of individual microplates were pushed with a glass tip manipulated by a micromanipulator (NI2, Eppendorf), manually triggering detachment of the plates from the substrate. Thus, a large amount of the microstructures can be produced in order. In the second approach, we incorporated the flexible joint (Fig. 2B) with a 3–5% gelatin sacrificial layer. In this case, microplates were detached and self-folded by CTF spontaneously as the gelatin layer dissolved at 37 oC, which is the temperature at the cell incubator. Consequently, many 3D microstructures were produced simultaneously. The optimum concentrations of the gelatin in both approaches were experimentally determined (Table 1).

2.2 Culturing the cells We used various types of cells, including NIH/3T3

cells (TKG299, RIKEN Cell Bank, Japan) and BAOSMCs (CAB35405, Cell Applications) that are commonly used as adherent cells to study CTFs. We prepared cells suspended in culture media at various concentrations (cells/ml). Then, we added 2 ml of the cell suspension into a petri dish containing our substrate with the microplates. The cell seeding concentration was 2 × 104 – 2 × 105 cells/ml, which corresponds to 4.2 × 103 – 41.5 × 103 cells/cm2. Non-adhered cells on the substrate were washed out after a 4 h culture period.

3 Results and Discussion Using the microplates, we successfully produced a dodecahedron (Fig. 3) with NIH/3T3 cells. The cells

coved inside the hollow microstructures. By changing the geometry of 2D developments, we are able to produce various shaped 3D microstructures.

To widen the field of practical applications of the cell origami technique toward cell-laden microstructures, the produced microstructures need to be reproducible and suitable for mass production.

For the microplates incorporating the flexible joint with a 3–5% gelatin sacrificial layer, we found that the microplates were self-folded by the CTF without any extra triggers, and parallel processing of self-folding 3D cell-laden microstructures was achieved. Cubic and tetrahedral microstructures were self-folded spontaneously, and as a result the 3D cell-laden microstructures were successfully mass-produced (data is not shown). Further complex structures can be produced by introducing mountain folds as well as valley folds.

4 Conclusion In this study, we exploit the CTF to drive the

folding of 2D microplates into 3D cell-laden microstructures. Cell origami is a highly biocompatible, simple, and efficient technique with a single step to encapsulate cells into the microstructures. It is particularly useful for producing hollow structures with cells in various shapes including cylindrical tubes and cubes.

Acknowledgements

This work was supported by Grant-in-Aid for Exploratory Research (24656161) from JSPS, Japan, and Grant-in-Aid for Scientific Research on Innovative Areas “Bio Assembler” (23106008) from MEXT, Japan.

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Fig. 3. Sequential images of various 3D cell-laden microstructures folded by CTF. Scale bars, 50 µm.