Cell Encapsulation and 3D Self-assembly Using
Multi-layered Polymeric Thin Films

3.1 Self-folding of polymeric bilayer film

The combination of silk fibroin with conformally deposited parylene-C functions as a mechanically heterogeneous bilayer [9]. After the Ca-alginate layer was dissolved by EDTA, the released bilayer films transformed into controlled 3D geometries (Fig. 3(a)). This dissolution initiates the formation of tubular structures through self-folding. The curvature radius of the self-folded micro-roll reaches an equilibrium value, which is delayed by more than 10 s after the dissolution of the sacrificial layer. Confocal reconstructed images show that the dimensions of the micro-patterns had a noticeable effect on the diameter of the resulting tubular structures.

In addition to the cylindrical shape, various 3D geometries can be controlled and self-folded depending on the 2D micro-pattern design. For instance, a unique doll-shaped 3D structure was self-folded by combining rectangles with cross-shaped hinges, where the rectangles correspond to the body and limbs and a circle forms the head (Fig. 3(b)). The cross portion connecting the arms retained the backbone shape, making it possible to maintain the 3D doll geometry. Hence, by incorporating the branched structure in the pattern interior, it is possible to produce more complex 3D shapes such as dolls, zigzags, and caged skeletal frames.

Fig. 3. Using 2D thin films to fabricate arbitrary 3D structures. (a) Fabrication of a cylindrical structure using a rectangular thin film. (b) 3D self-folding with the 3D doll geometry. Reprinted from Teshima et al. [9]. Copyright 2017.

We used Fourier transform infrared spectroscopy (FTIR) to characterize the molecular conformation of the self-folded micro-rolls [9]. Silk fibroin was methanol-treated to induce β-sheet conformation in its crystalline layer. The spectra ranging from 1450 to 1700 cm⁻¹ are assigned to the absorption of the peptide backbones in silk fibroin (Fig. 4(a)). All of the films containing the silk fibroin have spectra centered at 1625 cm^–1, which is characteristic of the antiparallel β-sheet conformation in crystalline silk. However, the folded micro-rolls exhibit a reduction in absorbance around 1650 cm⁻¹, suggesting a transition from the helical conformation of amorphous silk to a β-sheet-rich conformation. There are no particular differences in the peak wavelengths of the micro-rolls with varying curvature radii. In addition, thanks to its high elastic modulus, the β-sheet-rich matrix was maintained without a phase transition after the self-folding step. Accordingly, the inducement of the β-sheet-rich conformation is an essential process to maintain the 3D shape of the micro-rolls and make them sufficiently durable for long-term incubation.

We applied quantitative predictions of 2D designs to the final 3D geometry to allow us to control the micro-roll architecture [9]. Since the underlying mechanism of self-folding is the stiffness mismatch in the bilayer, the curvature of the micro-rolls (1/ρ) was calculated using Timoshenko’s bimorph beam theory [14] (Fig. 4(b)). This theory predicts the curvature radius (ρ) based on the elastic modulus, layer thickness, and strain. For simplicity, we assumed a constant thickness of silk fibroin. The experimental value ρ is plotted in the equilibrium state as a function of thickness of parylene from 30 to 400 nm. We confirmed that ρ mostly follows the trend predicted by the bimorph beam theory.

Fig. 4. Characterization of self-folded micro-rolls. (a) FTIR spectra of crystalline silk fibroin in micro-rolls. Resonance absorbances centered at 1650 and 1625 cm⁻¹ respectively indicate a random coil conformation in amorphous silk protein and the helical β-sheet conformation of annealed protein. (b) Plot of average curvature radius of free-floating, self-folded 200×400 μm² films for various parylene film thicknesses. Reprinted from Teshima et al. [9]. Copyright 2017.

3.2 Encapsulation and manipulation of cells

The key challenge with cell-laden micro-rolls is to fabricate tissue-mimicking tubular structures with non-cytotoxicity and to confirm their cellular function. We encapsulated human embryonic kidney (HEK) cells within the micro-rolls. When EDTA was added, the cells on the 2D film surface were encapsulated inside the 3D micro-rolls through the self-folding of the bilayer (Fig. 5). Confocal cross-sectional images revealed that the arrayed micro-rolls encapsulated numerous cells at a time, which were separated from the cells outside the patterned area. A live/dead assay revealed that more than 95% of the cells were still alive inside the micro-rolls after exposure to EDTA. This indicates that the self-folding and encapsulation process was sufficiently gentle to avoid cell damage. Since the curvature radius and length of the micro-rolls are controllable, the number of encapsulated cells can be easily controlled with the cell density and the volume of the micro-roll. The use of an array of micro-rolls potentially makes it possible to create highly parallel assays that can be used to investigate cellular behaviors including migration, proliferation, and differentiation.

Fig. 5. Encapsulation of cells. As the Ca-alginate gel layer is removed by EDTA, the thin film deforms into a tubular structure. At the same time, cells near the surface of the film are encapsulated inside it. Reprinted from Teshima et al. [9]. Copyright 2017.

The high biocompatibility and transparency of the micro-rolls enable stable culturing of encapsulated cells. During the incubation, the encapsulated cells migrated and proliferated only inside the micro-rolls without any migration out of the micro-rolls. This leads to the formation of cell-cell contacts along the inner surfaces of the micro-rolls. The micro-rolls with encapsulated cells not only provide the transparency needed for microscope observations, but they also have sufficient mechanical strength to withstand the cell traction force. HEK cells exhibit relatively strong cell-to-cell contact, so the cells in a micro-roll aggregated and filled the micro-roll cavity after one day of culturing. Specifically, the cells proliferated, filled the micro-roll cavity, and then migrated onto the outer silk fibroin surface of the micro-rolls. The shapes of the cell clusters differed depending on the micro-roll volume and the cell-line’s adhesive properties.

A pico-liter fluid flow done with a glass capillary enables manipulation of cell-laden micro-rolls [9]. The smooth withdrawal and release of cells from the capillary enables us to relocate cells to the target location (Fig. 6(a)). When the micro-cells were being conveyed inside the glass capillary, the micro-rolls stably maintained their cylindrical shapes without buckling or bending. Therefore, individual cells inside the micro-rolls were collected and transported to the targeted position to induce cell-to-cell contact (Fig. 6(b)). Note that the cells on micro-rolls were manipulated without stripping them from their growth surfaces, which allowed the adhesive state of the manipulated cells to be preserved. This has the potential to facilitate high-order cellular assembly for potential use in tissue engineering by enabling the manipulation of individual cell types into any desired multi-cell-line combination.

Fig. 6. (a) Manipulation of 400-μm-long cell-laden micro-rolls in culture medium. The micro-rolls can be withdrawn, relocated, and ejected with a picoliter flow using glass capillaries. (b) Micrograph of manipulated multiple cell-laden micro-rolls in culture medium.

3.3 Reconstruction of functional tissue-like structures

Physiological functions within the cell-laden micro-rolls were investigated using two types of primary cultured cells: cardiomyocytes and hippocampal cells [9]. In contrast to the cell lines, neither of these proliferate spontaneously, and their cell bodies tend to reside in the micro-rolls. After five days of culture, the cardiomyocytes aggregated to form a more than 2-cm-long tubular structure. A time-dependent displacement at corresponding pixel locations was estimated by subtracting the average intensity from the optical images (Fig. 7). The intensity corresponding to the displacement shows that all the connecting cells synchronously contracted in the same period at approximately 1 Hz. Time-lapse fluorescence image measurement of fluo-4-labeled cells shows that the oscillation of intracellular Ca²⁺ propagated and was synchronized within the micro-rolls. The microenvironment provided by micro-rolls of any length was able to regulate the cell migration and cell-cell interactions and drive intrinsic morphologies and functions.

Fig. 7. Reconstruction of minute myocardial tissue structures. (Left) Phase contrast micrographs of cardiomyocytes cultured inside tubular structures, and black and white images showing the displacement of the cells during a heartbeat. (Right) Graph showing the amount of displacement during a heartbeat. In the tubular structure containing the cardiomyocytes, the cells are bound to each other to form a fibrous biological tissue-like structure. The cardiomyocytes beat in unison with a rhythm that is synchronized and constant. Reprinted from Teshima et al. [9]. Copyright 2017.

Primary hippocampal cells were also encapsulated to reconstitute neural tissues. The encapsulated cells aggregated and subsequently extended their dendrites in the micro-roll direction. After three days of culturing, the neurite-mediated cell-cell connection between the paired cell aggregation resulted in the formation of unidirectional neural cell fibers (Fig. 8(a)). Somas and their neurites were both located inside the micro-rolls without any protrusions. Interestingly, the neurites were further extended outside the micro-rolls, where they connected with neighboring cells. Immunostaining of neurites and somas showed that both were positionally restricted within the micro-rolls. Some spheroids were located at the edge of the micro-rolls, and the others were evenly distributed inside the micro-rolls. Regardless of their location, the dendrites were extended and evenly distributed in the micro-rolls after long-term incubation.

To further investigate the accessibility of neurites to the outer surface, we examined cellular responses to external stimulation with added glutamic acid. When the glutamic acid receptors in the hippocampal cells were activated, the intracellular Ca²⁺ were detectable by calcium indicators using confocal laser scanning microscopy (Fig. 8(b)). Fluo-4 labeled cells exhibited spontaneous Ca²⁺ oscillations that were synchronized across the cells via synaptic connections (cells H, I, J), whereas the cells that were not connected via neurites showed no oscillation signals (cell K). This result indicates that the encapsulated cells come into direct contact and build up a cellular network with the cells outside of the micro-rolls through hollow structures in order to transmit and exchange information with their neighboring cells. After two weeks of culturing, the extended neurites from the manipulated cell-laden micro-rolls were connected and bridged to achieve cell-to-cell contact between neighboring micro-rolls. Daily medium exchanges and cell maintenance ensures the long-term culturing of encapsulated cells without cytotoxicity. Consequently, the relocated cells can recreate intercellular communication with the cells outside the micro-rolls, serving as building units of higher-order tissue assemblies and potential implantable grafts in vivo.

Fig. 8. (a) SEM images of hippocampal cell-laden micro-rolls cultured on poly-D-lysine/laminin-coated dishes. (b) Relative fluorescence intensity profiles of Ca²⁺ oscillations in hippocampal cells labeled with Fluo-4 and stimulated by adding glutamic acid in the segment shown in the confocal fluorescent image. Reprinted from Teshima et al. [9]. Copyright 2017.