We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.
Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.
While human tissues are composed of several assemblies of cells and can help to maintain homeostasis of the body, single cells by themselves also play important roles via cell-to-cell interaction. Therefore, it is important to elucidate how single cells can be stimulated by external signals and how they transfer such signals to other adherent cells. For this purpose, several methods have been established for the construction of single-cell-based 3-dimensional (3D) assemblies1,2,3,4,5,6,7,8. However, the materials that are used to construct cellular assemblies can still be improved. For example, synthetic gels and polymers including polyethylene glycol (PEG) possess certain chemical physicochemical properties and may affect target cells (e.g., toxicity).
We recently reported a novel system that could generate a single-cell-based 3D assembly of cells using dextran (DEX) by establishing stable cell-cell contact9. We considered that this technology could be useful in several research fields, including regenerative medicine and even cancer biology. In this report, we describe how we manipulate single cells and construct 3-dimensional (3D) cellular assemblies in the presence of various hydrophilic biomacromolecules including DEX without an artificial scaffold.
1. Preparation of Cells
2. Preparation of Dextran (DEX)
3. Preparation for Laser and Microscopy
4. Cell Manipulation using the Laser Trapping System
5. Construction of a 3-Dimensional (3D) Cell Structure
Figure 1 shows the microscope and software used in this study. Figure 2 is a schematic representation of the procedure for placing the sample solution containing cells. Figure 3 demonstrates the formation of a pyramid structure using double-beam optical tweezers. If the experiment is successful, these cellular assemblies remain stable even after the laser is switched off.
Figure 1: (a) The control system for the Laser Trapping System (NanoTracker2 (11)). The system is activated by turning on the laser switch following steps ①-③. (b) The software for controlling the Laser Trapping System. The camera, LED light, focus adjust, and moving stage are activated by clicking icons ①, ②, ③, and ④, respectively. The microscopic image is displayed in panel 1. The on/off control for the LED is in panel 2. The focus is controlled in panel 3. The laser beams are irradiated at Positions 1 and 2 by clicking icons I to IV. The details of this Laser Trapping Systemare provided in Ref. (12). Please click here to view a larger version of this figure.
Figure 2: Representative schematic for placing the slide glass. 20 µL of the sample (cell suspension containing dextran) is placed on the slide and used for laser manipulation. Please click here to view a larger version of this figure.
Figure 3: a) Assemblies of epithelial cells (NMuMG) of an intended shape in a medium with DEX (40 mg/mL): a pyramid is shown as an example of a 3D cluster. b) A schematic figure of the pyramid-shaped 3D cellular assembly is also shown. Please click here to view a larger version of this figure.
The present study shows a concrete application of our recent reports9,11 on the use of soluble polymers for the construction of 3D single-cell assemblies. Such assemblies are stably formed in the bulk solution when the number of cells is up to 10, and can be held by a single laser beam. Assemblies precipitate on the glass surface when there are more than 10 cells. Although the experiments are still in a primitive stage, we expect that the novel methodology could be a powerful tool for the construction of next-generation 3D single-cell assemblies, which are indispensable for progress in the fields of cell biology and regenerative medicine.
In a solution containing no polymer, cells repel each other due to the electrostatic repulsion arising from the surface charge, the hydration repulsion force, the glycocalyx repulsion effect, and membrane undulation. Our previous study showed that cell pairs can be stable for a long time when the cells are treated with PEG. More importantly, the successful transport of a cell pair to a region without PEG, after the cells had been held in contact for 5 minutes in PEG, suggests that cellular contact is maintained in a stable manner. This is well explained in terms of the depletion effect11, and essentially the same mechanism applies to the cellular assemblies generated using DEX9. Our current results suggest that other kinds of natural macromolecules could also be used to construct stable 3D cellular assemblies.
For the prompt transport of cells, the concentration of polymer is important. Generally, the viscosity of the solution drastically increases when the polymer is dissolved above the overlap concentration. Under this condition, it is difficult to manipulate cells using optical tweezers. Hence, the experiment should be performed below the overlap concentration. For a DEX solution, the overlap concentration is ca. 50 mg/mL (the kinetic viscosity is 5.5 mm2/s). As shown in Ref. 9, a stable cellular assembly was observed when the concentration of DEX was 10 mg/mL to 40 mg/mL. This result suggests that the depletion effect is sufficiently large to maintain stable cell-cell contact even when the DEX concentration is lower than the overlap concentration. It has been shown that the addition of DEX does not affect cell viability up to 40 mg/mL 9.
The establishment of a method for the construction of 3D cellular assemblies is important in the field of regenerative medicine, since mimicking an in vivo cellular microenvironment by structuring single cells may facilitate stem cell-derived tissue formation. So far, we have used the present protocol to construct cellular assemblies using Neuro2A cells9 in addition to NMuMG cells. We hope to establish an experimental methodology for constructing 3D cellular assemblies of a larger number of cells of various morphologies. The optical tweezers system developed by Ichikawa et al.13 would be applicable for this purpose since the orientation of the cells can be controlled. Further trials along these lines should be promising.
The authors have nothing to disclose.
The authors thank Shu Hashimoto, Aoi Yoshida, and Taeko Ohta at Doshisha University for their generous assistance with the experimental setup. This work was supported by KAKENHI (15H02121, 15K05400, 25103012, 50587441) and by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities. This study was also supported by a Polish grant from the KNOW (Leading National Research Centre) Scientific Consortium "Healthy Animal – Safe Food", decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015..
Microscope IX71 | Olympus | IX71 | |
Dextran(200,000; molecular biology-grade) | Wako | CAS.NO 9004-54-0 | |
Laser Trapping System (NanoTracker 2) | JPK Instruments | S/N T-05-0200 | |
Upper Objective Lens | Olympus | LUMPLFLN60XW | |
Lower Objective Lens | Olympus | UPLSAPO60XW | |
Top Cover Glass | MATUNAMI | C022401 | |
Intermediate Cover Glass (Spacer) | MATUNAMI | – | custom-made (size = 10mm×10mm, thickness = 0.17mm) |
Bottom Cover Glass | MATUNAMI | C030401 | |
Camera | The Imaging Source | DFK 31AF03 | |
Software | JPK Instruments | NanoTracker2 PFM software | |
NMuMG cells | RIKEN BRC | RCB2868 | |
PBS | Wako | 166-23555 | |
Cell banker | Nippon Zenyaku Kogyo | ZR621 | |
D-MEM | Wako Pure Chem. Ind., Japan | 044-29765 | |
FBS | Cell Culture Biosci., Nichirei Biosci. Inc., Japan | 172012-500ML | |
Trypsin | Thermo Fisher Scientific | 25200056 | |
Penicillin-Streptomycin | Wako Pure Chem. Ind., Japan | 161-23181 |