Rapid assembling organ prototypes with controllable cell-laden multi-scale sheets
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A native organ has heterogeneous structures, strength, and cell components. It is a big challenge to fabricate organ prototypes with controllable shapes, strength, and cells. Herein, a hybrid method is developed to fabricate organ prototypes with controlled cell deposition by integrating extrusion-based 3D printing, electrospinning, and 3D bioprinting. Multi-scale sheets were first fabricated by 3D printing and electrospinning; then, all the sheets were assembled into organ prototypes by sol–gel reaction during bioprinting. With this method, macroscale structures fabricated by 3D printing ensure the customized structures and provide mechanical support, nanoscale structures fabricated by electrospinning offer a favorable environment for cell growth, and different types of cells with controllable densities are deposited in accurate locations by bioprinting. The results show that L929 mouse fibroblasts encapsulated in the structures exhibited over 90% survival within 10 days and maintained a high proliferation rate. Furthermore, the cells grew in spherical shapes first and then migrated to the nanoscale fibers showing stretched morphology. Additionally, a branched vascular structure was successfully fabricated using the presented method. Compared with other methods, this strategy offers an easy way to simultaneously realize the shape control, nanofibrous structures, and cell accurate deposition, which will have potential applications in tissue engineering.
KeywordsOrgan prototypes 3D printing Electrospinning 3D bioprinting Multi-scale sheets
Organ prototypes based on scaffolds or cell-laden structures have been widely used in tissue engineering and regenerative medicine, because they could provide carriers for cell attachment and microenvironment similar to the extracellular matrix (ECM) for cell growth [1, 2, 3, 4]. To be close to native organs, an ideal organ prototype must mimic the structure features, cell-favorable environment, and cell distribution of the natural organs. Therefore, the corresponding requirements are put forward for the fabrication method: (1) The method should fabricate user-specific patterned structures for getting complicated tissues with controlled size and shape; (2) the method needs to produce microscale or nanoscale fibers for simulating ECM; (3) the method enables to obtain controlled cell deposition for building complicated tissues with multiple cell types and gradient ECM.
Currently, numerous methods have been reported for the fabrication of scaffold-based organ prototypes [5, 6, 7, 8], among which extrusion-based 3D printing is a widely used method because it can easily fabricate 3D customized structures by using thermoplastic biodegradable polymers, such as polylactic acid (PLA) and polycaprolactone (PCL) [9, 10, 11]. Although the fibers produced by 3D printing were relatively large (> 100 μm) for providing mechanical strength, they are not close to the scale of the extracellular matrix (ECM) or the cells (10–20 μm), thus cannot provide the cell-favorable environment for cell adhesion [12, 13]. Due to its ability to produce nanoscale fibers which is better for cell adhesion, electrospinning technology is widely used to fabricate scaffolds for simulating ECM [14, 15, 16]. However, electrospinning can only fabricate two-dimensional (2D) membranes. Although several strategies have been presented to fabricate 3D patterned electrospun nanofibrous structures [17, 18, 19], they are all limited by the poor mechanical strength. Moreover, it is a challenge for the existing scaffold fabrication methods to realize accurate cell deposition, namely, they fail to obtain controlled distribution of cell density and cell types. Recently, 3D bioprinting has attracted increasing attention in tissue engineering due to its ability to fabricate complicated cell-laden structures with multiple cell types and different cell densities [20, 21, 22]. Nevertheless, the strength of hydrogel is too low to meet the requirement of long-term cell culture and induction. Therefore, here comes a question: could we realize fabrication of organ prototypes with controllable shape, nanofibrous structures, and controllable cell deposition by combining these three techniques?
Here, we presented a novel method to fabricate organ prototypes with controllable cell-laden multi-scale sheets by integrating the merits of extrusion-based 3D printing, electrospinning, and 3D bioprinting. With this method, the macroscale structures could ensure 3D shape and strength support, the nanoscale structures could offer a favorable environment for cell growth, and cells could be deposited in a controlled manner by bioprinting. In this study, we demonstrated the feasibility of this method for the manufacture of multi-scale sheets with controlled cell deposition. Cell viability, cell proliferation, and cell morphology were systematically studied compared to 2D culturing condition to confirm the excellent biocompatibility of the fabricated structures. These studies indicated that this method is superior to other approaches in that it offers an easy way to simultaneously realize the shape control, nanofibrous structures, and cell accurate deposition in fabricating organ prototypes, which may open up new potential applications in tissue engineering.
Materials and methods
In this study, PLA filament (PLA 1.75, Alkht Co., Ltd., Beijing, China) was used to fabricate macroscale structures, and polycaprolactone (PCL, Mw = 80,000, Solvay company) was used to fabricate nanoscale structures. Sodium alginate, gelatin, and calcium chloride (CaCl2) were purchased from Sigma-Aldrich, and alginate/gelatin hydrogel was used to encapsulate cells to realize controlled deposition on multi-scale sheets. Red dye was added to the alginate/gelatin hydrogel for visualization. MEM, ECM, FBS, and PBS were all purchased from Tangpu Biological Technology Co., Ltd., Hangzhou, China.
Fabrication process of organ prototypes with controllable cell-laden multi-scale sheets
Cell culture and cell deposition
Mouse fibroblasts (L929) and human umbilical cord vein endothelial cells (HUVECs, labeled with red fluorescent protein (RFP)) were cultured in MEM and ECM, respectively, which were added with 10% fetal bovine serum, 1% penicillin (100 units/ml), and streptomycin (100 µg/ml). The cells were incubated at 37 °C in 5% CO2 in polystyrene tissue culture flasks. The culture media were changed every other day, and cells were passaged using trypsin–EDTA dissociation every 4 days.
When depositing cells directly on multi-scale sheets (PLA/PCL), the sheets were first immersed in 75% ethanol under UV light for 1 h, washed three times with PBS, and incubated in 24-well plates containing culture media overnight. The cell suspensions were seeded on the sheets (one sheet in each well of 24-well plates, at 2 × 104 cells/sheet in 1 ml MEM). The fibroblasts were incubated at 37 °C in 5% CO2, and the media were replaced every other days.
When depositing cells on multi-scale sheets using bioprinting (PLA/PCL/Hydrogel), alginate/gelatin hydrogel was used to encapsulate cells and CaCl2 was used as cross-linking agent. The preparation process of cell-laden hydrogel solution was described in our previous study  and resulted in a concentration of 2% alginate (w/v), 6% gelatin (w/v), and a cell density of 1 × 106 cells/ml or 2 × 106 cells/ml. Then, cells were deposited controllably on the multi-scale sheets by bioprinting.
Cell viability was analyzed using LIVE/DEAD assay reagents (KeyGEN BioTECH Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. Live and dead cells were stained by calcein AM (green, 6 µM) and propidium iodide (red, 24 µM), respectively, and imaged under a confocal fluorescence microscope (ZEISS LSM780). Then, the cell viability was calculated as the number of green stained cells/total cells × 100% using ImageJ software.
Cell proliferation was analyzed using cell counting kit-8 (CCK-8, Dojindo) according to the manufacturer’s instructions. Different groups of cell-seeding samples were washed three times with PBS. Then, 1450 μl MEM medium and 50 μl CCK-8 were added to each well of 24-well plates and incubated for 3 h. Finally, the solutions were transferred to a 96-well plate (200 μl per well) to read the OD values at a wavelength of 450 nm.
Cell morphology was analyzed by using TRITC phalloidin and DAPI to stain F-actin and nuclei, respectively, according to the manufacturer’s instructions. First, the samples were fixed in 4% paraformaldehyde for 30 min, followed by permeabilized with 0.5% Triton X-100 for 5 min. Then, they were stained with TRITC phalloidin (0.1 μM, YEASEN BioTECH Co., Ltd., Shanghai, China) for 30 min and stained with DAPI (10 μg/ml Solarbio, Beijing, China) for 10 min. The samples were washed three times with PBS before each step. Finally, the cell morphology was imaged under the confocal fluorescence microscope.
Results and discussion
Fabrication and characterization of multi-scale sheets with controlled cell deposition
Biocompatibility analysis of bioprinted L929 mouse fibroblasts
Comparison of cell proliferation and cell morphology in 3D and 2D culture
In order to compare cell growth in 3D and 2D culture within the multi-scale sheets, in this study, cells cultured in the hydrogel-embedded sheets (PLA/PCL/GEL) were acted as 3D culture model, and the cells seeded on the pure multi-scale sheets without hydrogel ((PLA/PCL) and tissue culture plates (TCPS) were regarded as 2D culture model (control groups). The cell proliferation, morphology, and interaction with the fibers were investigated during the culture period.
Fabrication of a multi-scale vascular structure
In this study, a novel method in which organ prototypes with controllable shapes, nanofibrous structures, and cell deposition was fabricated layer by layer by combining the technology of extrusion-based 3D printing, electrospinning, and bioprinting. In this structure, the macroscale structures could offer 3D shape and strength support, the nanoscale structures could offer a favorable environment for cell growth, and cells could be deposited in a controlled manner by 3D bioprinting. Through systematic experiments, we have demonstrated the feasibility of this method for the manufacture of organ prototypes with cell-laden multi-scale sheets and confirmed the excellent compatibility of the hydrogel-laden multi-scale sheets. Additionally, the success of manufacturing a branched vascular structure further confirmed the stability and versatility of this method. We believe that the hybrid fabrication of multi-scale sheets with controlled cell deposition will have wide applications in manufacturing organ prototypes.
This work was sponsored by the National Nature Science Foundation of China (Nos. 51805474, 51622510, U1609207), the Science Fund for Creative Research Groups of National Natural Science Foundation of China (No. 51821093), and China Postdoctoral Science Foundation (No. 2017M621915).
Compliance with ethical standards
Conflict of interest
Q.G, P.Z, R.Z, P.W, J.F and Y.H declare that they have no conflict of interest.
This paper does not contain any studies with human or animal subjects performed by any of the authors.
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