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Confined bioprinting and culture in inflatable bioreactor for the sterile bioproduction of tissues and organs – Scientific Reports

Conception and production of FUGU prototypes

The FUGU system is an intricate assembly of several components, each manufactured independently and showcased in Fig. 1-B. Key elements include, and will be discussed further in the following section:

  1. 1-

    Flexible Silicone Elastomeric Membrane: A 1 shore A Liquid Silicone Rubber (LSR) silicone elastomeric moulded membrane.

  2. 2-

    Needle Insertion Pore: This is 3D printed using a 70 shore A LSR. The dimensions of the part are 15 mm in height, 1 mm in outer diameter, and 7 mm in inner diameter.

  3. 3-

    Solid Bioprinting Platform, Membrane Holder, and Clamps: Initially, these parts were machined from stainless steel 316/316L. For disposability and compliance with single-use strategy in the bioprocess field11, we explored their production through 3D printing using medical-grade polycarbonate (MAKROLON®). The building platform surface has to be perfectly flat and smooth, to be compatible with initial bioprinter Z-axis calibration. The dimensions of the platform are 19 mm in height and 40 mm in outer diameter. The dimensions of the membrane holder are 5 mm in height, 42 mm in outer diameter, and 32 mm in inner diameter. The dimensions of the clamps are 11 mm in length, 4.5 mm in width, and 22.5 mm in height for the outer dimensions, and 5 mm in length, 4.5 mm in width, and 16.5 mm in height for the inner dimensions.

  4. 4-

    Fluidic Operations Ports: To aid fluid operations within the system, valved connectors were integrated directly into the printing bed.

The assembled FUGU device underwent steam sterilization. All components (both flexible and solid) and materials (silicone, stainless steel, MAKROLON® polycarbonate) of the FUGU device were able to withstand steam sterilization without compromising the geometry of the parts. However, the durability of the silicone membrane and MAKROLON® polycarbonate regarding this sterilization process was not assessed, as they are intended for single-use.

The inflatable silicone elastomeric membrane and its insertion pore are the corner stone of the FUGU system. These components ensure system compliance during 3D bioprinting and enable sterile needle insertion. Figure 2A demonstrates how homogeneous silicone membranes of varying thicknesses (1.5 and 3 mm) distort under different inflation pressures. Notably, thinner membranes require less pressure for comparable distortion, offering a broad range of internal volumes (from 0 to 460 mL, Fig. 2-A3). This variability allows for unprecedented adaptability in the internal size of the FUGU during bioprinting and culture. Of note, adding an insertion pore to the membrane had a similar effect to increasing the membrane thickness, resulting in a reduction in inflation capability.

Figure 2
figure 2

FUGU prototyping and characterization. (A) Characterisation of the inflation of the FUGU silicone membrane. A1/ 3D scans of a 1.5 mm thick FUGU membrane while it was being inflated using increasing pressure. A2/ Z-distortion of 3 FUGU silicone membranes of varying thicknesses according to the applied inflation pressure. A3/ Calculated (using 3D scan) internal volume of the FUGU according to the applied inflation pressure (data extracted from 3D scans). (B) Design and characterization of the FUGU insertion pore. B1/ 3D model cross-section of the pore. B2/Cross-section view of the 3D printed insertion pore. B3/ Bioprinting needle (1.6 cm long, 800 µm diameter) inserted into the insertion pore. B4/ Puncture test of the FUGU insertion pore. The arrows represent the insertion and removal of the needle in the case of the puncture FUGU. (C) Building plate production using 3D printed MAKROLON® polycarbonate on the FreeFormer 300-3X technology. C1/ Scanning electron microscopy images of MAKROLON®test parts cross-section, before and after printing optimisation. C2/ Mechanical characterization of the 3D printed MAKROLON® parts and comparison with the moulded reference. C3/ Images of the final FUGU MAKROLON® parts.

The needle insertion pore itself serves three main functions: pierceability, sealing after puncture, and barrier to microbiological contamination. Its design, detailed in Fig. 2-B1, incorporates a 1 cm diameter tube with 2 mm thick walls, placed between two 2 mm thick diaphragms. The present configuration, produced using a specific 3D printable LSR silicone formulation12, ensures its functionality and integrity. Its 70 shore A composition ensure its geometrical stability while the FUGU membrane inflate (Fig. 2-B3).

The sealing property of the insertion pore was assessed by monitoring the FUGU’s deflation rate before and after inserting an 800 µm diameter needle.

Regardless of whether the pore was pierced or not, an average deflation rate value of 0.4 cm3/hour was observed (Fig. 2-B4). This observation sheds light on two important behaviours: the punctured pore is efficiently sealed after removal of the needle, and the silicone membrane possesses a clear gas permeation. This last point is a key attribute for maintaining optimal oxygen and pH levels in a CO2 incubator during tissue cultivation. Indeed, pH is commonly maintained thanks to a buffering couple of dissolved CO2/bicarbonate. This permeability was evaluated using gas diffusion measurements through a 2 mm silicone membrane. The 1 shore A LSR silicone elastomeric membrane was found to have high permeability, with 177 ± 4 Barrer, 370 ± 2 Barrer and 1747 ± 20 Barrer, for N2, O2 and CO2, respectively.

Lastly, to achieve a fully disposable version of the FUGU system, all solid components- were 3D printed from MAKROLON®, a medical-grade polycarbonate, using the Freeformer technology. The optimized process yielded parts with mechanical properties closely matching those of injection-moulded MAKROLON®, as shown in Fig. 2-C1 and C2. Dense, homogeneous and watertight polycarbonate parts were obtained (Fig. 2-C1) with mechanical properties corresponding to 85% and 93% of the injected MAKROLON® Young’s modulus and stress at break, respectively (Fig. 2-C2).

Evaluation of FUGU capacity as an advanced versatile biofabrication platform

The aim of the advanced biofabrication platform was to allow for a wide range of geometries and sizes to be bioprinted in a sterile and confined environment. Thus, to describe and establish the boundaries of the bioprintable volume within the FUGU system, a detailed image analysis was conducted. Such analysis involved observing the movement of a standard micro-extrusion bioprinting needle within the FUGU system. To do so, a 1.6 cm long 800 µm diameter needle was inserted into the FUGU at various inflation volumes. Three different FUGU volumes (85, 140, and 180 cm3) were examined, as shown in Fig. 3-A. The maximum bioprintable volume was determined by identifying the largest cylinder that could fit within the needle’s range of motion without touching the FUGU’s silicone walls. The compliance of the FUGU system at various volume, during the movement, was easily noticed. This flexibility is crucial for two reasons: (1) it allows for the movement of the printing needle within the system, and (2) it helps maintain the internal volume during the bioprinting process.

Figure 3
figure 3

Range of motion and biopriting volumes in the FUGU device. (A) Deformation profiles extracted from live bioprinting pictures. A1/ Within a 180 cm3 inner volume FUGU. A2/ Within a 140 cm3 inner volume FUGU. A3/ Within an 85 cm3 inner volume FUGU. (B) Images of the live bioprinting within the FUGU. B1/ Within an 85 cm3 inner volume FUGU. B2/ Within a 140 cm3 inner volume FUGU. B3/ Within a 180 cm3 inner volume FUGU.

Through calculations, it was determined that the maximum bioprintable volume is 75 cm3 (forming a cylinder 4.5 cm in diameter and 4.7 cm high) when the FUGU is at its largest inflation (180 cm3). This volume represents 42% of the total FUGU volume. Interestingly, this efficiency ratio improves to 53% when using a 140 cm3 FUGU. These calculated bioprinted object volumes were not only theoretical but also practically validated through bioprinting experiments, as demonstrated in Fig. 3-B.

This analysis highlights the FUGU system’s potential in bioprinting applications, highlighting its ability to adapt to different sizes and shapes while maintaining high efficiency and effectiveness in the bioprinting process.

In order to evaluate the FUGU printing system versatility, 3 challenging shapes were printed using the DIY bioprinter: a 33 cm3 hemisphere (25% infill), a 4.5 cm long human ear and a 14 mm high vascular branching (Fig. 4). As can be seen, in all cases the compliance of the FUGU enabled the printing of the complex shapes without noticeable distortion. Videos of the printing can be found in Supplementary Video 1, 2 and 3.

Figure 4
figure 4

Versatility of the bioprinting in the FUGU device. (A) Bioprinting 33 cm3 25% porosity hemisphere. A1/ Printing G-code visualisation. A2/ The bioprint within the FUGU. A3/ The bioprint once the FUGU membrane removed. (B) Bioprinting of a human ear shape. B1/ Printing G-code visualisation. A2/ The bioprint within the FUGU. A3/ The bioprint once the FUGU membrane removed. (C) Bioprinting of a 14 mm high vascular branching. C1/ Printing G-code visualisation. C2/ The bioprint within the FUGU. C3/ The bioprint once the FUGU membrane removed.

To ensure compatibility across multiple bioprinting platforms, the FUGU system was designed to be compatible with a wide range of extrusion-based bioprinters. A crucial step in demonstrating this universality involved interfacing the FUGU with different bioprinters configurations. Indeed, each brands of bioprinter has its dedicated operating systems, which includes considerations such as motor torque and inner available space. To cover a large panel, we have chosen to work with the two most prevalent types of 3D bioprinter architectures: a Cartesian system13 and a 6-axis system14.

Identical FUGU systems in material composition, conception and size (85 cm3), were tested on these two types of bioprinters. The first was a DIY low-tech bioprinter (TOBECA®, France), and the second a sophisticated 6-axis robotic arm (Advanced Solutions Lifescience®, USA). In practice, the FUGU systems integrated seamlessly with both types of bioprinting setups (Fig. 5). Fundamental operations like needle insertion and deformation of the silicone membrane were executed smoothly, even with the relatively low torque (0.31 N.m) of the NEMA17 motor in the DIY bioprinter.

Figure 5
figure 5

FUGU integration with micro-extrusion bioprinters. (A) Integration of the FUGU within a Do-It-Yourself (DIY) bioprinter from TOBECA®. The integration was performed using a Puredyne® cavity pump micro-extrusion system. (B) Integration of the FUGU with a 6-axis robotic bioprinter BioassemblyBot® (Advanced Solution LifeScience). The integration was performed using a pneumatic micro-extrusion system.

The versatility of the FUGU was also challenged with the two micro-extrusion systems available in the field, namely pneumatic and cavity pumps. Both systems were found to seamlessly interface with the FUGU (Fig. 4). This compatibility across various bioprinting platforms and micro-extrusion systems highlights the FUGU system’s adaptability and potential for widespread application in the field of extrusion-based 3D bioprinting. Its design not only addresses the need for a universal bioprinting solution but also ensures ease of integration with existing technologies, paving the way for more innovative and flexible bioprinting approaches.

Challenging the FUGU confinement

The efficiency of the FUGU system’s confinement was tested through sterility assays. The methodology for these tests is outlined in Fig. 6-A, involving the controlled introduction of bacterial charges. In addition to the introduction of bacteria, the FUGU was also challenged by performing the experiment in a grade D environment (NF EN 17,141) using the TOBECA® DIY bioprinter. The FUGU was filled with a bacterial culture medium, and the insertion pore was swabbed with ethanol. Then, bioprinting was simulated by inserting the needle through the insertion pore and immersing it in the bacterial culture medium. Bioprinting simulation lasted for 5 min, followed by complete needle removal.

Figure 6
figure 6

Evaluation of the sterile confinement within the FUGU. (A) Schematic representation of the logical steps involved in challenging confinement. (B) Numeration of the bacteria contamination through the needle insertion and bioprinting process.

Two different scenarios were assessed. The best case scenario was performing the experiment with an autoclaved 800 µm needle ensuring its complete decontamination before use. The worst case scenario was a deliberate and severe contamination of the needle with 30 × 109 E. coli prior to its insertion through the pore.

Following bioprinting simulation, the culture medium inside the FUGU (Recovery Bottle RB) and remaining in the feeding Bottle (FB, acting as internal control) were incubated at 37 °C for 24 h. Culture media were plated on LB-agar solid medium for colony counting and incubated at 37 °C for 24 h. The results of grown bacteria enumeration are detailed in Fig. 6-B. Each aforementioned scenario was replicated three times.

In the best case scenario, despite the challenging conditions (no laminar flow, grade D environment, non-decontaminated DIY bioprinter), none of the three experiments resulted in contamination. This outcome demonstrates that even under strenuous conditions, simply swabbing the insertion pore with ethanol seems adequate to maintain sterility within the FUGU.

In the worst case scenario, two out of three experiments resulted in contamination within the FUGU. This indicates that even with a significantly high bioburden on the bioprinting needle, no detectable bacteria were transferred through the FUGU insertion pore in one case. This effect might be attributed to the elastomeric properties of the insertion pore, which could potentially clean the needle to some extent during its passage through the top and bottom diaphragms.

Overall, these results underscore the FUGU system’s capability in maintaining sterility, highlighting its potential for safe and effective use in various bioprinting environments even under less-than-ideal conditions.

Bioprinting and culturing within the FUGU

The FUGU system’s capability for bioprinting and culturing large tissues was demonstrated through bioprinting a human tissue model and culturing for 16 Days. This model, measuring 14.5 cm3 with 30% porosity, comprised colorectal cancer cells (HT29) and cancer-associated fibroblasts (CAF). The bioprinted tissue was cultured statically within the FUGU system in a 37 °C/5% CO2 environment. Notably, during this period, no intervention such as culture medium renewal, or active gas perfusion were performed. The initial addition of 50 ml of culture medium to the 14.5 cm3 tissue containing 15.5 million cells was sufficient for the entire culture period. Furthermore, the gas exchange capabilities of the silicone membrane (i.e. the permeability for O2 and CO2 as described above) might have been sufficient to maintain appropriate pH and oxygen levels inside the FUGU.

Tissue production with the FUGU system (Fig. 7) begins with the bioprinting of a cell-laden hydrogel composed of gelatine, alginate, and fibrinogen (Fig. 7-A), followed by the consolidation of this hydrogel using calcium, thrombin, and transglutaminase (Fig. 7-B). The process then enters the culture phase.

Figure 7
figure 7

Bioprinting and culturing human tumour model within the FUGU. (A) Image of the FUGU during the bioprinting step. (B) Image of the FUGU during the consolidation step. (C) Images of the FUGU during 16 days of culture. D16-C corresponds to a control experiment without cells in the bioprinted object. (D) and (E) Contrast phase images of the tumour model at day 16. (F) and (G) Hematoxylin and eosin (H&E) stained histological sections of the tumour model at day 16.

The longitudinal monitoring of the culture, as depicted in Fig. 7-C, reveals significant changes in both the medium and tissue colour of cell-laden hydrogels over the 16-day maturation period, whereas no such changes were observed without cells. The tissue appeared to shrink and densify, indicative of cell growth and tissue remodelling, as suggested by previous studies15. The alteration in the colour of the culture medium further supported the hypothesis that the bioprinted tissue was actively producing pH-altering metabolites like lactate16.

Optical microscopy observations, shown in Figs. 7D,E, confirmed that after 16 days of confined culture, the bioprinted hydrogel was densely populated with HT29 spheroids17,18. These spheroids and the CAF cells were distinctly identified using H&E histology19, as demonstrated in Figs. 7F,G.

This experiment successfully demonstrates the FUGU system’s effectiveness in facilitating the bioprinting and culturing of large-scale tissues under confined conditions, thus highlighting its potential for tissue engineering and regenerative medicine applications.