Summary

Over the past few years, bioprinting has emerged as a compelling field of study. The fusion of bioprinting technology with organ-on-chip platforms has opened novel avenues for advancing in vitro cell culture methodologies. This technical note explores intriguing possibilities arising from the synergistic integration of these two cutting-edge technologies.

 

Introduction

3D printing, or additive manufacturing, fabricates three-dimensional objects by depositing successive layers of material until the intended shape is formed. This technique is opposed to traditional subtractive manufacturing where the material is removed from a sold block to create a shape [1]. 3D printing first starts with the creation of the digital model of the object by computer-aided design software and then slicing the digital model into thin horizontal layers. Then these thin layers are printed sequentially to build the object.

This technique encompasses several advantages [2]:

• Design flexibility: 3D printing makes it possible to fabricate complex designs and geometries that are challenging or even impossible for traditional manufacturing techniques.
• Cost-effectiveness for prototyping: with this technique prototypes can be manufactured quickly and at a fraction of the cost, allowing for a faster iteration and refinement of designs.
• Reduced waste: since 3D printing is an addictive process, the material deposited is needed only, avoiding the waste material subtractive manufacturing has.
• On-demand manufacturing: with quick and cheaper prototyping, the products can be fabricated on-demand, without the need for large inventories or big storage space/costs.
• Complexity without assembly: with 3D printing most designs can be printed in a single whole piece, without the need to assemble multiple parts after printing, reducing the assembly time and labour, as well as weak points that can arise from the union points of the pieces.

 

Bioprinting is described as the controlled deposition of cells and biomaterial. It is encompassed inside the technology of 3D printing and was adapted to the construction of biology models. This technique allows the deposit of different biomaterials and cell lines in the same construct, translating into more complex tissue constructs with human-like features such as tissue functions.

Bioprinting on a microfluidic platform

Bioprinting technology has the potential to streamline microfabrication procedures, potentially tackling the challenges of throughput and reproducibility encountered by conventional organ-on-chip systems [3].

This technology can be used to:

• Develop a microenvironment that mimics the heterogeneity of cells and extracellular components found in nature.

Achieving an extracellular matrix-like heterogeneous composition of biomaterials in microfluidic devices has been a significant challenge. Therefore, consistently printing biomaterial formulations directly onto a microfluidic chip could revolutionize the creation of biomimetic microenvironments. Customizing bioink compositions makes it possible to produce heterogeneous tissue constructs with various cell types and substrates, employing multiple exchangeable printheads with different materials.

• Achieve consistent cell printing and patterning directly within the microfluidic devices.

Cells are typically manually seeded inside a microfluidic chamber, a process that is generally slow. Therefore, using a bioprinter for direct multiple-cell printing and/or patterning would enable high-throughput, high-precision production of consistent tissue constructs with minimal human intervention.

• Generate intricate 3D microstructures.

The versatility of bioprinting modes enables the deposition of biomaterial-cell spheroid-tissue strands into complex, free-form 3D structures on the chip, allowing the creating of cell culture models specifically designed for studying cell-cell and cell-matrix interactions.

• Simulate barriers and interfaces within the vascular system.

Achieving successful vascularization has been a significant challenge in creating functional tissues for in vitro models. Advanced microfluidic-assisted bioprinting techniques allow for the generation of multiscale hydrogel-based flow networks that closely mimic the form and function of real vessels. Bioprinting these vascular networks can enable fluid flow, facilitating nutrient transport, gas exchange, and waste removal.

 

Printing of microfluidic devices

Traditionally, soft lithography, photolithography, and etching techniques have been widely used to fabricate OOC devices, but these methods have significant limitations that hinder development and innovation in microfluidic applications. These limitations include 1) the necessity for numerous repeated processing steps; 2) the inability to create continuously curved structures and complex geometries; 3) long lead times and high labour requirements; 4) issues with reproducibility, dimensional accuracy, and surface quality; and 5) the need for separate cleanroom facilities and skilled users. Consequently, a rapid prototyping technique is required to overcome the limitations of these conventional methods [4].

3D printing of microfluidic devices presents an innovative approach to fabricating complex and precise structures with various applications in biotechnology, medical research, and chemical analysis. This technology allows for the creation of micro-scale channels, chambers, and intricate networks that are essential for manipulating small volumes of fluids in a controlled environment.

Suitable Printing Techniques

Several printing techniques are suitable for fabricating 3D microfluidic devices:

Stereolithography (SLA):

In stereolithography, a specific pattern of UV light or laser is directed over a liquid photopolymer, causing the polymer to cure and form three-dimensional structures. This patterning is achieved using digital micromirror arrays, which allow for the creation of highly complex, flexible, and scalable structures [5]. Unlike laser-assisted bioprinting, stereolithography employs a layer-by-layer process that enhances speed and resolution, achieving less than 25 µm resolutions. This technique supports the use of high-precision polymers such as acrylics and epoxies, although it is limited by the range of materials available and requires intense UV exposure and extensive post-processing times [6].

• Resolution: High, typically around 20-100 µm.
• Advantages: Offers excellent precision and surface finish. It can create complex geometries and is compatible with a range of materials, including biocompatible resins.
• Disadvantages: Limited to photopolymerizable materials, which may have restricted mechanical properties and biocompatibility.

Digital Light Processing (DLP):

Digital light processing is a layer-by-layer technique, similar to SLA but distinguished by the method of light projection onto the photosensitive material using digital light mirrors [7, 8].

• Resolution: Comparable to SLA, with resolutions down to 10 µm.
• Advantages: Faster than SLA as it cures entire layers at once. High precision and good material properties.
• Disadvantages: Similar material limitations as SLA, with potential issues in scaling up productions.

Fused Deposition Modelling (FDM):

Fused deposition modelling works by extruding thermoplastic filament through a heated nozzle, layer by layer, to build the desired structure [9]. The viscoelastic properties of the processed materials are crucial at each stage of the FDM process: predicting the printability of the molten material during extrusion and ensuring a continuous flow through the nozzle; regulating the deposition process of the moulded part and ensuring layer adhesion during the subsequent consolidation phase.

• Resolution: Lower than SLA and DLP, typically around 50-200 µm.
• Advantages: Cost-effective, wide range of materials, including thermoplastics like PLA and ABS. Suitable for rapid prototyping.
• Disadvantages: Lower resolution and surface finish, less suitable for highly detailed or complex microstructures.

Two-Photon Polymerization (2PP):

Two-photon polymerization is a specialized form of stereolithography that enables the creation of three-dimensional structures with resolutions beyond the diffraction limit. This technology is often utilized for fabricating microfluidic devices and replicating physiological microenvironments in vitro due to its high precision, achieving resolutions of less than 100 nm. However, the high resolution of 2PP results in extended fabrication times, making it impractical for constructing larger tissue analogues. Additionally, 2PP is restricted to using photosensitive polymers from the microelectronics industry, which typically have lower biocompatibility⁶.

• Resolution: Extremely high, down to 100 µm.
• Advantages: Can create nanoscale features with exceptional precision, ideal for intricate microfluidic structures.
• Disadvantages: Expensive equipment and slow process, limiting its use for large-scale production.

3D printing offers significant advantages in the field of microfluidics, offering distinct advantages over traditional methods like photolithography and soft lithography. One of these benefits is its design flexibility allowing for the creation of highly complex and customized microfluidic structures that are difficult or impossible to achieve with traditional techniques. Furthermore, 3D printing supports rapid prototyping, enabling quick iteration and faster development cycles, which are important for advancing research and development in microfluidics. Unlike traditional methods, 3D printing can integrate components such as valves, mixers, and sensors directly into the device during the fabrication process, reducing assembly time and potential points of failure. Additionally, the wide range of materials compatible with 3D printing, including biocompatible and transparent options, provides greater versatility to meet specific applications requirements. This makes 3D printing an interesting choice for the efficient and innovative fabrication of microfluidic platforms.

Despite its many advantages, 3D printing for microfluidics has some disadvantages compared to traditional methods. One key issue is resolution limitations; while some 3D printing techniques offer high resolution, others, such as Fused Deposition Modelling, may not meet the precision required for certain microfluidic applications. Additionally, there are material restrictions, as some printing techniques are limited to specific materials that might not possess the necessary mechanical or chemical properties for all applications while being biocompatible. Most of the materials used are not transparent, making cell culture visualization a challenge.

The cost of high-resolution printers and specialized materials can also make this technology less accessible for some researchers or small-scale production efforts. Scalability remains a challenge, with printing time and costs being significant barriers for large-scale production, although ongoing advancements in printing technology are gradually addressing these issues. Lastly, the final product often has a less refined appearance compared to those fabricated with injection molding, which may affect the aesthetic and functional quality of the microfluidic platforms.

 

Microfluidic platforms in bioprinting

The anatomical arrangement of human tissues at the micro, meso, and macro scales to form complex 3D hierarchical structures is essential for the proper function of individual tissues and organs, and consequently, the organism as a whole [10]. However, a significant limitation of current 3D bioprinting technologies is the lack of microscale precision and control over cellular composition or arrangement, particularly when using multiple cell types or achieving single-cell resolution during the printing process [11]. This loss of microscale precision during mesoscale fabrication hampers the organization of different cell types within a tissue assembly, limiting the reproducibility and application of artificial tissues [12]. To create artificial biological tissues that function as effectively as natural ones, it is crucial to position specific cell types in multiscale structures with hierarchical features that replicate the natural microscale tissue environment. Therefore, a multi-material, bottom-up biofabrication approach would be highly advantageous.

To enhance precision at the microscale, microfluidic platforms can be incorporated into biofabrication methods to leverage the capabilities for handling and manipulating cells and biomaterials. By exploiting the geometric constraints of fluids at the microscale within microfluidic devices, where forces like viscosity and surface tension predominate, laminar flow can be utilized to create precise patterns by controlling fluid containing biomolecules, cells, organisms, or chemical agents [11]. For example, diverse fluidic mechanisms such as focusing, concentrating, mixing, or separating can be employed within microfluidic devices to produce various structures like cell-laden droplets, microfibers, or cell sheets.

Moreover, the microfluidic environment featuring multiple inlets offers the potential for enhancing the printing process, thereby improving cell viability, and guiding primary cell differentiation. Additionally, it enables a smooth transition between materials. The microfluidic biofabrication differs from traditional bioprinting methods as the constructs remain internal to the chip rather than being extruded. Nonetheless, they can be seamlessly integrated with existing 3D bioprinting platforms by adapting the nozzle to incorporate microfluidic chips. This integration facilitates multi-material printing with enhanced control over material concentration, positioning, and composition. The application of microfluidic-enhanced 3D bioprinting (MF3D) holds promise for the fabrication of various organoids in a manner that more accurately reflects the structural and functional integrity of natural tissues. For example, to create a 3D multi-material organized tissue construct, cell-laden multi-layer filaments can be used to print vascularized hollow tissues that functional as blood vessels. These are interwoven with cardiac cells, which are printed as droplets within a hydrogel scaffold to form functional cardiac tissue.

 

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