Extrusion vs. Laser-Assisted Bioprinting: Which Suits Multi-Layered Organoid Fabrication?

Introduction

Miniature, simplified versions of organs produced by growing stem cells in the lab are known as organoids, and have become valuable in modeling disease, drug screening and regenerative medicine. They form self-organized structures with qualities of three dimensions, multilayered structures, particularly advantageous to investigation of the architecture and functionality of tissues. Nevertheless, bioprinting technologies that can deliver complex, multilayered geometry and cell-type additions with spatial control, cell viability and scalability will be important in the fabrication of organoids with such structures.

Organoid fabrication through two leading technologies extrusion-based bioprinting and laser-assisted bioprinting has been largely investigated. Each has its own distinctive plus and minus regarding the resolution, compatibility with different materials, and the complexity of a tissue. In this article we compare the performance of both methods in assembling functional multi-layered organoids. Interrogating their results on how best to resolve, cell viability and to scale, we wish to advise researchers on the best available bioprinters to use in their advanced tissue modeling requirements.

Understanding Multi-Layered Organoid Fabrication

Multilayered organ cavity construction has to do with the spatial distribution of various cell enough to form stratified building patterns. This is necessary towards simulating the biological structure of the tissues like brain cortex, intestinal lining, kidney nephrons and cardiac muscle. The cells within each layer might necessitate individual microenvironment, stiffness of the matrix and signaling signature.

The production of such structures is a success that is subject to three key factors:

• High Resolution: Layer boundaries between the various tissue layers by exact placement of the cell.
• High Cell Viability: Maintenance of cell functioning in the printing process and its post-printing period.
• Scalability and Throughput: Capability of creating many constructs either in experimentation or in treatment.

These parameters are the foundations of our comparative study of extrusion, compared to laser-assisted bioprinting.

Overview of Extrusion Based Bioprinting

In extrusion-based bioprinting, syringe-extrusion (i.e. continuous deposition through a nozzle where the pressure is pneumatic, piston, or screw-driven). Bioinks tend to be either hydrogels loaded with cells in order to be able to create stable filaments when extruded. This has increased the popularity of this method because it is simple and cheap and works with many materials.

Strengths of Extrusion Bioprinting

• Material Versatility: Extrusion accommodates a wide variety of viscosity of the hydrogel and this creates an opportunity to use bioinks including alginate, gelatin-methacrylate (GelMA, collagen, and fibrin).
• Cell Density: It is suitable to produce bulk tissues since they can incorporate high cell densities.
• Layering Capability: Basic three-dimensional structuring is achieved due to the possibility to layer substances.

The extrusion systems can also be modular and scalable and hence can be used in development of the tissue model at an early stage or prototyping.

Limitations in Organoid Applications

In spite of these benefits, extrusion has a number of disadvantages applied to the process of multilayered organoid construction:

• Low Resolution: The resolution is normally constrained by the nozzle diameter and ink rheology causing the overlapping of boundaries of adjacent layers of cells.
• Shear Stress: Mechanical stress applies to cells during extrusion, and is known to diminish cell viability, particularly at the delicate cell type including induced pluripotent stem cells (iPSCs) or neural progenitors.
• Limited Complexity: Complex geometries or close interfaces between cell type are challenging since the extruded bioinks tend to spread and merge.

Extrusion is perhaps adequate with more simple or large constructs, although it is not ideal when reproducing high fidelity organoids.

Overview of Laser Assisted Bioprinting

Laser-assisted bioprinting (LAB) The method is nozzle-free, and involves focus laser pulses to move the bioink droplets on a donor slide to a receiving substrate. It follows the laser-induced forward transfer (LIFT) mechanism, projecting microdroplets of character laden material in an extremely controlled way.

Strengths of Laser Assisted Bioprinting

• Hight Resolution: Droplet size and location can be adjusted on the micrometer scale and so it is suited to patterning complex tissues.
• No Shear Stress: Mechanical pressure is avoided in deposition preserving delicate cells and enhancing post-printing cell viability.
• Layer Precision: enables for the précised motion positioning of distinct cell types in a densely packed multilayered configuration.

 LAB is a precise technique and this quality has presented a unique opportunity to applications of organoids with a complex requirement of exact location of spots with little or no interference with cell physiology.

Limitations in Practice

Even laser-assisted bioprinting is not going without any challenges:

• Material Constraints: Needs low viscosity bioinks that can be easily converted to form droplets so it cannot be applied to structural or highly viscous hydrogels.
• Limited Throughput: Large constructions or batch production takes longer due to retardation in printing speed as compared to extrusion.
• Cost and Complexity: The technology is costly and technically rigorous and involves laser calibration and greatly specialized equipment.

Nevertheless, these concerns do not outweigh the fact that LAB is one of the few methods that can produce a print using a single cell accuracy.

Comparing Resolution in Multi Layered Organoid Fabrication

Resolution is the capability of the system to place the bioink on the desired spot and avoid diffusion and mixing of the nearby areas.

Commonly found resolutions on printing extrusion-based are 100 to 300 micrometers, by way of nozzle size, ink viscosity and printing speeds. This would be suitable to make bulk tissue or simple geometrical shapes but not suited to make organoids that need fine stratification of layers. Printed types of cells present in close vicinity tend to mix complicating the boundary between the tissue regions.

Laser-assisted bioprinting, in its turn, shows even greater resolution (10-50 micrometer range). It enables the cells and biomaterials to be positioned accurately into specific areas to enable the successful mimicking of multi-layered structure organoids such as the intestinal crypt-villus units or the brain cortical layers. This level of resolution is required where organoid development, or functionality are influenced by a position of identity.

Conclusion on Resolution: The use of laser-assisted bioprinting offers much superior resolution, which allows establishing a cell position in multi-layered organoid structures in a much better way.

Evaluating Cell Viability

Cell viability in printing and post-printing is the key success variable during fabrication of organoids, particularly cells that are sensitive during the process of self-assembly and differentiation.

Extrusion causes significant mechanical stress to the cells since the force must be used to extrude viscous bioinks. Strains might break the membranes, cause apoptosis or disrupt the long-time viability of cells. In optimal conditions, cell survival rates might be satisfactory (70 to 85 percent), yet too sensitive cell lines can be damaged.

LAB on the other hand shuns mechanical pressure. The movement of cells is achieved using laser whose energy is adjusted finely in order to prevent thermal or photochemical injury. These studies have observed that cell viability after printing rises to more than 90 percent even when the cells were in the form of pluripotent stem cells or the neurons. The great accuracy and sensitive manipulation promotes a healthier cellular state, which further promotes organoid growth and development of functional maturation.

Conclusion on Viability: Use of lasers in bioprinting is better than extrusion in maintaining cell viability, particularly in gentle or pluripotent cells.

Assessing Scalability and Throughput

To scale large batches of organoids to screen drugs, to treat individuals on a personalized basis, or to treat those who have injured tissues using regenerative medicine, scaling is needed.

Bio printing through extrusion is very scalable. It can be used in parallel with multi-nozzle systems, with automation platforms and robot arms and can be used to produce dozens through hundreds of constructs in parallel. The refill time and the labor associated with it is also minimized by the ability to fill the syringe with a lot of bioink.

LASER assisted systems are more restricted in this respect. When using droplets, the transfer of each has to be done separately thus taking longer and more manual. Although modern systems have enhanced their speed by automating the process, extrusion is still the quickest means of bulk productions.

Conclusion on Scalability: Bioprinting using extrusion would be where large scale or large volume of organoids can be produced.

Application Scenarios

When to Use Extrusion Bioprinting

Extrusion bioprinting is at most standard for:

• Creating an easy or bigger volume tissue models.
• Platforms where by the resolution of quality is not necessary.
• High-throughput screening which is quantity over precision.
• Integrating cells to high-viscosity scaffolds to provide structures.

Examples of applications are cartilage patches, liver tissue slabs or dermal substitutes where layering is relatively easy.

When to Use Laser Assisted Bioprinting

Laser auto-assisted bioprinting passes in:

• Recreating fine tissue structure having numerous layers.
• Making use of fragile or unique cell populations
• The creation of organoids that will need spatial control, i.e. brain, eye, or kidney models.
• Early developmental biology in which the micro-environmental signals are spatially context dependent.

A vivid example is the process of developing cortical organoids having a laminar structure or renal tubules with epithelial cells facing the lumen, which is more optimally conducted with the LAB.

Final Comparison Summary

Conclusion

Extrusion and laser-assisted bio-printing are distinct in the depth or strengths and weakness they present in multi-layered organoid production. Extrusion bioprinting has long been a proverbial workhorse of many labs, and gave simplicity, speed, and a scalable design. It is appropriate in the general tissue model and bulk production activities. Its moderate resolving power and cell stress induced by shear conditions however, make it less suitable to high-sensitivity or sensitive applications.

Laser-assisted bioprinting is more complicated and expensive, but it is exceptional with providing a high resolution, high cell survival rate, and direct architecture control on layered tissue. Such properties are essential in the fabrication of complex organoids in which every cell type needs to be at a particular location to perform its duty.

After all, the most effective method is the one that relates to the peculiarities of the research or therapeutic goal. Laser-assisted bioprinting is preferred over the others in case of high-resolution, layer-specific organoids which closely resemble in vivo tissue structure. Extrusion-based printing is good and manageable to use in larger scale, easy to print tissue constructs or in high-throughput research.