3D PRINTING OF GELS WITH INTEGRATED VASCULAR CHANNELS FOR CELL CULTURE USING A MICROFLUIDIC PRINTHEAD

Rana Attalla1 and P. Ravi Selvaganapathy1,2

1Department of Biomedical Engineering, 2Department of Mechanical Engineering, McMaster University, ON, CANADA  

ABSTRACT This work details the development of a three dimensional (3D) printing system with a modified

microfluidic print-head used for the generation of complex vascular tissue scaffolds. The print-head features an integrated coaxial nozzle that allows the fabrication of hollow, calcium-polymerized alginate tubes that can be easily patterned using 3D-bioprinting techniques. This microfluidic design allows the incorporation of a wide range of scaffold materials as well as biological constituents such as cells, growth factors, and ECM material. With this setup, gel constructs with embedded arrays of hollow channels can be created and used as a potential substitute for blood vessel networks.

KEYWORDS: 3D Printing, Biofabrication, Tissue Engineering, Coaxial Flow, Hollow Fibers

INTRODUCTION Classical tissue engineering methods involve fabrication of scaffolds made of either natural or

synthetic materials and cells of interests seeded on to them. These methods have been available for years, being used to restore and maintain the normal function of damaged tissues and organs. More recently, focuses have shifted towards the additive layer-by-layer robotic biofabrication processes used to construct 3D functional living macrotissues [1,2,3]. This cutting edge technology provides advantages over classical tissue engineering, including high precision of anatomic geometries as well as advanced control of cell seeding and alignment. However, one particular area that has proved to be technically challenging in both these approaches is the integration of vascular networks in the scaffolds. Some attempts have been made using microfluidic chips [4] as well as scaffold-free approaches based on tissue spheroids [5].

Here, we describe a method for fabrication of gel layers with interspaced hollow tubes directly on dry substrates in precise geometries using a custom built 3D printer with a microfluidic nozzle. With this system, we are able to construct multi-layered structures with precise control of tube position, spacing, and diameter. These factors can be simultaneously manipulated during printing in order to create a single gel structure with variable architecture. The hollow tubes are immediately perfusable and could serve as vascular networks for tissue engineering applications. The interiors of scaffold-engineered tissue grafts are usually associated with poor nutrient diffusion. Perfusion of these embedded channels allows the required nutrients to reach cells in the bulk region of the construct, thus increasing cell viability.

METHODS AND MATERIALS An open-source printer (RepRapPro Mendel) that included an x,y,z positioner and a heated printbed

(Fig. 1a) was modified with a custom built microfluidic printhead for coaxial hollow fiber extrusion (Fig. 1b). Standard 3D CAD (STL) files were converted to printing instructions through an open source g-code compiler software (Slic3r). Custom g-code files for specific line-by-line patterns were translated into movement commands using an open-source interpreter (Printrun).

The microfluidic printhead consists of an L-shaped cylindrical channel (2mm) with an embedded needle (700um). A mold for this device is created using 3D printing of acrylic plastic, which allows curved surfaces to be formed with ease. Polydimethylsiloxane (PDMS) pre-polymer was cast and two elastomeric replicas were bonded together using oxygen plasma with the needle aligned along the center. This produces a concentric flow of CaCl2 solution (100mM) surrounded by an annulus of sodium alginate (2% w/w). This concentric flow is extruded from the printhead as it is moved using a XYZ positioner leading to a patterned deposition on to a dry printbed.

978-0-9798064-7-6/µTAS 2014/$20©14CBMS-0001 479 18th International Conference on MiniaturizedSystems for Chemistry and Life Sciences

October 26-30, 2014, San Antonio, Texas, USA

 (a)                                (b)  

Fig. 1: (a) Modified 3D RepRap printer. (b) Coaxial extrusion nozzle schematic: 700um needle, 2mm L-shaped channel.

Ionic crosslinking is one of the most common gelling methods used for sodium alginate, particularly using Ca2+ from calcium chloride solution as the crosslinking agent. In our case, diffusion of calcium ions from the inner stream into the surrounding alginate causes gelation to occur in the annulus and results in the formation of a hollow tube. The concentration of calcium and the length of time allowed for diffusion to occur govern the crosslinking phenomenon. This means that the length of the printhead’s outlet channel and the flow rates used are both major factors affecting the level of diffusion, and therefore have implications on the dimensions of the resulting tubes. Ionically crosslinked gels can be dissolved by the release of crosslinker ions in exchange for physiologically available monovalent cations such as sodium, making them a viable choice for biological applications.

RESULTS AND DISCUSSION Inner and outer tube diameters can be changed by manipulation of respective flow rates (Fig. 2a-c).

An increase in inner flow rate results in an increase of inner diameter, while an increase in outer flow rate results in an increase of outer diameter. Similarly, a decrease in each of the flow rates causes a decrease in their respective diameters as well. Flow rates were tested between 1 to 20 mL/min.

       (a)       (b)      (c)  

Fig. 2: (a-c) Hollow tubes extruded into CaCl2 bath. Inner flow is constant at 10mL/min, while outer flow is 20, 10, and 5mL/min respectively. Tubes extruded into a bath cannot be precisely positioned on a

substrate. Also, extrusion into a bath solidifies the outer layer of the gel instantaneously and does not allow merging of adjacent tubes. Scale bars, 1mm.

Upon extrusion onto a dry substrate in a pattern depicted in Fig. 3a,b, the inner side of the annular alginate layer was crosslinked while the uncrosslinked gel on the outside spreads laterally resulting in fusion between one parallel tube to the next thereby forming a continuous solid gel layer with embedded, hollow channels as shown in Fig. 3c. Fig. 3d shows the photograph of the printed gel layers, illustrating the alignment of the adjacent hollow channels. Since the individual channels are extruded in a continuous fashion they are connected at the ends (Fig. 3e), which makes perfusion of the entire 3D gel layer straightforward. Multi-layer hydrogel structures can also be created with a connection from channels in one layer to the next, provided that printing is continuous (Fig. 3f). Fig. 3g,h,i show perfusion of the printed gel structures with solution containing fluorescent nanoparticles that indicate the structural integrity of the single and multilayer printed structures.

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                                                                                                                                                                          (a)                  (b)                            (c)  

                                                     (d)                  (e)                            (f  )  

                                                         (g)                  (h)                                (i)  

 

Fig. 3: Formation of a continuous layer of gel with embedded hollow channels due to extrusion onto a dry substrate (a) Single layer pattern used for extrusion. b) Dual layer pattern used for extrusion. (c)

Cross-section of continuous gel layer formed. Hollow tube can be seen embedded in gel layer. (d) Parallel tubes printed continuously using the pattern in (a)show good alignment (e) Bent connections between adjacent tubes when continuously printed. (f) Multi layer printing using pattern in (b). (g-h)

FITC- nanoparticle flow through tubes shown in d-f respectively. Scale bars, 1mm.

Hollow gel structures seeded with Escherichia Coli K12 cells were also printed to illustrate bacterial cell seeding and culture in these gel constructs (Fig. 4).

Fig. 4: Gel layer with embedded hollow tubes, seeded with GFP Escherichia Coli-K12. Scale bar, 2mm.

CONCLUSIONS The method demonstrated in this paper allows simple and fast 3D printing of hollow gel tubes

seeded with cells that can be immediately perfused. This printing process illustrates the promising potential for biofabrication of vascularized, self-sustaining tissue constructs using a biologically compatible hydrogel. Printing of gel structures seeded with human umbilical vein endothelial cells (HUVEC) are ongoing.

REFERENCES [1] Xu, T., et al. Hybrid printing of mechanically and biologically improved constructs for cartilage

tissue engineering applications, Biofabrication, 2013. 5(1):015005. [2] Hockaday LA, et al. Rapid 3D printing of anatomically accurate and mechanically heterogeneous

aortic valve hydrogel scaffolds, Biofabrication, 2012. 4(3):035005. [3] Mannoor MS, et al. A 3D Printed Bionic Ear, Nano letters, 2013. 13(6):2634-2639. [4] Kim S, et al. Engineering of functional, perfusable 3D microvascular networks on a chip, Lab Chip,

2013. 13(8): p.1489-1500. [5] Mironov V, et al. Tissue spheroids as building blocks. Biomaterials, 2009. 30(12): p.2164-2174.

CONTACT *Rana Attalla, tel:905-525-9140 ext. 21388; attallr@mcmaster.ca

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