Brief communication Cryogenic 3 D printing for tissue engineering

We describe a new cryogenic 3D printing technology for freezing hydrogels, with a potential impact to tissue engineering. We show that complex frozen hydrogel structures can be generated when the 3D object is printed immersed in a liquid coolant (liquid nitrogen), whose upper surface is maintained at the same level as the highest deposited layer of the object. This novel approach ensures that the process of freezing is controlled precisely, and that already printed frozen layers remain at a constant temperature. We describe the device and present results which illustrate the potential of the new technology. © 2015 Published by Elsevier Inc. Tissue engineering is a specialized field of biomedical engineering established to address failing human organs and tissues, by replacing them [2]. Recently, additional applications of tissue engineering have emerged, as replacement for animal use in research, in the form of micro-organs or organs on a chip [3]. A key element in tissue engineering is the manufacture of a tissue scaffold, an extracellular matrix [7]. Various methods are used for generating a tissue scaffold [1]. Freezing of a hydrogel, is the tissue scaffold engineeringmethod relevant to this work [5,10]. Herewe describe a new freezing technology for generating tissue scaffolds using 3D additive methods. 3D additive methods are of increasing interest in tissue engineering in general, and scaffold design in particular [4,6,8]. 3D printing produces a three dimensional object through the addition of layers. There are a variety of manufacturing methods that can be classified as 3D printing. The additive method of manufacturing, layer over layer, is a central element to all of them. One conventional 3D printing technology employs a printer head that delivers the material to be printed, in a molten form, at a controlled rate and temperature. The head has the ability to move in the XYplane and the printing table canmove on the z-axis, under computer control, allowing the manufacturing of complex shapes. The molten material is deposited on the printing table where it solidifies; the process continues until a layer is completed, then the printing table moves downwards and another layer is deposited on the previous. In conventional 3-D printing the material is a plastic and the printing process usually occurs in open air and at room temperature. The phase transition temperature of the molten plastic is higher than the room temperature. This study describes a new addition to 3D printing that, while simple to implement, facilitates 3D printing with freezing for tissue engineering. We found that the outcome of a freezing process during 3D printing can be controlled by immersing the 3D printed object in a liquid with a lower temperature than the phase transition temperature of the printed material. As consecutive layers are printed, the level of the immersion liquid should be continuously raised to be flush with the highest printed layer i.e. the dispensing printing head. Fig. 1, is a schematic showing the key elements of this 3-D printing method. A central element is the printing head that can dispense the printing fluid at a controlled rate, through a syringe. The printing head is attached to a carrier that can move in the xey plane. Both the xey motion and the motion of the syringe plunger are computer controlled. Another element is the printing surface. The printing surface has a computer controlled z-direction motion. A container for the immersion fluid and the printed object rests on the printing surface. The immersion fluid temperature should be controlled and lower than the freezing temperature. The immersion fluid level changes during the printing process to continuously match the top of the last printed layer. A valve is used to meter the addition of the immersion liquid, maintaining the desired level. To demonstrate the technology, we have modified a conventional 3D printer (FlashForge, Creator Model, Los Angeles, CA USA). The original printer head that melted and dispersed a plastic was replaced with a new printer head assembly. A photograph of the * Corresponding author. E-mail address: rubinsky@berkeley.edu (B. Rubinsky).