Development of FRESH

Explains the path in developing FRESH / embedded printing.

Luckily for me, for my Masters research, I was given a broken Fab@Home 2 and told to print gels. There was no way to print gels outside of direct ink writing (DIW), and most DIW results compared poorly to FDM in 2011. Consumer 3D printers were just becoming popular due to RepRap, MakerBot, Ultimaker, et al. making headlines and driving the maker movement. Since no relevant fluid for printing tissues would stack in layers in open air without modification, I looked into creating a medium that could support the fluid or enhance the rate of its gelation. It became obvious that I needed a yield-stress fluid, so I looked into hydrophilic gel slurries and ways for making them, realizing there were many different material choices - agar, clays, polyacrylic acid, alginate, gelatin, polyacrylamide, gellan, even structured fluids like liquid crystals. Choosing which gel was figuring out which was sustainable, biocompatible, effective, and harmless. I chose gelatin because it had the advantage of being very compatible with mammalian cells and produced a slurry that melts when incubated at standard 37° C. This made sense because I wanted to create a method that would support the printing of cells in a BSC (at ~20° C) and "release" a print in an incubator. I began the work of patenting the method and developing it for both commercialization and wider adoption.

To ensure researchers had access to the basic methodology, I started figuring out the easiest versions of the method using common tools and pushed for open access wherever possible. As part of my investigation of methods, slurries of gelatin were made using a ball mill, syringe back-and-forth pumping, a blender, a mortar and pestle, beakers/stirbars, etc. (see below) and I eventually developed the method used in the 2015 Science Advances paper, which required an Oster beehive blender and chilled block of gelatin. I spent months trying to name embedded printing with a catchy acronym that would effectively capture the idea, and I kept coming back to the phrase "reversible embedding", actively avoiding "embedded printing" since it was used by another technology at the time. I settled on Freeform Reversible Embedding of Suspended Hydrogels or FRESH. In all honesty, I wanted someone to say FRESH prints.

Syringe-pumped slurry print

Ball milled slurry prints

Blended slurry print

Between 2013 and 2017, many things added momentum to FRESH. More researchers began to work with me, allowing me to learn how to teach the method and expand its footprint. Before the 3D benchy became a standard for eyeballing 3D printer performance, there were simpler benchmarks such as the 20 mm calibration cube that allowed us to troubleshoot printing. The Elliptical Window Calibration (EWC - see below, download here) was designed to be the equivalent of the benchy or the 20 mm calibration cube, but for embedded printing, and it is still my go-to design for teaching newcomers how to do embedded printing well. With the EWC, I realized I needed a better bioprinting platform, so I created the Replistruder syringe pump extruder for the MakerBot Replicator. The resulting machine unlocked the ability to use G-code for syringe extrusion, and the inaugural print was a 20mm calibration cube of mayonnaise (see below). Now that I had access to G-code from all of the slicer softwares, the printing platform was no longer a bottleneck and we could essentially print gels with all the tricks available to desktop plastic printing, and we didn't have to worry about "overhangs" or printing "supports". Finally, we printed EWC's well.

The EWC benchmark for FRESH

Many small, failed EWC's

DIW with mayonnaise using 5D gcode and the first Replistruder

Good EWC's

Andrew Hudson and I began producing liters of gelatin microparticles using coacervation (sample figure below), supplying the entire Feinberg lab with a higher quality support bath. My custom Duet-based printers and Replistruder syringe pump extruders (v3 shown below) were getting better and easier to use. Every week, the firmwares (Sailfish, Jetty, Marlin, reprap firmware) were improving from community involvement, too. New, faster slicing softwares that were user friendly (Slic3r, KISSlicer, and Cura) saved us hours of time per week. Better printers, better support baths, and better slicing software meant we quickly saw better printing results.

Coacervation = more, better slurry

I built several modified desktop plastic printers to bioprint using syringes.

Replistruder syringe pump extruders enable retraction, which can be advantageous for extrusion printing.

The results started speaking for themselves - we were printing with better resolution than consumer 3D printers using many different tissue engineering gels. Advanced Biomatrix was aware of FRESH and working with us to facilitate positive results with collagen. FRESH was also on tour - we were doing posters, talks, and demos wherever we were allowed to take the printer. Here are some examples of early good FRESH prints.

Alginate "Dragon's Egg"

2016 collagen heart in a jar

NYT demo

WIRED demo

By 2016, I was working on my Ph.D. thesis, using FRESH to create small 3D tubes of engineered epithelium that invasive carcinomas could be placed into and invade in vitro. What I figured out was a method only FRESH could do - 2 material prints where lattices of alginate extrusions externally buttress collagen lumens so they can be handled and cultured with aggressive cell types, and eventually released from the alginate mesh for imaging. There are a few images of this shown below, with arguably the most complex print I've ever done - the "complex duct". During my postdoc, I focused on socializing the method and building expertise in the lab which led to a highly collaborative paper that finally got published in Science.

Example of collagen in alginate

A simplified duct

Printing a complex duct

A complex duct

Due to the hard work of the team involved in Lee et al 2019 Science, FRESH/embedded printing took off. Afterward, Drs. Shiwarski, Tashman, Bliley, Hudson, and Lee went on to further FRESH. Even now, because of the mountain of thought Josh Tashman put into the design, the Replistruder 4 (shown below) is the best extruder anyone can make for printing with a glass syringe. Kira Pusch produced the Large Volume Extruder (LVE), which later resulted in Eman Mirdamadi's full-sized heart print. Lulzbot made their bioprinter specifically targeting FRESH/embedded printing. Andrew Hudson's thoughtful VAPOR bioreactor utilizes FRESH printed fluidic constructs. The list keeps growing. It is entirely due to the hard work of the researchers furthering it. Some particularly nice examples of prints are shown.

The Replistruder 4 is a superior syringe pump extruder

Heart Tissue - Lee et al. 2019 Science

Collagen Heart - Lee et al. 2019 Science

Lifesize collagen ears like this are easy with LifeSupport and collagens like LifeInk 240

Following my postdoc, I took a gap year, traveled around the US and then to South America for 6 months. I then spent several years working at FluidForm, using embedded printing in impactful and unusual ways. Unlike the Feinberg lab, FluidForm is aimed at making embedded printing into products, so most of what we have done is confidential. I can say LifeSupport, FluidForm's powdered bath for embedded printing went through a multi-year development process involving hundreds of experiments that eventually resulted in a very reliable product. Our work includes medical devices, consumer electronics, a variety of biomaterial processes, implantables, and even high performance composites.

It's 2024; there are many different implementations of embedded printing/FRESH, with researchers often giving their unique flavors names like RLP, SWIFT, and CLASS. I am trying to figure out what's next for the community of embedded printing folks, and also bioprinting and biofabrication. I used to frown on DIW and the challenge of "printability", mostly because FRESH solved so much of that, but I came to realize an enormous amount of material research has come from trying to stack fluids for additive purposes. It is still my goal to provide access to embedded printing for everyone, given practically every bioink prints better using it, yet barriers to entry remain. I will continue to try and help anyone who wants assistance with embedded printing and open source bioprinting hardware. Hard to say what's next.

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