Open Source Bioprinter

The 'Nydus One Bioprinter' is an extrusion-based Bioprinter. It operates via a controllable mechanical press, to extrude cell-laden bioinks. Basis for the Bioprinter is a remixed version of the RepRap Prusa i3 MK2-X. The printer is capable of printing all three common cell sources: cell-lines, primary tissue and stem cells. To operate the Bioprinter, Dennis Ogiermann has written a "Composer". This open source pathplanning tool allows to dispense into standarized container and allows repetitive experiment designs. The 'Nydus One Bioprinter' was established using a experimental 'Microgel Approach'. Main idea of the approach is the use of a thermoreversible gelatin bath (called Slurry Bath) to print objects with complex geometry. The approach was published by Hinton et al. in 2015 (see Sources).

Development

To track and coordinate our development efforts we use an OpenProject repository: See OpenProject Homescreen

Composer issues

• depending on object(geometry) to be printed and the print settings, some prints might not work (trial and error, may work with some settings, but we don't know them)
• the pathplanning seems to be off (Old Composer issue?)
• If you change the number of objects-to-be-printed

Methods of Bioprinting

Preparations

Before you can start your print project, first you have to prepare the bioink and the slurry bath. The bioink consist of a hydrogel as well as cells (if necessary). While printing the bioink in the prepared slurry bath, you can higher the stability of your construct and avoid blur of the printed slices. Besides the "wet" steps, you have to create a .stl file with a CAD software and generate a custom gcode.

microgel/gelatine bath preparation

The gelatine is prepared directly in a mixer bin. A 4,5 % w/v gelatine solution in 0,1 M CaCl_2 solution is prepared (in this case: 6,7g gelatine and 150 mL 0,1 CaCl_2 solution) and heated in the microwave until the liquid starts to boil. The solution is stored in the fridge overnight to solidify. The next day a small amount of the 0,1 M CaCl_2 solution is poured on the gel and the gelatine is losened carefully with a spatula. Afterwards the bin is filled up with 0,1 M CaCl_2 solution until spilled to prevent air bubbles, and closed with the lid. The gelatine is stored for 30 min in a -20°C freezer to prevent heat production during blending and blended for 2 min afterwards. To remove residues of dissolved gelatine, the mixture is washed. For this purpose the blended gel is evenly distributed over falcon tubes and centrifuges at 3.000 rcf and 4°C for 2 min. The supernatent is discarded and the tube is filled up with cold 0,1 M CaCl_2 solution to the 40 mL mark.The gel is resuspended by shaking and vortexing. This step is repeated three times. After the third step, the supernatant is discarded and the tubes filled up with 0,1 M CaCl_2 solution to the 25 mL mark each. The gel is resuspended via shaking and vortexing and the contents of two tubes united, followed by an additional centrifugation step.These steps are repeated once.Finally, the supernatant is dicarded again, the tubes filed up to the 25 mL mark, followed by resuspension. The Gel is sterilised by exposure to UV light (1000 J/cm^2) twice with the tubes turned by 180° before the second exposure. The micogel is stored at 4°C and can be used up to two weeks after preparation.

Bioink Preparation

Preparation of a 4% alginate hydrogel solution (dissolve 1g of alginate in 25 ml ddH_2O). To prevent contamination, the filter has to be filtered with a 0,45 µm mesh. The temperature of the hydrogel should be 37°C before using and should be stored at 8°C if necessary. The concentration of the cells and the total amount of the bioink should be choosen depending on the individual experiment, as well as the ratio of cells and bioink. The following protocol should be a fine for many interrogations: 1 mio. cells/ml in a 1:1 cells/bioink ratio.

Print Process

Using sterile equipment is highly recommended. The microgel should be filled in the construct (petri dish or 6-well plate) in which the model has to be printed in, preferably without any bubbles, then left for around 15 minutes at room temp. to let the hydrogel to adjust in the dish. Before attach the bioink and the hydrogel dish to the printer, you have to run the calibrating routine on your printer. After the calibration is done, the ink and the dish can be placed at their places and run the print. When the print is finished, the dish should be placed in the incubator at 37°C for aroudn 20 minutes until the slurry bath is melted, so that the bath can be changed to (prewarmed) mHEBS medium

OSF Repository

To allow sharing the required soft- and hardware, we have created an OSF repository: Open Science Framework Bioprinter Project

Costs

Electronics

Component	Details	Amount	Price	Link
Motherboard	mini-Rambo v1.3 Kit	1	120 $	Ultimachine Shop
Mechanical Endstop	inlcuded	3	/	see above
LCD Display	2004 LCD	1	10$	AliExpress
Heatbed	Aluminium incl. Thermistor	1	45$	AliExpress
PINDA Probe	Reddit	1	4$	AliExpress
NEMA17	Servo-Kit incl. threaded rod	3 + 2	110$	AliExpress
Pressure Sensor	Interlink FSR 406	1	15$	Exp-Tech
Power Supply	???	1	17$	AliExpress
Thermistor	HotEnd, req.	1	6€	reprapteile

Mechanics

Component	Details	Amount	Price	Link
Aluminium frame	X: 360mm Y: 320mm Z: 330mm	X: x3 Y: 2x Z: 2x	30€	Alu Profil Technik
Y-Carriage	???	1	30 €	Ebay RepRap Universe
Threaded rod	E: 160mm (d:8mm)	1	?	Hamwa noch, oder?
Smooth guiding rods (d: 8mm)	X: 360mm Y: 360mm Z: 320mm E: 2x205mm + 1x182mm	X & Y: x4 Z: x2 E: x3	~10€ (ebay) ~kA (proKilo)	ebay / ProKilo
Linear Bearings	LM8UU + LM8LUU	Y&E(LM8LUU): x5 X&Z(LM8UU): x7	Y: 15€ X&Z: 9€	LM8LUU rayntec ebay LM8UU rayntec ebay
Toothed Belt	2 meter	1	2€	rayntec ebay
Pulley	GT2-16/623h bearing housing	2	6€	rayntec ebay
Return Pulley	623h bearing housing	2	8€	rayntec ebay

Parts (Screws etc.)

Component	Details	Amount	Price	Link
Winding Tube	10 meter	1	4$	AliExpress
Screws	Frame&Axis	Schraubengrößen in 50er Pacaks holen	?	Schraube&Mutter
Plastic Parts	Frame&Axis	Printed by Vitt	~30€ (white)	Check OSF Repository & Sources
Powersupply Cable	2-adrige	nachmessen am BP	???	Conrad

In summary: ~500-600€

Editable List

Sources

Basis

Frame Modifications

Assembly Guide

Extruder Parts

Microgel Approach