Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter

Summary

This article describes a method of transforming a low-cost commercial 3D printer into a bacterial 3D printer that can facilitate printing of patterned biofilms. All necessary aspects of preparing the bioprinter and bio-ink are described, as well as verification methods to assess the formation of biofilms.

Abstract

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced antibiotic resistance, which poses potential health dangers, but can also be beneficial for environmental applications such as purification of drinking water. The further development of anti-bacterial therapeutics and biofilm-inspired applications will require the development of reproducible, engineerable methods for biofilm creation. Recently, a novel method of biofilm preparation using a modified three-dimensional (3D) printer with a bacterial ink has been developed. This article describes the steps necessary to build this efficient, low-cost 3D bioprinter that offers multiple applications in bacterially-induced materials processing. The protocol begins with an adapted commercial 3D printer in which the extruder has been replaced with a bio-ink dispenser connected to a syringe pump system enabling a controllable, continuous flow of bio-ink. To develop a bio-ink suitable for biofilm printing, engineered Escherichia coli bacteria were suspended in a solution of alginate, so that they solidify in contact with a surface containing calcium. The inclusion of an inducer chemical within the printing substrate drives expression of biofilm proteins within the printed bio-ink. This method enables 3D printing of various spatial patterns composed of discrete layers of printed biofilms. Such spatially-controlled biofilms can serve as model systems and can find applications in multiple fields that have a wide-ranging impact on society, including antibiotic resistance prevention or drinking water purification, among others.

Introduction

There is currently an increasing need to develop environmentally-friendly, sustainable solutions for the production of spatially-patterned materials, due to the expanding number of markets for such materials¹. This article presents a simple, economical method for the production of such materials and therefore offers a large spectrum of future applications. The method presented here allows three-dimensional (3D) printing of spatially-patterned structures using a bio-ink containing living bacteria. Bacteria remain viable within the printed structures for over one week, enabling the bacteria to perform natural or engineered metabolic activities. P

Protocol

1. Conversion of a commercial 3D printer into a 3D bioprinter

1. Remove the extruder and the heater of a commercial 3D printer (Table of Materials) from the printer frame, and unplug the wiring controlling these elements from the main circuit board (Figure 1A). Since the sensor that controls the operational temperature of the printer needs to be functional to communicate with the printer software, remove from the printing software the algorithm that delays printing until operational temperature is reached.
2. Connect a pipette tip (200 µL tip) via silicon tubing (inner diameter of 1 mm) to a 5 mL syri

Results

The first step for successful 3D printing of biofilms is converting a commercial 3D printer into a bioprinter. This conversion is achieved by removing the extruder and heater of the printer, designed for printing with a polymeric ink, and replacing these with components appropriate for printing bio-ink containing living bacteria (Figure 1A). The extruder is replaced by a pipette tip (or tips, if multiple bio-inks will be used in the printing process) attached...

Discussion

The protocol presented here for 3D printing of engineered biofilms has two critical steps. First is the preparation of the agar printing surface, which is the most critical factor to producing a specific printing resolution. It is important to ensure that the printing surface is flat and that the pipette tip on the printhead is positioned at the correct height from the surface. If the surface is not flat, the working distance will change during the printing process. If the working distance is less than 0.1 mm, the CaCl

Materials

Name	Company	Catalog Number	Comments
3D printer	CoLiDo	3D-P Kit
3D printing software	CoLiDo	Print-Rite ColiDo Repetier-Host v2.0.1
Agar	Sigma-Aldrich	05040
CaCl2 dihydrate	Sigma-Aldrich	C7902
Centrifuge	Eppendorf	5810 R
Chloramphenicol	Sigma-Aldrich	3886.1
LB broth powder	Sigma-Aldrich	L3022
Orbital shaker	VWR	89032-092	Model 3500
Petri dish	VWR	25384-326	150 x 15 mm
Rhamnose	Sigma-Aldrich	83650
Silicon tubing	VWR	DENE 3100103/25
Syringe pump	ProSense B.V.	NE-300
Sodium alginate	Sigma-Aldrich	W201502
Sodium citrate monobasic	Sigma-Aldrich	71498
Sodium hydrooxide	VWR	28244.295