Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Summary

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Abstract

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting^1-4 (extrusion, dip pen and soft lithography), contactless bioprinting^5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization⁸. It can be used for many applications such as tissue engineering^9-13, biosensor microfabrication^14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types¹⁷. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.

Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 °C and a solid above its gelation temperature ~20 °C for 24.5% w/v solutions¹⁸. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

Introduction

Tissue engineering approaches have made much progress over the last years with respect to regeneration of human tissues and organs^19,20. However, until now, the focus of tissue engineering has been often limited to tissues that have a simple structure or small dimensions such as the bladder^21,22 or the skin^23-25. The human body, however, contains many complex three-dimensional tissues where cells and extracellular matrix are arranged in a spatially defined manner. To manufacture these tissues, a technique is required that can place cells and extracellular matrix scaffolding within a three-dimensional construct at specified positions. B....

Protocol

1. Preparation of the Poloxamer 407 Solution

If available, perform the preparation of the poloxamer solution in a cold room (4 °C). If not available, place a glass bottle in a beaker filled with ice-cold water. At higher temperatures the poloxamer will be above the gel point and will not dissolve properly.

1. Add 60 ml of ice cold PBS solution into a glass bottle and stir vigorously using a magnetic stirrer.
2. Weigh 24.5 grams of poloxamer and add it in small amounts to the cold PBS. Wait until the poloxamer has partially dissolved before adding more.
3. Stir the solution until all poloxamer has dissolved.

Results

The representative results show that the reverse mold technique (depicted in Figure 2) will create a structured gel that can be filled with a second material. At the beginning of every printing process the printing parameters are first optimized. Step-wise adjustments of the parameters will result in printed multilayered constructs depicted in Figure 3 and Figure 4 when single lines are printed. If the layer thickness (the needle lift after one printed layer) is too low,.......

Discussion

Here we present, for the first time, the use of a thermoresponsive polymer for a sacrificial mold that can be quickly eluted in cold water due to the gel-sol transition of poloxamer of ~20 °C. The speed of the entire process makes poloxamer interesting for the rapid creation of biopolymer structures which cannot be printed with adequate resolution. The technique described here can be used for patterning one hydrogel within another hydrogel or for the creation of microfluidic channels as has been previously reported f.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS
Poloxamer (Pluronic F127)	Sigma	P2443
PBS	Invitrogen	10010-015
CAD software	regenHU	BioCAD
Alginate methacrylate	Innovent e.V Technologieentwicklung Jena		Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292
Fibrinogen From Human Plasma, Alexa Fluor 488 Conjugate	Invitrogen	F13191
Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)	Innovent e.V Technologieentwicklung Jena		Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292
Agarose	Lonza	50004
EQUIPMENT
Bioprinter	regenHU	Biofactory
Valve	regenHU	300 μm Nozzel Diameter
Needle	regenHU	150 μm Inner Diameter
Zeiss Axioobserver with ApoTome	Zeiss
UV Light Source	UVP	Blak-Ray B-100AP High Intensity UV Lamp	100 W