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. Bioprinting has the potential to be such a technique where the vision of manufacturing complex three-dimensional tissues can be realized^10,11,26-28.

Bioprinting is defined as "the use of material transfer processes for patterning and assembling biologically relevant materials - molecules, cells, tissues, and biodegradable biomaterials - with a prescribed organization to accomplish one or more biological functions"⁴. It encompasses several different techniques that work at different resolutions and length scales, ranging from the sub-micron resolution of two-photon polymerization²⁹ to a resolution of 150 μm to 420 μm for extrusion printing^1,12,30. Not a single material or material combination will satisfy the requirements of each method³¹. For extrusion printing, the key parameters are viscosity and gelation time³², where high viscosity and rapid gelation are desirable.

3D printing is a technique which allows the easy creation of sacrificial molds for creating complex geometries^30,33,34. This process is based on the construction of a mold using a rapid prototyping technique such as an extrusion bioprinter. The created sacrificial mold is used to form complex structures from materials which are difficult to print due to their low viscosity and slow gelation time. The method presented here involves the creation of a sacrificial mold consisting of a material that dissolves quickly at low temperature and can be extruded accurately. The block copolymer poly(ethylene glycol)₉₉-poly(propylene glycol)₆₇-poly(ethylene glycol)₉₉ (also known as Pluronic F127 or poloxamer 407) fulfills these requirements. It has already been used in a modified version in extrusion printing¹ but, to our knowledge, has never been used for printing in its unmodified version due to its instability in liquid environments. Poloxamer 407 also shows an inverse thermal responsive behavior¹⁸ i.e. it changes from a gel to a sol upon cooling. Most importantly, it can be printed into complex arbitrarily curved structures with very high fidelity. This allows the creation of a structured hydrogel from a low viscosity material, in this case slow gelling agarose, by pipetting the solution into the printed sacrificial mold. The combination of printing the sacrificial mold with high fidelity and its quick elution from the casted structured hydrogel makes it a fast and flexible method to create molds with different geometries without the use of a mask or a stamp as it is often required in lithographic methods. The casted structured hydrogel can be further filled with another material that is not suitable for extrusion printing due to its low viscosity. This is in our case a low viscosity alginate methacrylate solution. Here we present the method of thermoresponsive reverse sacrificial molds for hydrogel patterning using the example of a pillar array.

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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.
4. Add cold PBS until a final volume of 100 ml is reached. The final concentration will be 24.5% w/v.
5. Stop stirring the solution and let it rest at 4 °C until bubbles and foam in the solution have disappeared. Bubbles that are trapped within the gel will be transferred to the printer cartridge and will lead to defects in the printed sacrificial molds.
6. Filter (0.22 μm filter) the solution directly into the printing cartridge to remove any unwanted particles that could clog the needle. The filtering step should be performed in a cold room (or if not available with cooled tips, filter etc.) to avoid gelling of the poloxamer in the filter. Keep the loaded cartridge at 4 °C until 30 min before the experiment.

2. Preparation of the 3D Printer

The 3D printer used in this work was the "BioFactory" from regenHU. The extrusion part of the system consists of several parts. A cartridge under pressure at the top is attached to a connector via a luer-lock adapter. The connector bridges the spaces between the outlet of the cartridge and the inlet of a solenoid valve. At the outlet of the solenoid valve, needles with different diameters can be used. The material is extruded onto a substrate that is held to a moving stage by vacuum. The major parts of the system are depicted in Figure 1. Other extrusion based systems can be used for the printing process, and the optimization process needs to be done for each system.

1. Place the solenoid valve (nozzle diameter 0.3 mm) and the needle (inner diameter 0.15 mm) in separate 1.5 ml test tubes filled with ultrapure water and place them in a heated ultrasonic bath to clean for 30 min. Rinse the cleaned valves with ethanol and dry them with a nitrogen gun.
2. Install the valve and needle in the printer as well as an empty, clean cartridge.
3. Apply 3 bar pressure to the system and blow out any residual liquids from the installed valve and needle with compressed air. For small needle diameters, it is recommended to have a filter (common syringe filter, 0.45 μm pore size) installed at the exit of the compressed air to avoid entry of small particles that could clog the needle.
4. Turn the pressure off and install the cartridge loaded with the poloxamer. The cartridge should be taken out of the refrigerator approximately 30 min before mounting the cartridge so the poloxamer can reach room temperature and gel.
5. Apply 3 bar pressure to the system and dispense poloxamer until it reaches the needle tip and is extruded in a continuous strand.

3. Optimization of the Printing Parameters

To create accurate 3D structures, the printing process has to be optimized for the chosen material and concentration. Depending on the viscosity and the 3D printing system each material will yield a specific dispensing volume and line thickness for a fixed set of parameters.

1. With a suitable CAD software (able to create ISO files from the drawings), draw a single line about the same length as the structure that you intend to print.
2. Place a microscope glass slide 25 mm x 75 mm x 1 mm or any other substrate in the printer and secure it by turning on the vacuum.
3. In the printer software, set the solenoid valve to a high frequency of 50 Hz and set a high pressure of 3 bar.
4. Print one layer of a single line with a stage speed of 300 mm/min.
5. Reduce the pressure until the desired line width is reached. You can also control the volume that is extruded via the opening time of the valve.
6. Reduce the frequency of the valve until no continuous line can be printed anymore. Choose a frequency above this value.

Note: Once the desired line width and continuous lines are achieved, determine the optimal stage speed and layer thickness i.e. the lift of the needle after one printed layer.

7. Print several layers on top of each other and see if the needle is in the right position above the previous layer after several printed layers. Adjust the layer thickness (needle lift) so that each layer is printed on top of the next one (Figure 3).
8. Decrease the stage speed of the stage from 300 mm/min step-wise so that extruded layers start and end at the same positions as the previous ones (Figure 4). Too high stage speeds cause the stage to be moving before the extruded material has touched the previous layer.
9. For printing the pillar structures follow steps 3.1.-3.8., but instead of drawing a single line draw a single point. The parameters to focus on when printing the pillars are the pressure (regulates layer thickness and pillar diameter of poloxamer), the opening time of the valve (extruded volume) and the residence time of the print head at the position where the pillar should be deposited.
10. When the parameters are optimized, printing several layers of a line should result in a solid wall, or in case of the points, a pillar. Save the parameters for later use.

4. Printing and Elution of the Reverse Mold

Use the parameters found during the optimization procedure from this point on.

1. Print the inner structure (here it is a pillar array) on a glass microscope slide and let it dry overnight. This a) reduces the size and thickness of the structures and b) provides better adhesion between the structure and the substrate, so lift-off during the backfilling can be avoided.
2. With the CAD software, draw a structure that consists of an outer wall surrounding the structure you intend to have eluted away and filled. Print the structure with poloxamer. The printing of the wall will take 6 min.

Attention: The wall has to be printed at least 3.5 mm away from the inner structure because of the dimensions of the needle. Otherwise the printing of the outer wall will destroy the inner structure

3. Prepare the solution you want to backfill your sacrificial mold with (here 1% agarose in deionized water). The agarose solution should have a temperature between 35 °C and 45 °C. Beneath this temperature, the agarose will solidify too quickly; above this temperature, it might destroy the printed pillars because the poloxamer structure will soften.
4. Slowly fill the sacrificial mold with the backfilling solution using a pipette. This should be done slowly to avoid destruction of the structure inside the wall.
5. Let the backfilled solution gel or crosslink it depending on the polymer used. In the case of agarose the solidification took place at 4 °C for 10 min.
6. Place the backfilled sacrificial mold in an ice bath for 10 min to elute the poloxamer structure.
7. Blot the backfilled structure with a paper tissue and place it on a new glass microscope slide. Press the structure carefully onto the glass microscope slide to avoid leakage of the third hydrogel from the void into the space between the backfilled structure and the glass microscope slide.

5. Filling of the Voids

1. To fill the voids left by the eluted poloxamer, fill the intended polymer solution into a syringe equipped with a 30 G needle. In this example, we used a 1% alginate methacrylate in 0.15 M NaCl solution with the addition of 0.05% w/v lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and 2.5% v/v of Alexa-488 conjugated fibrinogen. The Alexa-488 conjugated fibrinogen was added for visualization purposes.
2. Photopolymerize the polymer with a high intensity UV lamp (100 Watt, 365 nm, distance from substrate was 3.5 cm) for 5 min and image the construct using an epi-fluorescence or confocal microscope.

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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,...

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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...

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Materials

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