Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

Summary

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

Abstract

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).

To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.

First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

Introduction

The precise and programmable 3D printing of cell-laden hydrogel constructs that mimic soft tissues in vitro presents a major challenge. For instance, attempts based on the direct extrusion of soft hydrogels are inherently problematic, as the poor mechanical properties required to recapitulate the in vivo microenvironment lead to a lack of structural integrity, deformations of the predefined features, or the complete collapse of the fabricated structures. A conventional workaround for this issue is to print a supporting scaffold from a stiffer biocompatible material that allows the final construct to maintain its shape. However, this approach greatly limits the design possibilities and requires careful rheological fine-tuning of the adjacent inks.

To overcome the limitations of the traditional layer-by-layer extrusion-based 3D printing, embedded 3D printing has emerged in recent years as a powerful alternative for soft material and tissue fabrication^1,2,3,4,5,6. Instead of extruding the ink in ambient air on top of a surface, the ink is directly deposited through a syringe needle inside a support bath that is solid-like at rest but reversibly fluidizes around the moving needle tip to allow the precise deposition of soft cell-laden material. The deposited material is kept in place as the support resolidifies in the wake of the needle. As such, embedded 3D printing allows for the high-resolution freeform fabrication of intricate structures from soft biomaterials with expanded design possibilities^7,8.

Granular gels have been extensively explored as support bath materials for embedded 3D printing, since they can be formulated to exhibit smooth, localized, and reversible solid-to-liquid transitions at low yield stresses^9,10,11. While they show excellent rheological properties for high-resolution printing, granular gels have been restricted to a handful of biomaterials¹². The lack of diversity in granular gel formulations, which is particularly evident if one considers the wide range of biomaterials available for bulk hydrogel formulations, is caused by the need for the cost-effective generation of a large number of microgels using simple chemistries. Due to the limited biomaterial landscape of granular gel supports, the tuning of the extracellular microenvironment provided by the printing support presents a challenge in the field.

Recently, a modular approach has been developed for the generation of embedded 3D printing supports, termed self-healing annealable particle-extracellular matrix (SHAPE) composites¹³. This approach combines the distinct rheological properties of granular gels with the biofunctional versatility of bulk hydrogel formulations. The presented SHAPE composite support consists of packed alginate microparticles (granular phase, ~70% volume fraction) with an increased interstitial space filled with a viscous collagen-based ECM pregel solution (continuous phase, ~30% volume fraction). It has further been shown that the SHAPE support facilitates the high-resolution deposition of human neural stem cells (hNSCs) that, after the annealing of the support bath, can be differentiated into neurons and maintained for weeks to reach functional maturation. Embedded 3D printing inside the SHAPE support bath overcomes some of the major limitations related to conventional techniques for neural tissue biofabrication while providing a versatile platform.

This work details the steps for the embedded 3D printing of hNSCs inside the SHAPE support and their subsequent differentiation into functional neurons (Figure 1). First, alginate microparticles are generated via shearing during internal gelation. This approach allows the easy generation of large volumes of microparticles without the need for specialized equipment and cytotoxic reagents. Furthermore, alginate is a widely available and economical material source for the formation of biocompatible hydrogel substrates for a diverse range of cell types. The generated alginate microparticles are combined with a collagen solution to form the SHAPE composite support material. Then, the hNSCs are harvested and loaded into a syringe as a cellular bioink for 3D printing. A 3D bioprinter is used for the extrusion-based embedded printing of hNSCs inside the SHAPE composite. The 3D-printed cells are differentiated into neurons to give rise to spatially defined and functional human neural constructs. Finally, the protocol describes how the generated tissue constructs can be characterized using live-cell imaging and immunocytochemistry. Additionally, tips for optimization and troubleshooting are provided. Notably, both the components of the granular and continuous phases could be exchanged with other hydrogel formulations to accommodate different biofunctional moieties, mechanical properties, and crosslinking mechanisms, as required by other cell and tissue types beyond neural applications.

Protocol

1. Preparation of the buffers and reagents

1. Prepare cell growth medium by adding the following supplements to DMEM/F12 with L-alanyl-L-glutamine dipeptide: 30 mM glucose, 5 µM HEPES, 0.5% w/v lipid-rich bovine serum albumine, 40 µM L-alanine, 40 µM L-asparagine monohydrate, 40 µM L-aspartic acid, 40 µM L-glutamic acid, 40 µM L-proline, 1% N2 supplement, 1% penicillin-streptomycin, and 20 ng/L each of epidermal growth factor (EGF) and fibroblast growth factor (FGF). Perform these steps in a laminar air-flow (LAF) bench.
2. Prepare 1% w/v alginate solution in ultrapure water by stirring vigorously for 4 h at 60 °C. Sterile-filter the dissolved, hot alginate solution with a 0.45 µm pore-size filter in a LAF bench. Store it at 4 °C.
   NOTE: If the temperature of the alginate solution is below 60 °C, it will not be possible to pass it through the filter.
3. Prepare a 37 g/L NaHCO₃ solution in ultrapure water. Adjust the pH of the solution to 9.5 using NaOH, and filter-sterilize in a LAF bench. Store the solution at 4 °C.

2. SHAPE composite material preparation

1. Alginate microparticle generation
   1. Prepare a 2 mg/mL CaCO₃ solution in ultrapure water in a sterile beaker. Mix it 1:1 with the alginate solution, and stir for 1 h at room temperature using a magnetic stirrer.
   2. Add acetic acid in a 1:500 ratio, and leave stirring overnight at 650 rpm.
      NOTE: The alginate solution will start gelling immediately. Make sure to use a stirring magnet with the same length as the diameter of the glassware used to ensure optimal, homogeneous stirring of the forming gel. The next day, the solution generated via stirring during gelation will appear viscous (Figure 2A).
   3. Mechanically fragment the gelled alginate solution into microparticles by homogenizing it at 15,000 rpm for 10 min with a homogenizer placed in a LAF bench (Figure 2B).
   4. Centrifuge the microparticles at 18,500 × g for 20 min (Figure 2C) at room temperature.
   5. Carefully discard the supernatant inside the LAF bench, resuspend the particles in DMEM containing 2 mM NaOH and 1% P/S, and incubate overnight at 4 °C (Figure 2D).
      NOTE: The color of the suspension should go back to red. If the suspension stays yellow, add NaOH dropwise, and mix until it turns red (Figure 2E).
   6. Homogenize the particles at 15,000 rpm for 3 min, and centrifuge at 18,500 × g for 10 min at room temperature.
   7. Observe the pellet (Figure 2F), and remove the supernatant carefully.
      NOTE: The pellet of tightly packed alginate microparticles can be stored at 4 °C until further use. If the alginate solution was not filter-sterilized, the microparticles should be used up within 1 week.
2. SHAPE composite formulation
   1. The day before printing, resuspend the alginate microparticle pellet in twice the volume of growth medium containing 4% HEPES (1 M stock) and 4% NaHCO₃ solution in a LAF bench, and incubate it overnight at room temperature.
   2. Centrifuge the microgel suspension at 18,500 × g for 10 min at room temperature, and discard the supernatant.
   3. Neutralize the collagen to be mixed with the alginate microparticles in a LAF bench. Dilute the collagen stock solution to reach a final concentration of 1 mg/mL, and neutralize it by adding 4% HEPES and 4% NaHCO₃. For example, for 3 mL of SHAPE composite, mix 2 mL of alginate microparticles with 1 mL of neutralized collagen (0.12 mL of HEPES, 0.12 mL of NaHCO₃, 0.16 mL of growth medium, and 0.6 mL of collagen).
      NOTE: All the materials being mixed with collagen need to be handled on ice to avoid collagen polymerization. As soon as the collagen is neutralized, polymerization will slowly start.
   4. Generate the SHAPE composite by mixing the alginate microparticle pellet with the diluted and neutralized collagen in a 2:1 ratio. Mix the gel thoroughly by pipetting slowly up and down on ice in a LAF bench.
      NOTE: Do not vortex, as this will introduce bubbles into the support material.
   5. In a LAF bench, transfer the generated composite material into a cooled microwell plate or any suitable printing container, and use within 30 min (Figure 3E, F).

3. hNSC culture and bioink preparation

1. Culture the cells in a basement membrane extract-coated T75 flask in growth medium. Passage the cells at least two times after thawing before using them for a 3D printing experiment.
2. Before printing, dissociate the cells enzymatically using a 0.025% trypsin solution for 5 min at 37 °C, neutralize the trypsin with growth medium, and centrifuge the cell suspension at 400 × g for 5 min at room temperature. Following the centrifugation, aspirate the supernatant, and resuspend the pellet in 2-3 mL of growth medium.
3. Perform a cell count, centrifuge the cells at 400 × g, and resuspend the pellet in growth medium supplemented with 0.1% xanthan gum (to prevent the cells from sedimenting) at a final concentration of 9 × 10⁶ cells/mL.
4. Use a 21 G blunt metal needle to load 100 µL of tightly packed alginate microparticles into a syringe (Figure 3A).
   NOTE: Creating a plug with the alginate microparticles serves two purposes: it helps maintain extrusion stability during printing and allows for the complete extrusion of the loaded cellular ink (no dead volume).
5. Using the same needle, load the prepared cell suspension into the syringe (Figure 3B).
   NOTE: Take extra care not to introduce any air bubbles during the syringe-loading steps. Air bubbles will cause instabilities during printing.
6. Replace the loading needle with a 27 G blunt metal needle, which will be used for printing (Figure 3C).

4. Embedded 3D printing

1. Design a structure to print using the referenced software (see the Table of Materials).
   NOTE: Make sure to adjust the initial height of the printed structure to within the depth of the SHAPE composite support. Design suggestions can be found in Figure 4A (spiral and woodpile designs).
2. Generate a G-Code by clicking on Generate.
3. Insert the cell-laden glass syringe into a volumetric extrusion head on an extrusion-based bioprinter (Figure 3D).
4. Measure the needle length of the cell-laden glass syringe by clicking on Needle Length Measurement.
5. Place the microwell plate or container loaded with the SHAPE composite onto the printer.
   NOTE: Keep the cell plate or container at 4 °C until printing to prevent the premature crosslinking of the support.
6. Measure the surface height of an empty well within the same microwell plate or container in which the SHAPE gel is loaded by clicking on SHM (surface height measurement).
   NOTE: Alternatively, determine the surface height manually by measuring the height of the well with the needle.
7. Set the extrusion rate to 3.6 µL/min and the feed rate to 0.3 mm/s.
   NOTE: Make sure to test the extrusion before printing. Cell sedimentation can cause nozzle clogging and can be avoided by pre-extruding a small volume before the actual embedded printing or by adding a retraction volume to the volumetric syringe. In some cases, the feed rate needs to be kept below 0.5 mm/s when the needle is being inserted into or exiting the SHAPE gel to avoid the dragging of the ink.
8. Load the G-Code into the user interface of the printer.
   NOTE: A new G-Code needs to be generated every time changes are made to the designed structure.
9. Initiate the print procedure by clicking on Run (Figure 3G).
10. Immediately after printing, place the SHAPE gel at 37 °C in a cell culture incubator for 30 min for annealing.
11. Add growth medium gently on top of the annealed SHAPE gel support.
12. The next day, replace the growth medium with differentiation medium formulated as follows: DMEM/F12 with L-alanyl-L-glutamine dipeptide with 30 mM glucose, 5 µM HEPES, 0.5% w/v lipid-rich bovine serum albumine, 40 µM L-alanine, 40 µM L-asparagine monohydrate, 40 µM L-aspartic acid, 40 µM L-glutamic acid, 40 µM L-proline, 1% N2 supplement, 1% penicillin-streptomycin, 100 µM dibutyryl-cyclic adenosine monophosphate (dibutyryl-cAMP), and 2 ng/mL glial cell-line derived neurotrophic factor (GDNF).
    NOTE: Make sure not to damage the hydrogel during the medium changes. Tilt the plate, and remove the old medium gently. Add fresh medium dropwise to the wall of the well containing the gel rather than directly on top of the gel. Do not use an aspirator to remove the medium.
13. Refresh the differentiation medium every 2 days until the experimental endpoint.

5. Live-cell fluorescence imaging

1. Remove excess medium from the gel.
2. Add an equal volume of 20 µM Calcein AM (diluted in differentiation medium from the stock solution) to the volume of the support gel.
3. Incubate for 40 min at 37 °C.
4. Remove the Calcein AM solution, and add an appropriate volume of fresh differentiation medium.
5. Transfer the plate to the microscope for imaging.

6. Immunocytochemistry

1. Remove the excess medium from the gel.
2. Using a small spatula, transfer the gel to a larger container containing DPBS.
3. Wash three times for 20 min each time with DPBS, and transfer the plate to a fume hood.
4. Remove the DPBS, add enough 4% formaldehyde solution to cover the gel, and incubate for 1 h at room temperature.
5. Wash three times with DPBS for 20 min each time.
6. Prepare blocking solution consisting of 5% donkey serum, 0.25% detergent, and 0.02% sodium azide in DPBS.
   NOTE: Prepare three times the volume needed to cover the gel; it will be used later as the basis for the primary and secondary antibody solutions.
7. After washing with DPBS, add the blocking solution to the gel, and incubate for 6 h at room temperature to prevent unspecific binding. Rock the plate gently.
8. Prepare the primary antibody solution by diluting the TUBB3 antibody in blocking solution at a ratio of 1:1,000.
9. Remove the blocking solution from the gel, add the primary antibody solution, and incubate for 48 h at 4 °C. Rock the plate gently.
10. Wash three times with DPBS for 20 min each time.
11. Prepare the secondary antibody solution by diluting 4',6-diamidino-2-phenylindole (DAPI, 1:1,000) and the secondary antibody (1:200) in blocking solution.
12. After washing with DPBS, incubate the gel in the secondary antibody solution for 24 h at 4 °C. Rock the plate gently.
13. Wash three times with DPBS for 20 min each time, and store at 4 °C until imaging.
14. Before imaging, transfer the stained gel with a spatula to a dish or a well-plate with a thin imaging bottom.

Results

Alginate microgel preparation via shear thinning during internal gelation followed by mechanical fragmentation yields alginate microgels that are polydispersed in size and flake-like in shape as seen in Figure 2G. The size of these irregular particles ranges from less than 1 µm to approximately 40 µm in diameter. When tightly packed, the microparticles form a transparent bulk material that is only slightly more opaque than the corresponding cell culture medium (

Discussion

The SHAPE composite material approach provides a versatile route for the formulation of annealable and biofunctional support baths for the embedded 3D printing of cellular inks. While this protocol provides an example of the 3D printing of neural constructs, the SHAPE toolbox could easily be adapted to biofabrication with other cell sources for the precise engineering of a range of target tissue types. The printing approach would also allow for the precise patterning of multiple cell types to study their interaction or t...

Materials

Name	Company	Catalog Number	Comments
1 mL Gastight Syringe 1001 TLL	Hamilton	81320
3DDiscovery 3D bioprinter	RegenHU
Acetic acid	Sigma-Aldrich	A6283
AlbuMAX	ThermoFisher	11020021
Alexa Fluor 488 secondary antibody	ThermoFisher	A-11001	Goat anti-Mouse
Blunt Needle, Sterican (21 G)	Braun	9180109
Blunt Needle (27 G)	Cellink	NZ5270505001
BioCAD software	SolidWorks
Calcein AM	ThermoFisher	65-0853-39
Calcium carbonate	Sigma-Aldrich	C5929
Dibutyryl-cAMP sodium salt	Sigma-Aldrich	D0627
Cultrex Rat Collagen I (5 mg/mL)	R&D Systems	3440-100-01
DAPI	ThermoFisher	62248
DMEM/F-12, GlutaMAX	ThermoFisher	10565018
Donkey serum	Sigma-Aldrich	D9663
DPBS	ThermoFisher	14190094
EGF	R&D Systems	236-EG
FGF	R&D Systems	3718-FB
Formaldehyde solution 4%, buffered, pH 6.9	Sigma-Aldrich	100496
GDNF	R&D Systems	212-GD
Geltrex	ThermoFisher	A1569601
Glucose	Sigma-Aldrich	G7021
HEPES Buffer (1 M)	ThermoFisher	15630080
L-Alanine	Sigma-Aldrich	5129
L-Asparagine monohydrate	Sigma-Aldrich	A4284
L-Aspartic acid	Sigma-Aldrich	A9256
L-Glutamic acid	Sigma-Aldrich	G1251
L-Proline	Sigma-Aldrich	P0380
Magnetic stirrer RET basic	IKA	3622000
N-2 Supplement	ThermoFisher	17502048
Penicillin-Streptomycin	ThermoFisher	15140122
S25N-10G dispersing tool	IKA	4447100
Sodium Alginate (80-120 cP)	FUJIFILM Wako	194-13321
Sodium azide	Sigma-Aldrich	S2002
Sodium bicarbonate	Sigma-Aldrich	S5761
Sodium hydroxide	Sigma-Aldrich	S5881
T18 Digital ULTRA-TURAX homogenizer	IKA	3720000
Triton X-100	Sigma-Aldrich	X100
Trypsin/EDTA Solution	ThermoFisher	R001100
TUBB3 antibody	BioLegend	801213	Mouse
Xanthan gum	Sigma-Aldrich	G1253