3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Summary

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 µm can be obtained and collected for further culturing.

Abstract

Microcarriers are beads with a diameter of 60-250 µm and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 µm. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

Introduction

Microcarriers are beads with a diameter of 60-250 µm and a large specific surface area and are commonly used for large-scale culture of cells^1,2. Their outer surface provides abundant growth sites for cells, and the interior provides a support structure for spatial proliferation. The spherical structure also provides convenience in monitoring and controlling parameters, including pH, O₂, and concentration of nutrients and metabolites. When used in combination with stirred tank bioreactors, microcarriers can achieve higher cell densities in a relatively small volume compared to conventional cultures, thereby providing a cost-effective way to achieve large-scale cultures³. Microcarrier culture technology has become one of the main techniques in cytological research, and much progress has been made in the field of large-scale expansion of stem cells, hepatocytes, chondrocytes, fibroblasts, and other structures⁴. They have also been found to be ideal drug delivery vehicles and bottom-up units, therefore taking on an increasingly important role in clinical drug screening and in vitro tissue engineering repair⁵.

To meet mechanical property requirements in different scenarios, multiple types of hydrogel materials have been developed for use in the construction of microcarriers^{6,7,8,9,10,11}. Alginate and hyaluronic acid (HA) hydrogels are two of the most used microcarrier materials owing to their good biocompatibility and crosslinkability^12,13. Alginate can be easily cross-linked by calcium chloride, and its mechanical properties can be modulated by changing the cross-linking time. Tyramine-conjugated HA is cross-linked by the oxidative coupling of tyramine moieties catalyzed by hydrogen peroxide and horseradish peroxidase¹⁴. Collagen, due to its unique spiral structure and cross-linked fiber network, is often used as an adjuvant to mix into the microcarriers to further promote cell attachment^15,16.

Current methods for preparing microcarriers include microfluidic chips, inkjet printing, and electrospray^{17,18,19,20,21,22,23}. Microfluidic chips have been proven to be fast and efficient in producing uniform-sized microcarriers²⁴. However, this technology relies on a complex flow channel design and fabrication process²⁵. High temperature or excessive extrusion forces during inkjet printing, as well as intense electric fields in the electrospray approach, may adversely affect the properties of the material, especially its biological activity¹⁹. Besides, when applied to various biomaterials and diameters, the customized nozzles used in these methods result in limited processing complexity, high cost, and low flexibility.

To provide a convenient method for microcarrier preparation, a 3D printing technique called alternating viscous-inertial forces jetting (AVIFJ) has been applied to construct hydrogel microcarriers. The technique utilizes downward driving forces and static pressure generated during vertical vibration to overcome the surface tension of the nozzle tip and thus form droplets. Instead of severe forces and thermal conditions, small rapid displacements act directly on the nozzle during printing, causing a minor effect on the physicochemical properties of the bioink and presenting great attraction for bioactive materials. Utilizing the AVIFJ method, microcarriers of multiple biomaterials with diameters of 100-300 µm were successfully formed. Besides, the microcarriers were further proven to bind cells well and provide a suitable growth environment for adhered cells.

Protocol

1. Cell culture

1. Supplement high-glucose Dulbecco's modified Minimum Essential Medium (H-DMEM) with 10% fetal bovine serum (FBS), 1% nonessential amino acid solution (NEAA), 1% penicillin G and streptomycin, and 1% Glutamine supplement as culture media for A549 cells.
2. Culture A549 cells in a CO₂ incubator at 37 °C and with 5% CO₂
3. Dissociate cells for subculture using trypsin at approximately 80% confluence.
   1. Use 3 mL of trypsin to treat the cells in the T75 culture flask at 37 °C for 3 min, and then add 6 mL of culture medium to stop the trypsinization.
   2. Pipette the culture medium to harvest loosely adhered cells.
   3. Centrifuge at 72.5 x g for 3 min. Remove the supernatant and resuspend with 3 mL of fresh medium.
   4. Transfer 1 mL of cell suspension to a new T75 culture flask containing 10 mL of fresh medium. Change the culture medium every 2 days.

2. Preparation of nozzles

1. Load glass micropipettes onto the puller following the manufacturer's instructions. The outer diameter, inner diameter, and length of the micro-jet pipe are 1.5 mm, 1.1 mm, and 100 mm, respectively.
2. Set the pulling parameters for the puller. Specifically, the values of HEAT, PULL, VELOCITY, TIME, and PRESSURE are set to 560, 255, 255, 150, and 500 by default, respectively. Pull off the nozzle under these parameters.
   CAUTION: The nozzle obtained after pulling off is very sharp, and careless operation may cause injuries.
3. Cut off the resulting nozzle (step 2.2) at the designated diameter by a micro-forge device following the manufacturer's instructions to obtain a specific tip diameter. Specifically, locate the specified diameter of the nozzle onto the heated platinum wire. Heat the wire up to 60 °C for about 5 s and pull the nozzle apart.
4. Immerse the printhead of the nozzle in a hydrophobic agent for 30 min, followed by three cycles of rinsing with sterilized water.
5. Before printing, sterilize the nozzle in alcohol for 5 min and rinse it with sterilized water three times to remove the residual alcohol.

3. Preparation of hydrogel bioink

1. Dissolve NaCl (0.45 g) into 50 mL of sterile water to obtain a 0.9% (w/v) NaCl solution. Vibrate the solution to promote dissolution. After being completely dissolved, filter the solution through a 0.45 µm polyethylene terephthalate filter membrane.
2. Weigh low-viscosity sodium alginate powder (1 g) and dissolve it in 25 mL of NaCl solution (step 3.1) at a high temperature of 60-80 °C overnight to obtain a 4% (w/v) sodium alginate stock solution. The stock solution can be stored at 4 °C for 1 month.
3. Sterilize the sodium alginate stock solution (step 3.2) by heating it three times in an oven (70 °C) for 30 min.
4. Dilute proper volume of sodium alginate stock solution (step 3.3) in 0.9% NaCl solution (step 3.1) at concentrations of 2%, 1.5%, and 0.5% (w/v).
   NOTE: In addition to heating in the oven, other preparation processes of sodium alginate solution should be carried out in the hood.
5. Dissolve 1 g of gelatin powder in 25 mL of NaCl solution (step 3.1) at a high temperature of 60-80 °C overnight to obtain a 4% (w/v) gelatin stock solution. The stock solution can be stored at 4 °C for 1 month.
6. Sterilize the gelatin stock solution (step 3.5) by heating it three times in an oven (70 °C) for 30 min.
7. Dilute the proper volume of gelatin stock solution (step 3.6) in 0.9% NaCl solution (step 3.1) at a concentration of 1.5% (w/v).
   NOTE: In addition to heating in the oven, other preparation processes of gelatin solution should be carried out in the hood.
8. To promote cell binding on the microcarriers, prepare the alginate-collagen solution by mixing the 2% (w/v) sodium alginate solution and the Type I collagen solution from rat tail (4 mg/mL) at a ratio of 3:1. Adjust the solution to pH 7.2 with 0.1 M NaOH solution and use immediately after preparation.
9. Dissolve the tyrosine-modified HA flocculent solid in phosphate buffer saline (PBS) solution at 60-80 °C overnight to obtain a 0.8% w/w tyrosine-modified hyaluronic acid PBS solution. The stock solution can be stored at 4 °C for 1 month.
10. Sterilize the tyrosine-modified HA PBS solution by heating it three times in an oven (70 °C) for 30 min.
11. Dissolve horseradish peroxidase powder (500 enzyme activity unit/mg) in PBS solution to obtain 8 enzyme activity unit/mg horseradish peroxidase PBS solution.
12. Dilute hydrogen peroxide solution (30%, w/w) by DI water to 4 mM.
13. Prepare the modified HA-horseradish peroxidase solution by mixing tyrosine-modified hyaluronic acid PBS solution and horseradish peroxidase PBS solution at a ratio of 1:1.
    ​NOTE: In addition to heating in the oven, other preparation processes of HA-horseradish peroxidase solution should be carried out in the hood.

4. Microdroplets formation based on AVIFJ

1. Use a home-established cell printing system as previously reported (Figure 1A)²⁶. The electrical signal output by the waveform generator is first amplified and then drives piezoelectric ceramics to generate controllable deformation. The nozzle fixed on the piezoelectric ceramic thus produces controllable vibrations.
2. Sterilize the printing system by wiping with 75% (v/v) alcohol and ultraviolet (UV) exposure overnight.
3. Load 5 mL of bioink (step 3.1) into a disposable sterile syringe. Install the syringe on the syringe pump.
4. Connect the syringe (step 4.3) and nozzle (step 2.5) with a silicone hose of 1 mm inner diameter.
5. Fix the nozzle (step 4.4) to be used for printing by adjusting the tightness of the clamping screw. Keep the tip of the nozzle away from the substrate during installation to avoid damage and contamination.
   ​CAUTION: If the nozzle is broken during installation, please wear thicker gloves and carefully clean up glass fragments and residues.
6. Rapidly push the syringe pump and load the bioink (step 4.3) to the nozzle (step 4.5). Set the flow rate at 30 µL/min. Wipe off the excess material at the tip.
7. Set signal generator parameters. Import self-designed waveform containing 500,000 sampling points. Set the sampling rate and peak-to-peak voltage (Vpp) as 5 million samples/s and 10 V, respectively.
8. Clean slides or Petri dishes with deionized water. Place them under the nozzle as the printing substrate.
9. Preset the motion path and trigger mode of vibration as drop-on-demand.
10. Print the droplets following the pre-designed patterns.

5. Microcarriers formation based on AVIFJ

1. Repeat steps 4.1-4.4, except change the bioink to 2% (w/v) alginate solution (step 3.4), alginate-collagen solution (step 3.8), or modified HA-horseradish peroxidase solution (step 3.13).
2. Dissolve 3 g of calcium chloride into 100 mL of sterile water to obtain 3% (w/v) calcium chloride solution. After being completely dissolved, filter the solution through a 0.45 µm polyethylene terephthalate filter membrane.
3. Add 5 mL of cross-linking solution (calcium chloride solution or hydrogen peroxide solution) into a Petri dish. Place the Petri dish under the nozzle to work as a substrate.
4. After printing, cross-link the microcarriers for 3 min.
5. Collect microcarrier suspension in a centrifuge tube, and enrich microcarriers by centrifugation at 29 x g for 3 min. Resuspend the microcarriers in culture media at approximately 600 microcarriers/mL.

6. Inoculating cells on the surface of microcarriers

1. Stain A549 cells with Cell Tracker Green CMFDA to facilitate the observation of cells. Specifically, remove culture media, add 5 mL of serum-free medium containing 10 µM Cell Tracker dye, and incubate cells for 30 min in an incubator. Then, replace the dye solution with fresh medium.
2. Resuspend the A549 cell suspension at the density of 1.6 x 10⁶ cells/mL.
3. Add 1 mL of alginate-collagen microcarrier suspension (step 5.3) and 1 mL of A549 cell suspension (step 6.1) into a low-adherent 6-well culture plate.
4. Place the plate onto a shaker at 30 rpm in an incubator at 37 °C and 5% CO₂.
5. Take the plate off the shaker and wait for 30 min to let the microcarriers settle down. Half change the culture medium every 2 days.

7. Analysis of microdroplets/microcarriers formation

1. Observe and measure the nozzle (step 2.2), the Petri dishes (step 4.10 and step 5.4), and cells with microcarriers (step 6.5) under a bright field microscope or confocal microscope. Specifically, the objective magnification is 4x, 10x, or 20x and the eyepiece magnification is 10x.
2. Measure the diameter of the microcarriers by ImageJ software. According to the scale bar that comes with the microscope when shooting in the picture, set the actual size of the scale in ImageJ, and draw 10 lines of the radius or semi-major axis of the microcarriers. ImageJ can get the average size and standard deviation of these line segments.

Results

Printheads of varied convergence rates and diameters were fabricated to achieve the printing of multiple types of materials. The nozzles obtained with increasing pull strength are shown in Figure 1B. The nozzles were divided into three areas: reservoir (III), contraction (II), and printhead (I). The reservoir was the unprocessed part of the nozzle, in which the liquid provided static pressure and bioink input for printing. The contraction area was the main part for generating downward drivin...

Discussion

The protocol described here provides instructions for the preparation of multi-types of hydrogel microcarriers and subsequent cell seeding. Compared to microfluidic chip and inkjet printing methods, AVIFJ approach to constructing microcarriers offers greater flexibility and biocompatibility. An independent nozzle enables a wide range of lightweight nozzles, including glass micropipettes, to be used in these printing systems. The highly controllable processing enables parameters including the volume of the reservoir, the ...

Materials

Name	Company	Catalog Number	Comments
A549 cells	ATCC	CCL-185	Human non-small cell lung cancer cell line
Bright field microscope	Olympus	DP70
Confocal microscope	Nikon	TI-FL
Fetal bovine serum, FBS	BI	04-001-1ACS
Gelatin	SIGMA	G1890
Glass micropipettes	sutter instrument	b150-110-10
GlutaMAX	GIBCO	35050-061
H-DMEM	GIBCO	11960-044	Dulbecco's modified eagle medium
Horseradish peroxidase powder	SIGMA	P6782
Hydrophobic agent	3M	PN7026	Follow the manufacturer's instructions and use after dilution
Micro-forge device	narishige	MF-900
Non-essential amino acids, NEAA	GIBCO	11140-050	non-essential amino acids
Penicillin G and streptomycin	GIBCO	15140-122
Petri dish	SIGMA	P5731-500EA
Puller	sutter instrument	P-1000
Sodium alginate	SIGMA	A0682
Trypsin	GIBCO	25200-056
Type I collagen solution from rat tail	SIGMA	C3867