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 cult....

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 ....

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