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3D printed alginate bead generator for high-throughput cell culture.

ABSTRACT: Alginate hydrogel beads are a common platform for generating 3D cell cultures in biomedical research. Simple methods for bead generation using a manual pipettor or syringe are low-throughput and produce beads showing high variability in size and shape. To address these challenges, we designed a 3D printed bead generator that uses an airflow to cleave beads from a stream of hydrogel solution. The performance of the proposed alginate bead generator was evaluated by changing the volume flow rates of alginate (Q_Alg) and air (Q_A), the diameter of device nozzle (d) and the concentration of alginate gel solution (C). We identified that the diameter of beads (D = 0.9 -2.8 mm) can be precisely controlled by changing Q_A and d. Also the bead generation frequency (f) can be tuned by changing Q_Alg. Finally, we demonstrated that viability and biological function (pericellular matrix deposition) of chondrocytes were not adversely affected by high f using this bead generator. Because 3D printing is becoming a more accessible technique, our unique design will allow greater access to average biomedical research laboratories, STEM education and industries in cost- and time-effective manner.

SUBMITTER: Lee D

PROVIDER: S-EPMC8312329 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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3D printed alginate bead generator for high-throughput cell culture.

Lee Donghee D Greer Sydney E SE Kuss Mitchell A MA An Yang Y Dudley Andrew T AT

Biomedical microdevices 20210405 2

Alginate hydrogel beads are a common platform for generating 3D cell cultures in biomedical research. Simple methods for bead generation using a manual pipettor or syringe are low-throughput and produce beads showing high variability in size and shape. To address these challenges, we designed a 3D printed bead generator that uses an airflow to cleave beads from a stream of hydrogel solution. The performance of the proposed alginate bead generator was evaluated by changing the volume flow rates o ...[more]

PMID: 33821331

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Project description:Mechanical stimuli have been shown to play a large role in cellular behavior, including cellular growth, differentiation, morphology, homeostasis, and disease. Therefore, developing bioreactor systems that can create complex mechanical environments for both tissue engineering and disease modeling drug screening is appealing. However, many of existing systems are restricted because of their bulky size with external force generators, destructive microenvironment control, and low throughput. These shortcomings have preceded to the utilization of magnetic stimuli responsive materials, given their untethered, fast, and tunable actuation potential at both the microscale and macroscale level, for seamless integration into cell culture wells and microfluidic systems. Nevertheless, magnetic soft materials for cell culture have been limited due to the inability to develop well-defined 3D structures for more complex and physiological relevant mechanical actuation. Herein, we introduce a facile fabrication process to develop magnetic-PDMS (polydimethylsiloxane) porous composite designs with both well-defined and controllable microlevel and macrolevel features to dynamically manipulate 3D cell-laden gel at the scale. The intrinsic stiffness of the magnetic-PDMS porous composites is also modulated to control the deformation potential to mimic physiological relevant strain levels, with 2.89-11% observed in magnetic actuation studies. High cell viability was achieved with the culturing of both human adipose stem cells (hADMSCs) and human umbilical cord mesenchymal stem cells (hUCMSCs) in 3D cell-laden gel interfaced with the magnetic-PDMS porous composite. Also, the highly interconnected porous network of the magnetic-PDMS composites facilitated free diffusion throughout the porous structure showcasing the potential of a multisurface contact 3D porous magnetic structure in both reservoir and 96-well plate insert designs for more complex dynamic mechanical actuation. In conclusion, these studies provide a means for establishing a biocompatible, tunable magnetic-PDMS porous composite with fast and programmable dynamic strain potential making it a suitable platform for high-throughput, dynamic 3D cell culture.

Project description:BackgroundIntradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model.MethodsAfter EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin-eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (MMP1, MMP13, ADAMTS5, IL6, NOS2), anabolic (ACAN), and hNPC marker (SOX9, KRT19) genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis.ResultsTreatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment.ConclusionsWJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.

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3D printed alginate bead generator for high-throughput cell culture.

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3D printed alginate bead generator for high-throughput cell culture.

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