Researchers from Los Alamos National Laboratory (LANL) have developed a 3D printed perfusion bioreactor (3D-PBR) created to improve how human bone marrow-derived mesenchymal stem cells (MSCs) grow and differentiate.
With contributions from the University of New Mexico (UNM), the device also supports co-culture with vascular cells. Designed to address the limitations of basic cell culture models while capturing the complexities of real tissue environments, this compact device aims to make cellular differentiation studies more practical and precise.
Published in Nature, the 3D-PBR is made using Formlabs 3B Low Force Stereolithography (SLA) 3D printer, relying on a biocompatible resin composed of methacrylate monomer and urethane dimethacrylate. After printing, the researchers carefully cleaned the components with isopropyl alcohol and cured them under UV light at 60°C for 30 minutes, a process that not only ensures structural stability but also enhances clarity for imaging.
Dual-compartment setup supports cell interaction
What sets this device apart is its design, featuring two compartments separated by a porous polyethylene terephthalate (PET) membrane with a pore size of 0.4 µm. This membrane allows media transport and cellular interaction between the compartments without compromising their separation.
One compartment, designed for vascular cells, includes Luer-lock ports for controlled fluid flow and has a rectangular channel measuring 11.8 mm × 4.8 mm × 0.8 mm. The other compartment, intended for MSCs, has an oval-shaped channel measuring 15.8 mm × 4.8 mm × 0.8 mm. Despite its intricate design, the entire system, including the peristaltic pump and 3D printed media reservoir, fits within a compact volume of 60 cubic inches.
Researchers chose this resin-based polymer over the commonly used polydimethylsiloxane (PDMS), which often struggles with issues like molecular absorption and manufacturing constraints. The new approach provides greater flexibility and durability, allowing for more complex structures that are better suited for various experimental conditions.
During their experiments, the team cultured MSCs within a collagen-fibrin gel, a material selected for its ability to provide structural support and mimic extracellular matrix conditions. They prepared the gel with MSC concentrations of 3.8 × 10⁴ cells/mL for bone differentiation and 2.6 × 10⁵ cells/mL for fat differentiation.
Meanwhile, human umbilical vein endothelial cells (HUVECs) were introduced to the vascular compartment at a concentration of 1 × 10⁶ cells/mL, forming a confluent monolayer over a period of 21 days. To further enhance the differentiation process, researchers applied flow rates of 23 µL/min and 100 µL/min, which are known to promote bone-generating conditions.
The team then used fluorescent microscopy to evaluate how well the MSCs differentiated within the 3D-PBR. Imaging markers such as VE-Cadherin, ActinRed™ 555, and NucBlue provided clear visuals of cell structures and interactions. The results were encouraging, with MSCs demonstrating high viability, 92% for MSCs and 91% for HUVECs.
More importantly, the cells cultured within the 3D-PBR exhibited enhanced differentiation compared to static conditions, showing greater complexity and maturity in bone cell structures along with more consistent fat cell differentiation.
To verify the device’s structural integrity, researchers conducted µCT scans, confirming that the compartments were well-sealed and suitable for extended experiments. This step was crucial to ensuring the reliability of the device’s design.
While the results are promising, the researchers noted a few limitations. Their focus was mainly on biocompatibility and differentiation, without examining how signals from vascular cells might influence MSC growth at a molecular level.
The need for different media types meant vascular cells were introduced only after MSCs had begun differentiating, which the team aims to address by developing a common medium and conducting gene expression analysis.
Moving forward, researchers believe the 3D-PBR’s compact design, compatibility with standard lab equipment, and adaptability make it a valuable tool for studying bone formation, fat differentiation, and other tissue engineering applications requiring realistic environments.
Advances in 3D printed bioreactors
3D printing has improved bioreactor development by allowing researchers to quickly create compact, efficient designs that support better cell growth and interaction.
For instance, scientists from Massachusetts Institute of Technology (MIT) and Indian Institute of Technology (IIT) Madras developed a 3D printed microfluidic bioreactor for growing human brain tissue using SLA 3D printing and dental resin. Priced at just $5, the reusable device served as a low-cost alternative to commercial culture dishes for drug testing and treatment research.
During tests, stem cells cultured in the bioreactor showed enhanced proliferation and viability compared to those grown in conventional dishes. The cells developed into a neocortex-like structure with no decline in viability over seven days. Researchers plan to enhance the device with valves and pumps, aiming for efficient, affordable drug testing and pathogen interaction studies.
Argentinian biotechnology firm Stämm Biotech closed a $17 million Series A funding round to advance its 3D printed bioreactor development using its proprietary Brick Printing Technology and Sclereid 3D printer. The technology aimed to miniaturize traditional bioreactors into desktop-sized units, enhancing productivity by approximately 70 times.
Funds were intended to support workforce expansion to around 200 employees and international growth. Led by Varana with participation from several investors, the funding brings Stämm’s total to $20 million at the time, with pilot-scale commercialization planned in 2022.
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Featured image shows the fully assembled 3D-PBR system with the modular peristaltic pump and 3D printed cell culture media reservoir. Photo via LANL.
