In a recent study published in Trends in Biotechnology, researchers developed a novel hollow fiber bioreactor (HFB) to generate chicken skeletal muscle tissues, exemplifying large-scale tissue engineering. The bioreactor enables centimeter-scale production of tissues with active perfusion through vessel-mimicking channels that facilitate myoblast differentiation.
Automating the HFB technology could revolutionize lab-grown meat production, paving the way for large-scale engineering of body organs and biohybrid robots.
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Introduction
Large-scale skeletal muscles can address food and environmental issues in cellular agriculture. Creating well-distributed vascular networks is critical for large-scale tissue reconstruction.
Existing perfusable networks are limited in size, sparsity, and uniformity, affecting nutrient supply. Current HFBs lack anchors to prevent tissue shrinkage and achieve uniform spacing.
In highly cellularized thick tissues that lack an integrated vascular system, nutrient diffusion may slow down, potentially leading to necrosis. Consequently, the thickness of tissues without integrated vascular networks is limited to less than 1.0 mm. The limited tissue thickness makes large-scale tissue engineering challenging.
About the Study
In the present study, researchers present a top-down approach for creating centimeter-scale cultured chicken meat using a hollow fiber bioreactor with myoblast-laden hydrogels and uniformly spaced hollow fibers, nurturing the tissue uniformly.
The team used high-resolution stereolithography to develop an HFB with 50 semipermeable polyethersulfone hollow fibers. They subjected the fibers to high internal pressures to form closely packed myotubes for nutrient delivery. Microfabricated anchors held the hollow fibers in a three-dimensional lattice-style framework.
In the lattice, cells were present within a 350 μm range from the outer surface of the hollow fibers. The fibers' internal diameter was 200 μm-250 μm, and their outer diameter was 300 μm-350 μm.
The distance between the centers of adjacent fibers (pitch distance) was 0.90 mm. Hydrogel precursors filled spaces between lattice frames, anchoring the tissue.
The lattice underwent morphological, functional, and nutritional analyses for cultured meat applications. The team analyzed cell density to determine optimal tissue perfusion with 15 μl/minute, 100 μl/minute, and 500 μl/minute flow rates.
They performed myosin heavy chain (MyHC) immunostaining to study tissue morphology and analyzed α-actinin expression to assess protein function.
To evaluate performance, the researchers manually assembled HFBs to produce 2 cm × 1 cm × 5 mm chicken meat tissues. To assess scalability, they constructed a robotically assembled 1125-fiber bioreactor measuring 7.00 cm × 4.05 cm × 2.25 cm, imaged using microcomputed tomography (μCT).
The HFB produced whole-cut chick meat from the University of Minnesota, St. Paul/DF-1 chicken embryo fibroblast cells (UMNSAH/DF-1). Electrical stimulations investigated the contractile function of the fabricated meat. Texture profile analysis (TPA) and free amino acid (FAA) analysis indicated texture and flavor.
Results
The lattice geometry of the closely arranged hollow fibers held in place by microfabricated anchors enables unidirectional tension for myotube alignment and uniform nutrient delivery throughout the tissue.
The HFB technology enables leak-free perfusion of the fibers under a high internal pressure of 3.8 kPa. This feature is crucial as it enhances cell activity near the hollow fibers, ensuring that cells receive adequate oxygen and nutrients for their growth and function.
The team achieved high cellular nuclei densities (more than 103 cells per mm2) at a flow rate of 500 μl/minute. Active perfusion enhanced protein expression and sarcomere formation across the biologically fabricated chicken muscle tissue, enhancing its texture and flavor.
The perfusion system enhances the structural integrity and functionality of the biofabricated tissues, which is particularly important for applications in cultured meat. In addition, the fabricated tissue exhibited improved metabolic activity, as indicated by significant glucose consumption and lactate production during culture.
Electrical stimulations confirmed contractile function, a property affected by cell alignment and optimized by the microfabricated anchors. The fabricated meat closely mimics the qualities of traditional meat products, making it more appealing for consumption.
The robot-assisted assembly system produced 11g of centimeter-scale whole-cut chicken meat, demonstrating scalability crucial for large-scale tissue production to meet the demands of the cultured meat industry and other tissue engineering applications.
The HFB system demonstrates more uniform nutrient distribution and tissue morphology than traditional methods. The microscale uniformity in fibers reduces cell death (necrosis) in large-scale perfusion cultured tissues, a common challenge in tissue engineering.
The spatial arrangement enhances the morphological consistency of the tissues to levels far exceeding those previously achieved.
Conclusions and Future Outlook
The study demonstrated the top-down approach for fabricating centimeter-scale chicken skeletal muscle tissues using HFB. Compared to the bottom-up technique of modular assembly that relies on tissue glues to gather smaller tissue modules, the proposed top-down technique offers a more efficient fabrication solution.
The top-down method enables the one-stop production of cultured meats with uniformly distributed muscle fibers and perfusable channels that mimic in vivo interstitial flow, a significant advancement in tissue engineering.
Using edible fibers and recyclable materials could improve the practicality and environmental impact of the HFB technology.
To improve cost-effectiveness, researchers must optimize bioreactor layouts for cell expansion and tissue growth, reduce dependence on costly inputs like fetal bovine sera, and minimize single-use culture equipment.
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