Decellularised plant scaffolds facilitate porcine skeletal muscle tissue engineering for cultivated meat biomanufacturing.

Cultivated meat (CM) offers a sustainable and ethical alternative to conventional animal agriculture, involving cell maturation in a controlled environment. To emulate the structural complexity of traditional meat, the development of animal-free and edible scaffolds is crucial, providing vital physical and biological support during tissue development. The aligned vascular bundles of the decellularised asparagus scaffold were selected to facilitate the attachment and alignment of murine myoblasts (C2C12) and porcine adipose-derived mesenchymal stem cells (pADMSCs). Muscle differentiation was assessed through immunofluorescence staining with muscle markers, including Myosin heavy chain (MHC), Myogenin (MYOG), and Desmin. The metabolic activity of Creatine Kinase in C2C12 differentiated cells significantly increased compared to proliferated cells. Quantitative PCR analysis revealed a significant increase in Myosin Heavy Polypeptide 1 (MYH1) and MYOG expression compared to Day 0. These results highlight the application of decellularised plant scaffold (DPS) as a promising, edible material conducive to cell attachment, proliferation, and differentiation into muscle tissue. To create a CM prototype with biological mimicry, pADMSC-derived muscle and fat cells were also co-cultured on the same scaffold. The co-culture was confirmed through immunofluorescence staining of muscle markers and LipidTOX staining, revealing distinct muscle fibres and adipocytes containing lipid droplets respectively. Texture profile analysis conducted on uncooked CM prototypes and pork loin showed no significant differences in textural values. However, the pan-fried CM prototype differed significantly in hardness and chewiness compared to pork loin. Understanding the scaffolds' textural profile enhances our insight into the potential sensory attributes of CM products. DPS shows potential for advancing CM biomanufacturing.

A pump-free tricellular blood-brain barrier on-a-chip model to understand barrier property and evaluate drug response.

Disruption of the blood-brain barrier (BBB) leads to various neurovascular diseases. Development of therapeutics required to cross the BBB is difficult due to a lack of relevant in vitro models. We have developed a three-dimensional (3D) microfluidic BBB chip (BBBC) to study cell interactions in the brain microvasculature and to test drug candidates of neurovascular diseases. We isolated primary brain microvascular endothelial cells (ECs), pericytes, and astrocytes from neonatal rats and cocultured them in the BBBC. To mimic the 3D in vivo BBB structure, we used type I collagen hydrogel to pattern the microchannel via viscous finger patterning technique to create a matrix. ECs, astrocytes, and pericytes were cocultured in the collagen matrix. The fluid flow in the BBBC was controlled by a pump-free strategy utilizing gravity as driving force and resistance in a paper-based flow resistor. The primary cells cultured in the BBBC expressed high levels of junction proteins and formed a tight endothelial barrier layer. Addition of tumor necrosis factor alpha to recapitulate neuroinflammatory conditions compromised the BBB functionality. To mitigate the neuroinflammatory stimulus, we treated the BBB model with the glucocorticoid drug dexamethasone, and observed protection of the BBB. This BBBC represents a new simple, cost-effective, and scalable in vitro platform for validating therapeutic drugs targeting neuroinflammatory conditions.

Rapid construction of mechanically- confined multi- cellular structures using dendrimeric intercellular linker.

Tissue constructs that mimic the in vivo cell-cell and cell-matrix interactions are especially useful for applications involving the cell- dense and matrix- poor internal organs. Rapid and precise arrangement of cells into functional tissue constructs remains a challenge in tissue engineering. We demonstrate rapid assembly of C3A cells into multi- cell structures using a dendrimeric intercellular linker. The linker is composed of oleyl- polyethylene glycol (PEG) derivatives conjugated to a 16 arms- polypropylenimine hexadecaamine (DAB) dendrimer. The positively charged multivalent dendrimer concentrates the linker onto the negatively charged cell surface to facilitate efficient insertion of the hydrophobic oleyl groups into the cellular membrane. Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular structures within minutes. The cells exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and other functions in the constructs. We constructed defined multi- cellular structures such as rings, sheets or branching rods that can serve as potential tissue building blocks to be further assembled into complex 3D tissue constructs for biomedical applications.