Global food security is a critical concern, demanding sustainable protein sources. Cultured meat offers a promising alternative to traditional animal agriculture, reducing environmental impact. However, identifying the optimal cell type for large-scale cultured meat production remains a challenge. Satellite cells, traditionally used, have limited proliferation and differentiation capabilities. Embryonic and induced pluripotent stem cells offer high proliferation but present challenges in differentiation and potential genetic modification concerns. Mesenchymal stromal cells (MSCs), abundant in adipose tissue (AT), are emerging as a promising alternative due to their proliferation potential and multi-lineage differentiation capacity, including myogenic differentiation. Adipose tissue is readily available, making MSC isolation from this source appealing. While various enzymatic methods exist, their efficiency in isolating bovine AT-MSCs has not been systematically compared. This study aimed to optimize the enzymatic digestion protocol for isolating bovine AT-MSCs to maximize cell yield, a crucial factor for cultured meat production. The focus is on enzymatic methods due to their advantages over explant cultures in terms of reproducibility, cost, speed, and automation potential. The study evaluated various enzymes, concentrations, and incubation times based on a literature review to determine the most effective protocol.

A literature search revealed that Collagenase type I is the most frequently used enzyme for isolating AT-MSCs across various species, with typical concentrations around 1 mg/mL and incubation times of 3 hours. Other enzymes reported include trypsin, Collagenase types II and IV, and Liberase™. Some studies combined Collagenase I with trypsin. This review informed the selection of 32 different conditions to be tested in this study, systematically varying the enzyme, concentration, and incubation time to optimize the isolation process for bovine AT-MSCs. The goal was to identify protocols that yielded a sufficient number of viable cells for downstream applications, such as myogenic differentiation for cultured meat production.

Subcutaneous adipose tissue samples were collected from eight adult female Belgian White Blue cows. The tissue was minced and digested using 32 different enzymatic conditions: four enzymes (Collagenase type I, Collagenase type I + Trypsin, Liberase™, and Collagenase type IV), two concentrations (0.1% and 0.04%), and four incubation times (3 h, 6 h, overnight, and 24 h). Successful isolation was defined by the observation of cells within 21 days, a minimum cell yield of 1 x 10⁶ cells/g AT at passage 1 (P1), and cell viability exceeding 95%. Spindle-shaped, plastic-adherent cells were observed under microscopy. Cell yield was quantified. Protocols achieving high yields (at least 35 x 10⁶ cells/g AT from at least five donors) were selected for further characterization. The proliferation potential was assessed by calculating the population doubling time across eight passages. Tri-lineage differentiation potential (adipogenic, osteogenic, and chondrogenic) was evaluated using histological staining and image analysis. Immunophenotyping was performed using multi-color flow cytometry to assess the expression of MSC surface markers. Finally, myogenic differentiation was induced in cells isolated using the optimal protocol, using 5-aza-2'-deoxycytidine and galectin-1. Myogenic differentiation was assessed by changes in cell morphology, mRNA expression of myogenic regulatory factors (MYF5, MYOD1, MYF6, MYOG, and PAX3), and immunofluorescence staining for desmin, tropomyosin, and myosin heavy chain.

The highest cell yield combined with a low population doubling time was achieved using Liberase™ at 0.1% for 3 hours. This condition yielded cells from all eight donors, ranging from 30.48 x 10⁶ to 67.1 x 10⁶ cells/g AT. Other conditions also yielded sufficient cells for further analysis but not consistently across all donors. No significant differences were observed in proliferation potential, tri-lineage differentiation capacity, or immunophenotype between the selected high-yield conditions. Myogenic differentiation was successful, showing decreased PAX3 mRNA expression and increased expression of MYF5, MYOD1, MYF6, and MYOG compared to undifferentiated MSCs. Immunofluorescence confirmed the presence of desmin, tropomyosin, and myosin heavy chain in differentiated cells. Collagenase type IV consistently showed lower yields, possibly because collagen type IV is less abundant in subcutaneous bovine AT. Neither enzyme concentration nor incubation time significantly affected cell yield, although longer incubation times (overnight and 24 hours) generally resulted in lower cell yields. The population doubling time using Liberase™ 0.1% for 3 and 6 hours remained low across eight passages, indicating that the proliferation potential was maintained. The study demonstrated that bovine AT-MSCs isolated with the optimized protocol can undergo successful adipogenic and myogenic differentiation, supporting their potential as a cell source for cultured meat.

The results demonstrate that Liberase™ at 0.1% for 3 hours is the optimal enzymatic condition for isolating bovine AT-MSCs, maximizing both cell yield and maintaining their differentiation potential. The superior performance of Liberase™ compared to other enzyme mixtures might be attributed to its balanced composition, effectively degrading the extracellular matrix without excessive damage to the cells. The consistency of results across donors highlights the robustness and reproducibility of this protocol. The successful myogenic differentiation of these cells confirms their suitability as a potential cell source for cultured meat production. Future work should focus on scaling up the process for industrial applications and further optimizing the myogenic differentiation towards mature, well-structured muscle fibers to enhance the texture and quality of cultured meat products.

This study provides a significantly improved protocol for the efficient isolation of bovine adipose tissue-derived mesenchymal stromal cells (MSCs) suitable for cultured meat production. The use of 0.1% Liberase™ for 3 hours yields a high cell number from all donors, preserving proliferation and differentiation potential. Future research should investigate the scalability of this method for industrial applications and explore strategies to enhance myogenic differentiation and mature muscle fiber formation.

The study was limited to subcutaneous adipose tissue from a specific breed of cattle (Belgian White Blue). The generalizability of the findings to other adipose tissue depots or cattle breeds may require further investigation. While the myogenic differentiation was successful, the resulting myotubes were immature, indicating a need for further optimization of the differentiation protocol to achieve mature muscle fibers.