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. 2024 Aug 21;11(8):854.
doi: 10.3390/bioengineering11080854.

Bovine Placentome-Derived Extracellular Matrix: A Sustainable 3D Scaffold for Cultivated Meat

Affiliations

Bovine Placentome-Derived Extracellular Matrix: A Sustainable 3D Scaffold for Cultivated Meat

Cemile Bektas et al. Bioengineering (Basel). .

Abstract

Cultivated meat, an advancement in cellular agriculture, holds promise in addressing environmental, ethical, and health challenges associated with traditional meat production. Utilizing tissue engineering principles, cultivated meat production employs biomaterials and technologies to create cell-based structures by introducing cells into a biocompatible scaffold, mimicking tissue organization. Among the cell sources used for producing muscle-like tissue for cultivated meats, primary adult stem cells like muscle satellite cells exhibit robust capabilities for proliferation and differentiation into myocytes, presenting a promising avenue for cultivated meat production. Evolutionarily optimized for growth in a 3D microenvironment, these cells benefit from the biochemical and biophysical cues provided by the extracellular matrix (ECM), regulating cell organization, interactions, and behavior. While plant protein-based scaffolds have been explored for their utilization for cultivated meat, they lack the biological cues for animal cells unless functionalized. Conversely, a decellularized bovine placental tissue ECM, processed from discarded birth tissue, achieves the biological functionalities of animal tissue ECM without harming animals. In this study, collagen and total ECM were prepared from decellularized bovine placental tissues. The collagen content was determined to be approximately 70% and 40% in isolated collagen and ECM, respectively. The resulting porous scaffolds, crosslinked through a dehydrothermal (DHT) crosslinking method without chemical crosslinking agents, supported the growth of bovine myoblasts. ECM scaffolds exhibited superior compatibility and stability compared to collagen scaffolds. In an attempt to make cultivate meat constructs, bovine myoblasts were cultured in steak-shaped ECM scaffolds for about 50 days. The resulting construct not only resembled muscle tissues but also displayed high cellularity with indications of myogenic differentiation. Furthermore, the meat constructs were cookable and able to sustain the grilling/frying. Our study is the first to utilize a unique bovine placentome-derived ECM scaffold to create a muscle tissue-like meat construct, demonstrating a promising and sustainable option for cultivated meat production.

Keywords: collagen; cultivated meat; dehydrothermal crosslinking (DHT); extracellular matrix; scaffolds; tissue engineering.

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Conflict of interest statement

Author Mohit Bhatia was employed by the company Atelier Meats. This study was partially supported by Atelier Meats, Corporation. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Figures

Figure 1
Figure 1
Schematic illustration of isolation of collagen and ECM from bovine placentomes.
Figure 2
Figure 2
Characterization and fabrication of isolated collagens and ECM. (A) The collagen content of isolated collagen or ECM was determined by hydroxyproline assay. The collagen content (%) = weight of calculated collagen/dry weight of sample. Data are shown as mean ± SD (n = 4). (B) A schematic representation of the crosslinking process is presented. (C) Images display collagen and ECM scaffolds both before and after crosslinking. (D) Bright-field images showcase the morphologies of ECM scaffolds before and after crosslinking. (E) Fluorescence staining against bovine collagen type I shows the fibrillar structure of the scaffolds after crosslinking. Scale bar = 100 µm. (F) Representative SEM images of non-crosslinked (non-XL) and DHT crosslinked (XL) scaffolds. Scale bar = 150 µm.
Figure 3
Figure 3
Bovine myoblast culture on collagen and ECM scaffolds. (A) A schematic representation of the bovine myoblast culture on the scaffolds. (B) The viability of bovine myoblasts cultured in crosslinked (XL) or non-crosslinked (NXL) scaffolds were monitored using alamarBlue assay for 14 days. The viability was expressed as the fluorescent intensity (arbitrary units). Data shown are mean ± SD (n = 3). * p < 0.05, ** p < 0.0, *** p < 0.005 compared with the viability of ECM + XL. (C) The viability of cells in crosslinked collagen scaffold (upper) and ECM scaffold (lower) was detected by CalceinAM staining. (D) The shapes of the scaffolds over time. The crosslinked scaffolds with cells were cultured for 1 day and 14 days. (E) ECM scaffolds were crosslinked using thermal crosslinking (Thermo. XL) or transglutaminase (Transgluta. XL). Viability of cells in crosslinked ECM scaffolds was monitored over time. Data shown are mean ± SD (n = 3) * p < 0.05, ** p < 0.01. (F) Images of scaffolds containing cells on Day 17. Scale bar = 200 µm.
Figure 4
Figure 4
Steak-shaped 3D-printed mold and cell culture in the scaffold. (A) Schematic representation of the fabrication process for the steak-shaped scaffold. (B) Images depict steak-shaped ECM scaffolds both before and after crosslinking (scale bar is 10 mm), along with their appearance during cell culture for 51 days (scale bar is 5 mm). (C) Calcein AM (green, (a)), Desmin (red, (b)), and Nuclei (blue, (c)) staining on Day 30. (D) Desmin (green, (a)) and Nuclei (blue, (b)) staining on Day 51. The scale bar is 100 μm.
Figure 5
Figure 5
Illustration of the proof-of-concept of frying lab-cultivated meat with cooking oil. (A) The cultivated meat before cooking (one small piece below was cut and removed from the whole piece for other testing). (B) Schematic illustration of frying. The representative image of the cultivated meat during (C) and after (D) frying.

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Grants and funding

This research was partially funded by Atelier Meats Corporation.

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