Aggregating in vitro-grown adipocytes to produce macroscale cell-cultured fat tissue with tunable lipid compositions for food applications
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Curated by eLife
eLife assessment
This manuscript describes an approach to creating fat tissue in culture for food applications. Specifically, the efforts of growing cultivated meat focus mostly on growing skeletal muscle. However, the taste component of such artificial meat would be determined by fat content. There is a significant desire and motivation to cultivate fat tissues in vitro for the purpose of the replacement of animal products. This paper provides new technological approaches to expand adipocytes and aggregate them into structures that resemble fat.
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Abstract
We present a method of producing bulk cell-cultured fat tissue for food applications. Mass transport limitations (nutrients, oxygen, waste diffusion) of macroscale 3D tissue culture are circumvented by initially culturing murine or porcine adipocytes in 2D, after which bulk fat tissue is produced by mechanically harvesting and aggregating the lipid-filled adipocytes into 3D constructs using alginate or transglutaminase binders. The 3D fat tissues were visually similar to fat tissue harvested from animals, with matching textures based on uniaxial compression tests. The mechanical properties of cultured fat tissues were based on binder choice and concentration, and changes in the fatty acid compositions of cellular triacylglyceride and phospholipids were observed after lipid supplementation (soybean oil) during in vitro culture. This approach of aggregating individual adipocytes into a bulk 3D tissue provides a scalable and versatile strategy to produce cultured fat tissue for food-related applications, thereby addressing a key obstacle in cultivated meat production.
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eLife assessment
This manuscript describes an approach to creating fat tissue in culture for food applications. Specifically, the efforts of growing cultivated meat focus mostly on growing skeletal muscle. However, the taste component of such artificial meat would be determined by fat content. There is a significant desire and motivation to cultivate fat tissues in vitro for the purpose of the replacement of animal products. This paper provides new technological approaches to expand adipocytes and aggregate them into structures that resemble fat.
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Reviewer #1 (Public Review):
This paper provides new technological approaches to expand adipocytes and aggregate them into structures that resemble fat. The authors use two cell types: a mouse cell line, as well as primary porcine cells. They demonstrate excellent lipid droplet accumulation in the mouse cell line however, this does not have translational relevance. So they go on to also perform those same experiments with the porcine cell line. The results are also encouraging especially if the cultivation is carried out over a period of 97 days.
The authors also demonstrate similar mechanically mechanical properties of their cultivated fat to the native fat as well as the ability to aggregate it using two different approaches.Overall, I think this is a thorough manuscript in the area of food bioengineering. The limitations remain the …
Reviewer #1 (Public Review):
This paper provides new technological approaches to expand adipocytes and aggregate them into structures that resemble fat. The authors use two cell types: a mouse cell line, as well as primary porcine cells. They demonstrate excellent lipid droplet accumulation in the mouse cell line however, this does not have translational relevance. So they go on to also perform those same experiments with the porcine cell line. The results are also encouraging especially if the cultivation is carried out over a period of 97 days.
The authors also demonstrate similar mechanically mechanical properties of their cultivated fat to the native fat as well as the ability to aggregate it using two different approaches.Overall, I think this is a thorough manuscript in the area of food bioengineering. The limitations remain the ability to fully remove FBS during this production process.
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Reviewer #2 (Public Review):
This work describes a new method to create three-dimensional macroscale fat tissues derived from adipocytes cultured in two-dimensional monolayers. By scraping the differentiated adipocytes from the tissue culture plastic and mixing them with an edible binding material, they have created fat tissues that demonstrate similar mechanical properties to native animal tissue. Additionally, using lipidomics, the authors demonstrate that lipid treatment of the cultured adipocytes modifies their fatty acid composition in the triglyceride as well as the phospholipid portions. The fatty acid profiles of the cultured adipocytes are then compared to those of native animal fat tissues.
Strengths:
This paper addresses the relevant issue of the development of a hypoxic and necrotic core during the culture of large …
Reviewer #2 (Public Review):
This work describes a new method to create three-dimensional macroscale fat tissues derived from adipocytes cultured in two-dimensional monolayers. By scraping the differentiated adipocytes from the tissue culture plastic and mixing them with an edible binding material, they have created fat tissues that demonstrate similar mechanical properties to native animal tissue. Additionally, using lipidomics, the authors demonstrate that lipid treatment of the cultured adipocytes modifies their fatty acid composition in the triglyceride as well as the phospholipid portions. The fatty acid profiles of the cultured adipocytes are then compared to those of native animal fat tissues.
Strengths:
This paper addresses the relevant issue of the development of a hypoxic and necrotic core during the culture of large three-dimensional structures. The authors describe a straightforward method to bypass the three-dimensional cell culture by assembling their macroscale fat tissues after the adipocytes have fully differentiated in a two-dimensional monolayer.
The authors use two different binders to assemble their fat tissues, alginate, and microbial transglutaminase, both GRAS-registered. As the authors recognized, in the field of cultivated fat production for food consumption, it is essential to use materials that result in an edible product. Importantly, the authors demonstrate with mechanical testing that the binder material is of more significance to the mechanical properties of the macroscale fat tissue than the degree of lipid accumulation of the adipocytes.
The authors describe a detailed fatty acid composition profile of murine and porcine cultured adipocytes, treated and untreated with Intralipid, and native fat tissues. This dataset gives valuable insight into the effect of lipid treatment on fatty acid composition.
Weaknesses:
In the introduction, the authors hypothesize that their approach reproduces the taste of native fat and describe that fatty acid composition provides insight into flavor. The paper does not provide an analysis of taste to test this hypothesis and the lipidomics data does not provide data on the flavor profile of the aggregated macroscale fat tissues. In the abstract, the authors describe that the 3D fats were visually similar based on uniaxial compression tests. However, this test does not describe visual similarity.
The authors describe that detachment of adipocytes during differentiation was avoided by carefully replacing media and adipocytes had to be scraped off the flask even after increased lipid accumulation as a result of Intralipid treatment in the porcine adipocytes. Cell detachment of adipocytes on tissue culture plastic is a common phenomenon limiting the long-term culture of adipocytes in 2D. It could be useful for the field if the authors could describe in more detail how they avoided cell detachment during adipocyte differentiation or if they could hypothesize why they did not observe this phenomenon.
The authors compare the fatty acid composition of cultured adipocytes to that of native animal fat tissue. In the discussion, the authors describe that genetics and diet likely have an influence on the fatty acid composition profile of animal fat tissue. To be able to understand better what the effect is of Intralipid treatment, and to determine if this treatment brings the fatty acid composition of cultured adipocytes closer to their native counterpart, the authors could have cultured adipocytes in vitro from cells derived from the same animals as those that provided the native animal fat tissue.
In the discussion, the authors claim that the aggregate of adipocytes after scraping looked like fat tissue. This claim is not supported by lipid staining of cryosections of these aggregates, which makes it not possible to visually compare to the images of cryosectioned native animal tissue.
At the end of the discussion, the authors imply that their macroscale aggregation concept can be applied to scalable bioreactor-based cell culture strategies. However, the authors do not demonstrate how their method of scraping adipocytes from a tissue culture flask (low degree of scalability) applies to the potential of combining large amounts of adipocytes cultured on microcarriers in suspension bioreactors (high degree of scalability). The authors have not addressed the limited scalability of monolayer cell expansion which is a significant part of their approach.
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