Acetate cellulose fibrous scaffold is suitable for cultivated fat production

Abstract

Fat is an essential component of meat which contributes to its sensory characteristics. Therefore, producing cultivated fat is essential to replicate the texture, flavor, and juiciness of conventional meat. One of the challenges in obtaining cultivated fat is that once adipocytes reach differentiation in culture, they tend to float. In this study, we tested whether immortalized pre-adipocytes could be viable, grow, and differentiate when cultivated onto a fibrous scaffold produced by the electrospun of cellulose acetate. Our results demonstrated that the cells attach, proliferate, colonize, and differentiate into mature adipocytes in the three-dimensional fibrous structure during the culture period. Moreover, when layers of the scaffold containing differentiated cells were stacked, it acquired a characteristic similar to conventional animal fat. Therefore, this research suggests that fibrous scaffolds produced using cellulose acetate are a promising substrate for producing cultivated fat.

Keywords: Adipocytes; Adipogenic differentiation; Cellulose acetate; Cultivated fat; Electrospinning; Scaffold.

Graphical abstract

Fig. 1

Survival rate of 3T3-L1 cells on the CAN scaffold analyzed at 24 (A–C) and 72 h (D–F). Live/dead staining (green = live cells; red = dead cells). Scale bar: 100 μm. (G) - Cell viability through the MTT assay of 3T3-L1 cells on the CAN scaffold over 24h, 48h and 72 h. Mean ± SD ∗∗∗∗p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Cell infiltration of 3T3-L1 cells growth on CAN scaffolds. SEM images after 1day (A), 3 days (D), 7 days (G), 10 days (J) and 15 days (M) of culture, CAN: arrow; cells: arrow head, scale bar: 50 μm. Bodipy Fluoresnce imagens showing nucleus (blue), CAN and lipid drops (both green) after 1day (B), 3 days (E), 7 days (H), 10 days (K) and 15 days (N) of culture, CAN: arrow; lipid drops: arrow head, scale bar: 50 μm. Confocal Z-stack images showing nucleus (blue), CAN and lipid drops (both green) after 1day (C), 3 days (F), 7 days (I), 10 days (L) and 15 days (O) of culture. Cell infiltration quantification on CAN, through analysis of the range of DAPI fluorescence of 1, 2, 3, 7, 10, 12 and 15 days of culture in the z-axis using confocal microscopy (P). Results are presented as mean ± SD, p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Confocal images of 3T3-L1 adipogenic differentiation on CAN scaffold on 7 (A), 10 (B), 12 (C) and 15 days (D). Bodipy Fluoresnce imagens (green lipid drops and green nanofibers) with DAPI (blue nuclei) lipid drops: arrow; undifferentiated cell nucleus: arrowhead. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

– Adipogenic differentiation of 3T3-L1 in monolayer (A, B, C, D, E) and CAN scaffold (G, H, I, J, K) evaluated by Oil red O staining at different differentiation times - Day 4 (A, G), Day 7 (B, H), Day 10 (C, I), Day 12 (D, J), Day 15 (E, K). Inverted light microscope image (magnification 20×) and magnifying glass, scale bar: 2 mm. Percentage of lipid accumulation by quantification of Oil Red O in monolayer (F) and CAN scaffold (L) after 4, 7, 10, 12 and 15 days. Results are presented as mean ± SD, p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

SEM image of 3T3-L1 cells in CAN scaffold during adipogenic differentiation. Images of 1 (A), 5 (B), 7 (C), 10 (D), 12 (E) and 15 days (D). Arrows: flat cells, Arrows head: spherical cells. Scale bar: 50 μm.

Fig. 6

– Expression levels of adipogenic-specific genes in 5, 10 and 15 days were compared to expression levels at 1 day in GM. Data is represented as mean ± SEM; ∗∗p < 0.01 and ∗∗∗p < 0.001.

Fig. 7

Tissue development by stacking CAN scaffolds seeding with 3T3-L1 cells after 15 days of cultivation. (A) Macroscopic image of stacking. (B) Tissue fat fried. Scale bar: 2 mm.