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.2021 Aug 24;12(1):5059.
doi: 10.1038/s41467-021-25236-9.

Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting

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Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting

Dong-Hee Kang et al. Nat Commun..

Abstract

With the current interest in cultured meat, mammalian cell-based meat has mostly been unstructured. There is thus still a high demand for artificial steak-like meat. We demonstrate in vitro construction of engineered steak-like tissue assembled of three types of bovine cell fibers (muscle, fat, and vessel). Because actual meat is an aligned assembly of the fibers connected to the tendon for the actions of contraction and relaxation, tendon-gel integrated bioprinting was developed to construct tendon-like gels. In this study, a total of 72 fibers comprising 42 muscles, 28 adipose tissues, and 2 blood capillaries were constructed by tendon-gel integrated bioprinting and manually assembled to fabricate steak-like meat with a diameter of 5 mm and a length of 10 mm inspired by a meat cut. The developed tendon-gel integrated bioprinting here could be a promising technology for the fabrication of the desired types of steak-like cultured meats.

© 2021. The Author(s).

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Conflict of interest statement

The authors D.-H.K., F.L., S.I., S.K., and M.M. declare the following financial competing interests: D.-H.K. and M.M. have filed the patent (PCT/JP2021/014104) related to the cell-printing technique described in the paper. F.L., S.I., S.K., and M.M. have filed the patents (PCT/JP2018/041659 and PCT/JP2020/012476) related to the adipogenesis of adipose-derived stem cells. F.L., S.K., and M.M. have filed the patent application (JP2020-196209) related to the endothelial differentiation of adipose-derived stem cells described in this paper. This research was also supported by TOPPAN INC. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Overview of the work.
a Structure of steak. (i, ii) H&E- and (iii) Azan-stained images of a piece of steak. Representative images from three independent experiments are shown. All scale bars denote 100 μm (iv) Schematic of a hierarchical structure in muscle.b Schematic of the construction process for cultured steak. The first step is cell purification of tissue from cattle to obtain bovine satellite cells (bSCs) and bovine adipose-derived stem cells (bADSCs). The second is supporting bath-assisted printing (SBP) of bSCs and bADSCs to fabricate the muscle, fat, and vascular tissue with a fibrous structure. The third is the assembly of cell fibers to mimic the commercial steak’s structure. *SVF stromal vascular fraction.
Fig. 2
Fig. 2. Verification of purified bovine stem cells.
a, b Proliferation rate (n = 3 independent samples) (a) and differentiation ratio (n = 4 independent areas examined over three independent samples) on day 5 of differentiation (b) of bSCs from passage 3 (P3) to P12 cultured on a tissue culture plate. Red and blue lines are a slope from P3 to P8 and from P8 to P12, respectively.c Representative immunofluorescence images of differentiation induced bSCs at P7 and P9 stained for myosin II heavy chain (MHC) (green) and nucleus (blue) from at least three independent experiments. Scale bars, 1 mm.d Adipogenesis ratio (left) of 3D gel-drop-cultured bADSCs derived by 12 combinations of free fatty acids (middle) in DMEM on days 5, 9, and 13 (n = 4 independent experiments, two-way ANOVA paired for the time and unpaired for the treatment with a Tukey’s HSD post test).e, f Lipid-droplet production in 3D-cultured bADSCs, depending on the concentration of ALK5i on day 7 (e) and culture day (f) in the #1 combination of free fatty acids and 5 μM (n = 5 (e) and 3 (f) independent experiments, unpaired (e) and paired (f) one-way ANOVA with a Tukey’s HSD post test).g, h Representative immunofluorescence images from three independent experiments (g) and mRNA expression levels (h) of 3D bADSCs tissue cultured with the media containing seven free fatty acid mixture (#1) and 5 μM ALK5i (n = 3 independent experiments, paired one-way ANOVA with a Tukey’s HSD post test).i, j CD31 immunostaining quantitation of bADSCs in 2D, depending on serum conditions in DMEM (i) and base media (j) on day 7 (n = 3 independent experiments, unpaired one-way ANOVA with a Tukey’s HSD post test (i) and unpaired two-way ANOVA with a Šidák post test (j)).k Representative immunofluorescence images of bADSCs depending on serum conditions on day 7 stained for CD31 (magenta) and nucleus (blue) from three independent experiments. Scale bars, 100 μm. The used bADSCs were extracted from subcutaneous fat. *p<0.05, **p<0.01, ***p<0.001; error bars represent mean ± s.d. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. The characterization of bSC tissue fabricated by SBP.
a, b Optical (left), phase contrast (middle), and fluorescence (right, green: live cells and red: dead cells) images of the bSC tissues printed inside granular gellan gum (G-GG) (a) and granular gelatin (G-Gel) (b) followed by bath removal. Scale bars, 500 µm.c Shape change of bSC tissue fabricated by SBP inside G-Gel from the fibrous form right after printing and bath removal to globular form on day 6 of suspension culture.d Schematic (left), size change in accordance with culture day (middle), and phase-contrast images (right) of needle fixed culture of printed bSCs tissues. Error bars represent mean ± s.d. Scale bars, 500 µm.e 3D-fluorescence images (upper, red: actin and green: MHC) and cell alignment measurements (lower) of the bSC tissues printed inside G-GG and G-Gel and in suspension and needle-fixed cultures on day 3 of differentiation (after six days), respectively. Representative images from at least two independent experiments are shown. Scale bars, 200 µm. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Tendon-gel integrated bioprinting (TIP) for muscle, fat, and vascular tissue fabrication.
a The schematic of TIP for cell printing.b Optical (upper) and phase-contrast (lower) images of the bSC tissue printed by TIP, keeping the fibrous structure on day 3. The images were taken after fixation. Scale bar, 1 mm.c The H&E-stained image of half of collagen gel (dotted black line)—fibrous bSC tissue (dotted red line) and a magnified image of the fibrous bSC tissue (right). Scale bars, 2 mm (left) and 50 µm (right).d 3D fluorescence image (left) and cell alignment measurement (right) of the TIP-derived bSC tissue stained with actin (red), MHC (green), and nucleus (blue) on day 3 of differentiation. Scale bar, 50 µm.e SEM images of TIP-derived bSC tissue on day 3 of differentiation. Scale bars, 10 µm and 100 µm (inset).f MHC mRNA expression levels of bSCs before printing and TIP-derived bSC tissue on day 3 of differentiation (n = 3 independent samples, pairwise t-test comparison).g Fluorescence image of TIP-derived bSCs tissue stained with actin (red), MHC (green), and nucleus (blue) on day 14 of differentiation. Scale bar, 50 µm.h The optical images of multiple tissue fabrication (25 ea.) by multiple printing. Black arrows indicate printed cell fibers.i, j mRNA levels (i) and protein expression levels (j) of TIP-derived fat tissues before printing and at day 14 of differentiation (at day 17 of total culture) (n = 3 independent samples, pairwiset-test comparison).k Whole fluorescence (left), optical (inset), and magnified (right) images of muscle (on day 4 of differentiation, green: MHC & blue: nucleus), fat (on day 14 of differentiation, red: lipid and blue: nucleus), and vascular (on day 7, magenta: CD31 and blue: nucleus) tissues fabricated by TIP. Scale bars, 1 mm (left) and 100 µm (right).l, m DNA amount per weight (light-gray bars: day 1, and dark-gray bars: day 6 in muscle fiber and day 17 in fat fiber) and (l) compressive modulus (m) of muscle and fat fibers in the commercial meat (white bar) and TIP-derived (gray bars). The modulus of the muscle fiber on day 3 of differentiation (after 6 days) and the fat fiber on day 7 of differentiation (after 10 days) was measured (n = 3 independent samples, paired one-way ANOVA with a Tukey’s HSD post test (l) and pairwiset-test comparison (m)). *p<0.05, **p<0.01, ***p<0.001; error bars represent mean ± s.d. Representative images from at least two independent experiments are shown. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Assembly of fibrous muscle, fat, and vascular tissues to cultured steak.
a Assembly schematic- (right) based sarcomeric α-actinin (blue) and laminin- (brown) stained image (left) of the commercial meat. It is assumed that the diameters of the fibrous muscle, fat, and vascular tissues are about 500, 760, and 600 µm, respectively. Scale bar, 1 mm.b, c Optical images of the cultured steak by assembling muscle (42 ea.), fat (28 ea.), and vascular (2 ea.) tissues at (b) the top and (c) cross-section view of the dotted-line area. Muscle and vascular tissue were stained with carmine (red color), but fat tissue was not. Scale bars, 2 mm.
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