. 2021 Sep 21;10(9):2499. doi: 10.3390/cells10092499.
Affiliations
Affiliations
- 1 Intractable Disease Research Center, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
- 2 Department of Biochemistry and Biophysics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo 1130-8510, Japan.
- 3 Institute of Research, Tokyo Medical and Dental University (TMDU), Tokyo 113-8510, Japan.
- 4 Department of Research and Development for Organoids, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
- 5 Division of Gastroenterology, Hepatology and Nutrition, Division of Developmental Biology, Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children Hospital Medical Center, Cincinnati, OH 45229-3039, USA.
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Yuna Naraoka et al. Cells. 2021.
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. 2021 Sep 21;10(9):2499. doi: 10.3390/cells10092499.
Affiliations
- 1 Intractable Disease Research Center, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
- 2 Department of Biochemistry and Biophysics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo 1130-8510, Japan.
- 3 Institute of Research, Tokyo Medical and Dental University (TMDU), Tokyo 113-8510, Japan.
- 4 Department of Research and Development for Organoids, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
- 5 Division of Gastroenterology, Hepatology and Nutrition, Division of Developmental Biology, Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children Hospital Medical Center, Cincinnati, OH 45229-3039, USA.
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Abstract
The current process of meat production using livestock has significant effects on the global environment, including high emissions of greenhouse gases. In recent years, cultured meat has attracted attention as a way to acquire animal proteins. However, the lack of markers that isolate proliferating cells from bovine tissues and the complex structure of the meat make it difficult to culture meat in a dish. In this study, we screened 246 cell-surface antibodies by fluorescence-activated cell sorting for their capacity to form colonies and their suitability to construct spheroid “meat buds”. CD29+ cells (Ha2/5 clone) have a high potency to form colonies and efficiently proliferate on fibronectin-coated dishes. Furthermore, the meat buds created from CD29+ cells could differentiate into muscle and adipose cells in a three-dimensional structure. The meat buds embedded in the collagen gel proliferated in the matrix and formed large aggregates. Approximately 10 trillion cells can theoretically be obtained from 100 g of bovine tissue by culturing and amplifying them using these methods. The CD29+ cell characteristics of bovine tissue provide insights into the production of meat alternatives in vitro.
Keywords: CD29; Ha2/5; adipogenic differentiation; culture meat; flow cytometry; mesenchymal stem/stromal cells.
Conflict of interest statement
The authors declare no competing interest.
Figures
Figure 1
Isolation of colony-forming cells by…
Figure 1
Isolation of colony-forming cells by cell surface markers. ( a ) Representative flow-cytometric…
Figure 1
Isolation of colony-forming cells by cell surface markers. (a) Representative flow-cytometric profiles of bovine muscle tissue stained with CD29 (Ha2/5 clone) antibody. Phase-contrast micrographs of Ha2/5 positive cells. Scale bar = 300 μm. (b) Analysis of colony-forming capacity of the Ha2/5+ fraction. Two thousand CD29+, CD29–, and all living cells were sorted and cultured in 10 cm dishes for 14 days. The cells were stained with crystal violet, and the colonies were counted. (c) CD29+ cells were cultured on fibronectin-coated (line) and non-coated (dot-line) culture dishes. The graph shows the cultured cell numbers during 21 days. Results are expressed as mean ± SE (n = 3). * p < 0.05. (d) Differentiation capacity of CD29+ cells. Scale bar = 500 μm. (e) The expression of CD56 and CD90 cell surface markers in CD29 positive fraction (red). Isotype control is used for negative samples (blue).
Figure 2
Differentiation of CD29+ cells to…
Figure 2
Differentiation of CD29+ cells to myogenic linage. ( a ) Scheme of CD29+…
Figure 2
Differentiation of CD29+ cells to myogenic linage. (a) Scheme of CD29+ cell differentiation. (b) Immunohistochemical analysis of induced cell differentiation. Desmin (a marker of muscle cells) is shown in red; BODIPY (indicating lipid droplets) is shown in green; and Hoechst-stained nuclei are shown in blue. (c,d) Relative expression of myogenic genes: DESMIN, MYOD, MYOSIN, PAX7, and adipogenic genes: CEBPα, CEBPβ, PPARγ, LEPTIN and ADIPONECTIN in CD29 cells by real-time RT-PCR (muscle: CD29+ cells from muscle tissue cultured without passage, adipo; CD29+ cells from adipo tissue cultured without passage, normal; non-induced cells, muscle differentiation; muscle-induced cells). Using one-way ANOVA with a Bonferroni correction, * p < 0.05 or ** p < 0.001; compared with muscle cells, †p < 0.05, ††p < 0.001; compared with control. (n = 4).
Figure 3
An analysis of the spheroid-forming…
Figure 3
An analysis of the spheroid-forming capacity of Ha2/5+ cells. ( a ) Ha2/5…
Figure 3
An analysis of the spheroid-forming capacity of Ha2/5+ cells. (a) Ha2/5 cells (1 × 105) were seeded in 96-well round-bottom plates, centrifuged at 400× g, and then cultured for 24 h. Ha2/5+ cells and unpurified cells (all living cells) were seeded, and photomicrographs taken one week later are shown. The scale bar represents 200 μm. The brown mass (in PI-negative cells) was hematopoietic cells. (b) Images of spheroid-forming cells at 0, 1, 2, 3, 4, 5, 6, and 24 h after seeding the cultured Ha2/5 cells. (c) Analysis of Ha2/5 spheroids by immunostaining. Muscle marker: Pax7 are shown in green. Cyto-skeletal marker (Phalloidin, red) and cell nuclei (Hoechst, blue) are shown. The white square is enlarged on the lower panel. Allowhead indicate the localization of Pax7. (d) Negative controls in Pax7 staining were displayed. The scale bar represents 100 μm.
Figure 4
Induction of lipid accumulation and…
Figure 4
Induction of lipid accumulation and lipid measurement in meat buds. ( a )…
Figure 4
Induction of lipid accumulation and lipid measurement in meat buds. (a) Scheme of CD29+ cells with adipogenic differentiation. (b,c) Spheroids prepared from Ha2/5+ cells were differentiated into adipose differentiation medium for two weeks after five days of differentiation into muscle. Control spheroid (b) and spheroids induced to adipose differentiation (c) are shown. Desmin (red), BODIPY (green), and Hoechst (Blue). The scale bar represents 100 μm. (d) Relative expression of bovine meat bud by real-time RT-PCR. RNA was extracted from adipose-differentiated bovine meat buds (two weeks) for gene expression analysis of adipose differentiation markers (CEBPα, CEBPβ, PPARγ, LEPTIN, and ADIPONECTIN), and (e) muscle markers (DESMIN and PAX7). (n = 4), * p < 0.05.
Figure 5
Construction of collagen gel tissue…
Figure 5
Construction of collagen gel tissue incorporating bovine meat buds. ( a ) Photographs…
Figure 5
Construction of collagen gel tissue incorporating bovine meat buds. (a) Photographs of mass culture of micro-spheroids by spheroid generation plates. Well diameter/depth (500/200). (b) Culture of collagen gel incorporating Ha2/5 meat buds. Collagen gel after 24 h (left) and three days (right). (c) Phase photographs of the incorporated Ha2/5 meat buds are shown. Time course of collagen gel incorporating Ha2/5 meat buds. Phase photographs of the incorporated spheroids are shown. The scale bar represents 200 μm. (d) Calculation of how many meat buds can be made from 100 g of meat in 21 days.
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