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. 2023 Nov 16:14:1288809.
doi: 10.3389/fphys.2023.1288809. eCollection 2023.

Effect of standard and physiological cell culture temperatures on in vitro proliferation and differentiation of primary broiler chicken pectoralis major muscle satellite cells

Caroline R Gregg  1 , Brittany L Hutson  1 , Joshua J Flees  1 , Charles W Starkey  1 , Jessica D Starkey  1
Affiliations

Effect of standard and physiological cell culture temperatures on in vitro proliferation and differentiation of primary broiler chicken pectoralis major muscle satellite cells

Caroline R Gregg et al. Front Physiol. .

Abstract

Culture temperatures for broiler chicken cells are largely based on those optimized for mammalian species, although normal broiler body temperature is typically more than 3°C higher. The objective was to evaluate the effects of simulating broiler peripheral muscle temperature, 41°C, compared with standard temperature, 38°C, on the in vitro proliferation and differentiation of primary muscle-specific stem cells (satellite cells; SC) from the pectoralis major (PM) of broiler chickens. Primary SC cultures were isolated from the PM of 18-day-old Ross 708 × Yield Plus male broilers. SC were plated in triplicate, 1.8-cm2, gelatin-coated wells at 40,000 cells per well. Parallel plates were cultured at either 38°C or 41°C in separate incubators. At 48, 72, and 96 h post-plating, the culture wells were fixed and immunofluorescence-stained to determine the expression of the myogenic regulatory factors Pax7 and MyoD as well as evaluated for apoptosis using a TUNEL assay. After 168 h in culture, plates were immunofluorescence-stained to visualize myosin heavy chain and Pax7 expression and determine myotube characteristics and SC fusion. Population doubling times were not impacted by temperature (p ≥ 0.1148), but culturing broiler SC at 41°C for 96 h promoted a more rapid progression through myogenesis, while 38°C maintained primitive populations (p ≤ 0.0029). The proportion of apoptotic cells increased in primary SC cultured at 41°C (p ≤ 0.0273). Culturing at 41°C appeared to negatively impact fusion percentage (p < 0.0001) and tended to result in the formation of thinner myotubes (p = 0.061) without impacting the density of differentiated cells (p = 0.7551). These results indicate that culture temperature alters primary broiler PM SC myogenic kinetics and has important implications for future in vitro work as well as improving our understanding of how thermal manipulation can alter myogenesis patterns during broiler embryonic and post-hatch muscle growth.

Keywords: apoptosis; broiler chicken; cell culture temperature; muscle satellite cell; myogenesis; myogenic regulatory factor.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Effect of culture temperature on myogenic regulatory factor expression in primary broiler chicken pectoralis major satellite cell cultures after 96 h. Representative inverted fluorescence images of 4′,6-diamidino-2-phenylindole-stained nuclei (blue; left) and Pax7 (green; center) and MyoD (red; right) protein expression in cultures grown at 38°C (A) or 41°C (B).
FIGURE 2
FIGURE 2
Effect of culture temperature on primary broiler chicken pectoralis major satellite cell myogenic regulatory factor heterogeneity over time. n = 9 wells per treatment of either 38 or 41°C incubation were immunofluorescence-stained to detect Pax7 and MyoD expression at 48, 72, and 96 h post-plating. Populations are presented as a percentage of total nuclei. Total nuclei describes every nucleus expressing the 4′,6-diamidino-2-phenylindole counterstain regardless of the status of other target proteins. Error bars represent the highest standard error of the pairwise mean comparison. Least square means accompanied by a * signify that the proportion of Pax7+:MyoD+ cells differs (p < 0.05) between 38°C and 41°C, # signify that the proportion of Pax7-:MyoD+ cells differs (p < 0.05) between 38°C and 41°C, and $ signify that the proportion of Pax7+:MyoD- cells differs (p < 0.05) between 38°C and 41°C.
FIGURE 3
FIGURE 3
Representative inverted fluorescence images showing a TUNEL-positive nuclei indicated with an arrow among normal nuclei (A) in primary broiler pectoralis major satellite cell cultures with a corresponding positive control image (B) after DNase I treatment. Images show 4′,6-diamidino-2-phenylindole-stained nuclei (blue; left), TUNEL assay (green; center), and merged channels (right).
FIGURE 4
FIGURE 4
Effect of culture temperature on the density of nuclei relative to myotubes in primary broiler chicken pectoralis major satellite cell cultures after 168 h. n = 9 wells per treatment of either 38°C or 41°C incubation were immunofluorescence-stained to detect Pax7 and myosin heavy chain (MHC) expression. Total nuclei describes every nucleus expressing the 4′,6-diamidino-2-phenylindole counterstain regardless of the status of other target proteins; internal nuclei were located within an area expressing MHC protein; external nuclei did not overlap with MHC expression. Error bars represent the highest standard error of the pairwise mean comparison. a,bBars with different superscripts within a cell population differ p ≤ 0.05.
FIGURE 5
FIGURE 5
Effect of culture temperature on myotube formation in primary broiler chicken pectoralis major satellite cell cultures after 168 h. Representative inverted fluorescence images at ×200 magnification of myosin heavy chain-defined myotubes (red) and 4′,6-diamidino-2-phenylindole-stained nuclei (blue) in cultures grown at 38°C (A) or 41°C (B). The scale bar depicts 100 µm.
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Grants and funding

The authors declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the United States Department of Agriculture National Institute of Food and Agriculture (USDA-NIFA) Competitive Grant Nos 2018-67017-27556 and 2020-67015-30824 awarded to JS, the USDA Hatch Program funds, and the Alabama Agricultural Experiment Station.

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