A simplified but robust method for the isolation of avian and mammalian muscle satellite cells.
A simplified but robust method for the isolation of avian and mammalian muscle satellite cells.
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DOI：
10.1186/1471-2121-13-16
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发表时间：
2012-06-21
期刊：
影响因子：
--
通讯作者：
Chang KC
中科院分区：
文献类型：
--
作者：
Baquero-Perez B;Kuchipudi SV;Nelli RK;Chang KC
目前从不同动物物种中分离肌肉卫星细胞的方法差异很大，使得物种间的比较存在问题。这种变化主要源于使用不同的蛋白水解酶从肌肉组织中释放卫星细胞（有时使用单一酶，但通常优选酶的组合）和用于包被培养器皿的不同细胞外基质蛋白。此外，卫星细胞的分离通常是费力的，并且有时可能需要将细胞制备物预接种在未涂覆的烧瓶上或Percoll离心以去除污染的成纤维细胞。体外分离培养卫星细胞的方法可以决定成肌细胞融合成多核肌管的能力。这些终末分化的肌管类似于体内肌肉组织中的成熟肌纤维，因此最佳融合是体外肌肉培养的关键。因此，非常需要一种能够产生高融合率的不同脊椎动物物种的肌肉卫星细胞分离和培养的简单方法。我们在这里展示了一个相对简单和快速的方法，分离高度富集的肌肉卫星细胞从不同的鸟类和哺乳动物物种。简而言之，将肌肉组织机械解离，用单一酶（链霉蛋白酶）消化，用10-ml移液管研磨，过滤并直接铺在胶原包被的烧瓶上。根据该方法并在优化细胞培养条件后，在鸭、鸡、马和牛（具有超过50%的细胞融合）中实现了优异的融合率，并且在较小程度上在猪中实现了优异的融合率，这表明链霉蛋白酶是从肌肉组织释放卫星细胞的高度合适的酶。我们的简化方法提供了一种快速和简单的替代分离高度富集的肌肉卫星细胞培养物，随后可以迅速分化成发育良好的初级肌管。使用相同的分离方案可以更好地进行肌肉卫星细胞的种间比较。在所有研究的农场动物物种中，收获的鸡肌肉细胞显示出最高的肌肉卫星细胞百分比，马肌肉细胞呈现出最高的融合指数，达到令人印象深刻的77%。猪细胞显示出最低量的卫星细胞，但仍达到了1.41%的适度融合率。
Current methods of isolation of muscle satellite cells from different animal species are highly variable making inter-species comparisons problematic. This variation mainly stems from the use of different proteolytic enzymes to release the satellite cells from the muscle tissue (sometimes a single enzyme is used but often a combination of enzymes is preferred) and the different extracellular matrix proteins used to coat culture ware. In addition, isolation of satellite cells is frequently laborious and sometimes may require pre-plating of the cell preparation on uncoated flasks or Percoll centrifugation to remove contaminating fibroblasts. The methodology employed to isolate and culture satellite cells in vitro can critically determine the fusion of myoblasts into multi-nucleated myotubes. These terminally differentiated myotubes resemble mature myofibres in the muscle tissue in vivo, therefore optimal fusion is a keystone of in vitro muscle culture. Hence, a simple method of muscle satellite cell isolation and culture of different vertebrate species that can result in a high fusion rate is highly desirable. We demonstrate here a relatively simple and rapid method of isolating highly enriched muscle satellite cells from different avian and mammalian species. In brief, muscle tissue was mechanically dissociated, digested with a single enzyme (pronase), triturated with a 10-ml pipette, filtered and directly plated onto collagen coated flasks. Following this method and after optimization of the cell culture conditions, excellent fusion rates were achieved in the duck, chicken, horse and cow (with more than 50% cell fusion), and to a lesser extent pig, pointing to pronase as a highly suitable enzyme to release satellite cells from muscle tissue. Our simplified method presents a quick and simple alternative to isolating highly enriched muscle satellite cell cultures which can subsequently rapidly differentiate into well developed primary myotubes. The use of the same isolation protocol allows better inter-species comparisons of muscle satellite cells. Of all the farm animal species investigated, harvested chicken muscle cells showed the highest percentage of muscle satellite cells, and equine muscle cells presented the highest fusion index, an impressive ≈ 77%. Porcine cells displayed the lowest amount of satellite cells but still achieved a modest fusion rate of ≈ 41%.
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影响因子：
--
作者：
BISCHOFF, R
通讯作者：
BISCHOFF, R
影响因子：
2.1
作者：
Cassar-Malek, I;Langlois, N;Geay, Y
通讯作者：
Geay, Y
影响因子：
5.6
作者：
ALLEN, RE;RANKIN, LL;PIERCE, PR
通讯作者：
PIERCE, PR
DOI：
10.1073/pnas.78.9.5623
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
BLAU, HM;WEBSTER, C
通讯作者：
WEBSTER, C
影响因子：
3.4
作者：
GIBSON, MC;SCHULTZ, E
通讯作者：
SCHULTZ, E