Intestinal crypts reproducibly expand in culture
Intestinal crypts reproducibly expand in culture
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DOI：
10.1016/j.jss.2012.03.037
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发表时间：
2012-11-01
影响因子：
2.2
通讯作者：
Helmrath, Michael A.
中科院分区：
文献类型：
--
作者：
Fuller, Megan K.;Faulk, Denver M.;Helmrath, Michael A.
背景：肠隐窝和单肠干细胞的体外生长技术最近得到了描述，但几个翻译重要性的问题仍未得到解决。本研究的目的是首先评估肠隐窝是否在体外可重复扩张；第二，确定年龄和肠道区域对体外隐窝生长的影响；第三，确定冷冻保存对体外隐窝生长的影响。方法和材料：4周龄、6-8周龄、12-14周龄、18-20周龄的C57BL/6J小鼠（n = 4-6只），取小肠近端、中端和远端5 cm处的隐窝进行培养。对于每个区域，我们测定了隐窝形成肠球（第1天）和进展到肠样（第7天）的效率。随后，肠样体传代并冷冻保存以确定这些操作是否改变了生长。结果：43 - 99%的肠隐窝形成肠球，在近端小肠和年轻小鼠中效率更高。25 - 64%的肠球在7天内发展成芽殖肠样。近端肠样的体外扩张更大。这种扩展以对数方式继续进行，与分离的肠隐窝芽的复制效率相似，达到97%。冷冻保存后，大约90%的肠样细胞恢复了正常的增殖能力。结论：肠隐窝培养是一种有效的、可重复的肠组织扩张方法。区域和年龄的生长差异可能反映了不同的干细胞特征或支持细胞的差异。体外培养和扩展肠道组织的能力为理解和治疗短肠综合征患者提供了一种潜在的转化方法。(C) 2012爱思唯尔公司版权所有。
Background: In vitro growth techniques for intestinal crypts and single intestinal stem cells have been recently described, but several questions of translational importance remain unaddressed. The purpose of this study was to first, evaluate if intestinal crypts reproducibly expand in vitro; second, determine the impact of age and region of intestine on crypt growth in vitro; and third, determine the effects of cryopreservation on crypt growth in vitro.Methods and materials: Crypts were harvested from 5 cm of proximal, middle, and distal small intestine of C57BL/6J mice aged 4 wk, 6-8 wk, 12-14 wk, and 18-20 wk (n = 4-6 animals) and cultured. For each region, we determined the efficiency of crypts forming enterospheres (day 1) and progressing to enteroids (day 7). Subsequently, enteroids were passaged and cryopreserved to determine if growth was changed by these manipulations.Results: Forty-three to 99% of intestinal crypts formed enterospheres, with higher efficiency in proximal small intestine and in younger mice. Twenty-five to 64% of enterospheres progressed to budding enteroids within 7 d. In vitro expansion was greater in proximal enteroids. This expansion continued in a logarithmic fashion, with similar to 97% replating efficiency of isolated enteroid crypt buds. Following cryopreservation, similar to 90% of enteroids recovered normal proliferative capacity.Conclusions: Intestinal crypt culture is efficient and significantly expands intestinal tissue in a reproducible manner. Regional and age growth differences may reflect distinct stem cell characteristics or differences in support cells. The ability to culture and expand intestinal tissue in vitro provides a potential translational approach toward understanding and treating patients with short bowel syndrome. (C) 2012 Elsevier Inc. All rights reserved.