MG 由国家卫生研究院赠款 K08DK101608 和 R03DK111473 支助, 3月的硬币基金会赠款 5-FY17-79, 华盛顿大学儿童发现研究所和圣路易斯儿童医院和部圣路易斯华盛顿大学医学院儿科。CJL 是由 R01DK104946 (PI:), 华盛顿大学儿童发现研究所和圣路易斯儿童医院支持。

这项研究的所有动物程序都是由圣路易斯大学动物保育和使用委员会 (20160187 号议定书) 或匹兹堡大学机构动物护理和使用委员会 (14103918 号议定书) 批准的。人类胎儿小肠在不到24周的妊娠是根据匹兹堡大学解剖组织采购指南后, 机构审查委员会批准 (礼宾 PRO14100537) 从大学匹兹堡健康科学组织银行通过一个诚实的经纪系统。未经匹兹堡大学机构审查委员会的同意, 获得了不确定的人母乳或巴氏杀菌捐赠母乳。
1. 从新生小鼠或早产人小肠 Enteroids 的隐窝分离和建立
<ol><li>媒体准备 注: 介质应在盖窝隔离前的前一天准备好。<ol>
<li>准备 1.11 L 文化媒介。从1升 Dulbecco 的改良鹰的培养基 (DMEM) 与4.5 克/升葡萄糖和 l-谷氨酰胺, 并添加110毫升胎牛血清 (血清) (最终浓度 10%), 11 毫升青霉素-链霉素 (最终浓度 1%), 1.1 毫升无菌胰岛素 (最终浓度 0.1%)。</li>
		<li>准备500毫升的 PBS-抗生素混合物。开始与500毫升 1x Dulbecco 的磷酸盐缓冲盐水 (DPBS) 与10% 的血清和添加5毫升庆大霉素 (最终浓度 1%), 1 毫升两性霉素 B (最终浓度 0.2%), 和5毫升青霉素-链霉素 (最终浓度 1%)。</li>
		<li>准备30毫升细胞分裂介质 #1。首先准备30毫升培养基。添加600µL 0.5 米乙二胺四乙酸 (EDTA) (最终浓度10毫米), 300 µL 庆大霉素 (最终浓度 1%), 60 µL 两性霉素 B (最终浓度 0.2%)。</li>
		<li>准备30毫升细胞分裂介质 #2。首先准备30毫升培养基。添加300µL 0.5 米 EDTA (最终浓度5毫米), 300 µL 庆大霉素 (最终浓度 1%), 60 µL 两性霉素 B (最终浓度 0.2%)。</li>
		<li>准备50毫升的隐窝培养基, 无生长因子。测量33.4 毫升1x 先进 DMEM/F-12 和添加10毫升血清 (最后浓度 20%)。<ol>
<li>添加500µL 青霉素-链霉素 (最终浓度 1%), 500 µL l-谷氨酰胺 (最终浓度 1%), 500 µL 庆大霉素 (最终浓度 1%), 100 µL 两性霉素 B (最后浓度 0.2%), 2.5 µL 1 M N-2-hydroxyethylpiperazine-N-2-ethane磺酸 (HEPES) (最终浓度0.05 毫米), 500 µL 100 毫米 n-乙酰半胱氨酸 (最终浓度1毫米), 500 µL 100x N-2 补充剂, 1 毫升50x 无血清补充剂 (例如, B-27) 减去维生素 A, 500 µL 1 毫米 100x Y-27632。</li>
		</ol>
</li>
		<li>用生长因子制备1毫升的隐窝培养基。首先准备1毫升无生长因子的隐窝培养基。添加2.5 µL 40 µg/毫升 Wnt3a (最终浓度 100 ng/毫升), 1 µL 100 µg/毫升 (最终浓度 100 ng/毫升), 2 µL 250 µg/毫升 R Spondin (最终浓度 500 ng/毫升), 0.5 µL 100 µg/毫升 EGF (最终浓度 50 ng/毫升)。</li>
	</ol></li>
	<li>隐窝隔离
	<ol>
<li>将可溶性基底膜萃取物 (见材料表) 放在冰层开始解冻前。 注: 将基底膜放在冰上, 否则会聚合。</li>
		<li>按照机构动物护理和使用委员会的协议, 为老鼠超过14天的年龄, 弄死小鼠与两种方法的安乐死, 第二个是一个物理程序, 以确保死亡。 注: 对于14天以下的小鼠, 斩首是使用剪刀的标准程序。</li>
		<li>使用剪刀和镊子, 垂直切开腹部中线, 通过皮肤和腹膜, 为整个腹部的长度。用剪刀将小肠从胃部切除到盲肠, 用镊子将肠系膜切除。 注: 两周大的雄性和雌性 C57BL/6J 鼠幼崽, 每重 5.5-7 克, 被用于隐窝隔离。任何年龄或大小的鼠标都可以用于隐窝隔离。</li>
		<li>准备新鲜收获的小肠为地穴隔离, 位置肚腑平直, 并且切开肚腑沿它的整个长度使用剪刀。 注意: 老鼠和人肚腑可以相似地被准备。</li>
		<li>用镊子在 PBS-抗生素混合填充培养皿中来回轻轻摇动肠道, 清洁大便。</li>
		<li>添加30毫升细胞中断介质 #1 到50毫升圆锥管。将肠道切成0.5 厘米长的块, 直接入管。把管子放在冰桶里, 以最低的速度在振动筛上轻轻搅动15分钟。</li>
		<li>当这些组织在振动筛上时, 如果将 enteroids 用于 RNA、DNA 或蛋白质的分离, 则获得24或48的钢板来生长墓穴。使用8井室幻灯片生长的地窖, 如果 enteroids 将用于整个安装共聚焦成像。</li>
		<li>在引擎盖中, 在24或48井板的每个井的底部添加15µL 的基底膜。如果使用腔片, 在每个腔的底部添加9µL 的基底膜。 注: 如有需要, 根据板材尺寸调整基底膜容积。</li>
		<li>用吸管尖迅速地将基底膜传播, 使基底膜在井底形成薄薄的层。把盘子放在37摄氏度的孵化箱里30分钟, 让基底膜聚合。 注: 如果基底膜不是薄层, 或基底膜含有气泡, 则该墓穴不会附着。</li>
		<li>15分钟后, 在一个振动筛, 过滤组织通过一个100µm 过滤器。丢弃流。</li>
		<li>添加30毫升细胞中断介质 #2 到一个新的50毫升圆锥管。从过滤器中取出组织并添加到管中。</li>
		<li>把管子放在冰桶里, 以最低的速度在振动筛上轻轻搅动15分钟。</li>
		<li>经过15分钟的振动筛, 过滤组织通过一个100µm 过滤器。在这一步保持流量, 以防在以后的步骤中产量低。 注: 所有流过管子的管道必须放在冰上。</li>
		<li>添加15毫升 DMEM 与4.5 克/升葡萄糖和 l-谷氨酰胺到一个新的50毫升锥管。从过滤器中取出组织并添加到管中。十年代用手大力摇动管子. 过滤通过100µm 过滤器和收集流程通过。重复, 直到流不包含微粒。</li>
		<li>在引擎盖中, 过滤从上一步到一个新的50毫升圆锥管的流动, 使用70µm 过滤器为小鼠组织或100µm 过滤器为人体组织。 注: 在待机状态下有额外的过滤滤器, 慢慢地倒入过滤体将堵塞杂物。</li>
		<li>离心管在 200 x g 为8分钟在4°c 并且直接地在冰桶在敞篷。用吸管除去上清液, 不干扰颗粒。并用重悬颗粒与总期望数量的隐窝培养基培养基没有生长因子。 注: 富集的墓穴是颗粒的 "沙质" 层。每一个井都需要250µL 的隐窝文化媒体, 没有生长因子。</li>
	</ol></li>
	<li>enteroids 的建立
	<ol>
<li>将墓穴介质混合物直接放在基底膜上。</li>
		<li>用显微镜检查地穴, 确保隔离过程成功。 注意: 细胞的细胞和球是需要的。大约100个单独地窖被镀每井。</li>
		<li>在37摄氏度处孵化出3小时的钢板, 让墓穴附着在基底膜上。 注: 这种孵化是与隐窝培养基培养基没有生长因子。</li>
		<li>慢慢地移除没有使用 P-200 吸管连接的介质和多余的墓穴。 注: 请勿扰乱基底膜, 因为附着的墓穴可能会脱落。</li>
		<li>慢慢地添加250µL 的隐窝培养基和生长因子给每个井。 注: 对于 24, 48 井板, 或会议厅幻灯片, 250 µL 的隐窝培养基培养基与生长因子是足够的。</li>
		<li>每2天用新鲜的地穴培养基和生长因子在治疗前5天更换培养基, 以获得最佳附着和生长。</li>
	</ol></li>
</ol>2. 鼠标母乳收集
<ol><li>在产后 7-12 天从幼崽中分离出一 C57BL/6J 泌乳大坝, 在挤奶前6小时。</li>
	<li>麻醉的泌乳大坝与异氟醚通过鼻锥, 并管理皮下注射催产素0.15 的体重。在尝试挤奶过程前等待3分钟。</li>
	<li>在挤奶过程前用70% 乙醇清洁奶嘴, 让干燥。</li>
	<li>一次性从奶嘴中提取乳汁, 使用带有硅胶导管的电动人乳泵, 将小鼠奶嘴放入5毫升收集管中。 注: 最佳的母乳泵有两个设置, 一个为速度和一个为吸力。最好从每一个最低的设置开始, 并慢慢增加, 直到牛奶被提取。理想情况下, 总产量将是500µL-1 毫升的小鼠母乳每泌乳大坝。</li>
	<li>立即整除母乳和存储在-80 摄氏度。 注意: 避免冻结/解冻循环。</li>
	<li>治疗 enteroids 5 天与50µL/毫升的小鼠母乳每毫升的隐窝培养基与生长因子24小时在37摄氏度。</li>
</ol>3. Enteroids 的全装染色
<ol><li>
试剂的制备
	<ol>
<li>每8井准备16毫升 1x PBS。</li>
		<li>在 1x PBS 中准备2毫升4% 多聚甲醛 (粉煤灰), 每8口井。</li>
		<li>在 1x PBS 中准备2毫升0.1% 海卫 X-100 每8口井。</li>
		<li>准备24毫升的 pbs (PBST 是0.1% 吐温20在 1x PBS) 每8口井。</li>
		<li>2毫升10% 正常驴血清 (nds) 在 0.1% PBST (10% NDS/PBST) 每8口井。</li>
		<li>在 0.1% PBST (1% NDS/PBST) 中, 每8个井准备4毫升 1% nds。</li>
	</ol>
</li>
	<li>
染色日1
	<ol>
<li>吸掉治疗, 用 PBS 洗 enteroids。每井加250µL 4% 粉煤灰。在转子上放置 1-2 小时, 在4摄氏度。用吸管取出4% 只粉煤灰。</li>
		<li>洗4次与250µL/井 1x PBS。在室温下 (RT) 在转子上放置10分钟。 注: 如果需要, 可以在4摄氏度储存 1-2 天的板材。</li>
		<li>添加250µL/井0.1% 海卫 X-100。在 RT 中孵化1小时. 用吸管去除 0.1% X-100。</li>
		<li>洗4次与250µL/井 1x PBS。在 RT 上放置15分钟的旋转。</li>
		<li>添加250µL/井 10% NDS/PBST。在 RT 中孵化45分钟. 用吸管去除 10% NDS/PBST。</li>
		<li>添加250µL/井原抗体, 稀释 1/250 1% NDS/PBST, 并在4摄氏度过夜。 注: 最佳抗体稀释根据制造商而异。</li>
	</ol>
</li>
	<li>
染色日2
	<ol>
<li>洗涤至少5次为 4-5 h 与250µL/井 PBST。在每个洗涤之间的4°c 放置在转子上。</li>
		<li>添加250µL/井次抗体, 稀释 1/1000 1% NDS/PBST。包装在箔板和孵化隔夜在4摄氏度。</li>
	</ol>
</li>
	<li>
染色日3
	<ol>
<li>添加250µL/井核染色 4 ', 6-diamidino 2-苯基吲哚 (DAPI) 15 分钟。 注意: 把盘子放在黑暗中。</li>
		<li>洗6次与250µL/井 PBST。在每个洗涤间, 在4摄氏度的旋转处放置10分钟。</li>
		<li>将盖玻片安装在带安装介质的室内滑动上。</li>
		<li>在光栅图形编辑器 (请参阅材料表) 中, 采用40X 目标共聚焦显微镜和同样渲染图像。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Patel, R. M., Denning, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+use+of+prebiotics,+probiotics,+and+postbiotics+to+prevent+necrotizing+enterocolitis:+what+is+the+current+evidence?.">Therapeutic use of prebiotics, probiotics, and postbiotics to prevent necrotizing enterocolitis: what is the current evidence?.</a> Clin Perinatol. 40 (1), 11-25 (2013).</li><li>Caplan, M. S., Jilling, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+concepts+in+necrotizing+enterocolitis.">New concepts in necrotizing enterocolitis.</a> Curr Opin Pediatr. 13 (2), 111-115 (2001).</li><li>Henry, M. C., Moss, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necrotizing+enterocolitis.">Necrotizing enterocolitis.</a> Annu Rev Med. 60, 111-124 (2009).</li><li>Lin, P. W., Stoll, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necrotising+enterocolitis.">Necrotising enterocolitis.</a> Lancet. 368 (9543), 1271-1283 (2006).</li><li>Neu, J., Walker, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necrotizing+enterocolitis.">Necrotizing enterocolitis.</a> N Engl J Med. 364 (3), 255-264 (2011).</li><li>Bartick, M., Reinhold, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+burden+of+suboptimal+breastfeeding+in+the+United+States:+a+pediatric+cost+analysis.">The burden of suboptimal breastfeeding in the United States: a pediatric cost analysis.</a> Pediatrics. 125 (5), e1048-e1056 (2010).</li><li>Bisquera, J. A., Cooper, T. R., Berseth, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+necrotizing+enterocolitis+on+length+of+stay+and+hospital+charges+in+very+low+birth+weight+infants.">Impact of necrotizing enterocolitis on length of stay and hospital charges in very low birth weight infants.</a> Pediatrics. 109 (3), 423-428 (2002).</li><li>Leaphart, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+TLR4+in+the+pathogenesis+of+necrotizing+enterocolitis+by+modulating+intestinal+injury+and+repair.">A critical role for TLR4 in the pathogenesis of necrotizing enterocolitis by modulating intestinal injury and repair.</a> J Immunol. 179 (7), 4808-4820 (2007).</li><li>Gribar, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+expression+and+signaling+of+TLR4+and+TLR9+in+the+pathogenesis+and+treatment+of+necrotizing+enterocolitis.">Reciprocal expression and signaling of TLR4 and TLR9 in the pathogenesis and treatment of necrotizing enterocolitis.</a> J Immunol. 182 (1), 636-646 (2009).</li><li>Good, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+fluid+inhibits+Toll-like+receptor+4+signaling+in+the+fetal+and+neonatal+intestinal+epithelium.">Amniotic fluid inhibits Toll-like receptor 4 signaling in the fetal and neonatal intestinal epithelium.</a> Proceedings of the National Academy of Sciences. 109 (28), 11330-11335 (2012).</li><li>Good, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+milk+protects+against+the+development+of+necrotizing+enterocolitis+through+inhibition+of+Toll-like+receptor+4+in+the+intestinal+epithelium+via+activation+of+the+epidermal+growth+factor+receptor.">Breast milk protects against the development of necrotizing enterocolitis through inhibition of Toll-like receptor 4 in the intestinal epithelium via activation of the epidermal growth factor receptor.</a> Mucosal Immunol. 8 (5), 1166-1179 (2015).</li><li>Yazji, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+TLR4+activation+impairs+intestinal+microcirculatory+perfusion+in+necrotizing+enterocolitis+via+eNOS-NO-nitrite+signaling.">Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS-NO-nitrite signaling.</a> Proc Natl Acad Sci U S A. , (2013).</li><li>Lundberg, J. O., Weitzberg, E., Gladwin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nitrate-nitrite-nitric+oxide+pathway+in+physiology+and+therapeutics.">The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics.</a> Nat Rev Drug Discov. 7 (2), 156-167 (2008).</li><li>Bober-Olesinska, K., Kornacka, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+glutamine+supplemented+parenteral+nutrition+on+the+incidence+of+necrotizing+enterocolitis,+nosocomial+sepsis+and+length+of+hospital+stay+in+very+low+birth+weight+infants.">Effects of glutamine supplemented parenteral nutrition on the incidence of necrotizing enterocolitis, nosocomial sepsis and length of hospital stay in very low birth weight infants.</a> Med Wieku Rozwoj. 9 (3 Pt 1), 325-333 (2005).</li><li>Li, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamine+decreases+lipopolysaccharide-induced+intestinal+inflammation+in+infant+rats.">Glutamine decreases lipopolysaccharide-induced intestinal inflammation in infant rats.</a> Am J Physiol Gastrointest Liver Physiol. 286 (6), G914-G921 (2004).</li><li>Good, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+milk+oligosaccharide+2'-fucosyllactose+attenuates+the+severity+of+experimental+necrotising+enterocolitis+by+enhancing+mesenteric+perfusion+in+the+neonatal+intestine.">The human milk oligosaccharide 2'-fucosyllactose attenuates the severity of experimental necrotising enterocolitis by enhancing mesenteric perfusion in the neonatal intestine.</a> Br J Nutr. 116 (7), 1175-1187 (2016).</li><li>Jantscher-Krenn, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+milk+oligosaccharide+disialyllacto-N-tetraose+prevents+necrotising+enterocolitis+in+neonatal+rats.">The human milk oligosaccharide disialyllacto-N-tetraose prevents necrotising enterocolitis in neonatal rats.</a> Gut. 61 (10), 1417-1425 (2012).</li><li>Akin, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+lactoferrin+to+prevent+nosocomial+sepsis+and+necrotizing+enterocolitis+of+premature+neonates+and+effect+on+T-regulatory+cells.">Oral lactoferrin to prevent nosocomial sepsis and necrotizing enterocolitis of premature neonates and effect on T-regulatory cells.</a> Am J Perinatol. 31 (12), 1111-1120 (2014).</li><li>Amin, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+supplementation+prevents+necrotizing+enterocolitis+in+the+premature+infant.">Arginine supplementation prevents necrotizing enterocolitis in the premature infant.</a> J Pediatr. 140 (4), 425-431 (2002).</li><li>Rodgers, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+aspects+of+milk+collection+in+the+rat.">Practical aspects of milk collection in the rat.</a> Lab Anim. 29 (4), 450-455 (1995).</li><li>DePeters, E. J., Hovey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+collecting+milk+from+mice.">Methods for collecting milk from mice.</a> J Mammary Gland Biol Neoplasia. 14 (4), 397-400 (2009).</li><li>Willingham, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milk+collection+methods+for+mice+and+Reeves'+muntjac+deer.">Milk collection methods for mice and Reeves' muntjac deer.</a> J Vis Exp. (89), (2014).</li><li>Saxena, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Intestinal+Enteroids:+a+New+Model+To+Study+Human+Rotavirus+Infection,+Host+Restriction,+and+Pathophysiology.">Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology.</a> J Virol. 90 (1), 43-56 (2015).</li><li>Zachos, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Enteroids/Colonoids+and+Intestinal+Organoids+Functionally+Recapitulate+Normal+Intestinal+Physiology+and+Pathophysiology.">Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology.</a> J Biol Chem. 291 (8), 3759-3766 (2016).</li><li>Drummond, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteroviruses+infect+human+enteroids+and+induce+antiviral+signaling+in+a+cell+lineage-specific+manner.">Enteroviruses infect human enteroids and induce antiviral signaling in a cell lineage-specific manner.</a> Proc Natl Acad Sci U S A. 114 (7), 1672-1677 (2017).</li><li>Ettayebi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+human+noroviruses+in+stem+cell-derived+human+enteroids.">Replication of human noroviruses in stem cell-derived human enteroids.</a> Science. 353 (6306), 1387-1393 (2016).</li></ol>

作者没有什么可透露的, 也没有利益冲突。

肠道上皮是由许多细胞亚型组成的, 负责为病原体提供宿主防御, 维持肠道屏障完整性, 并可在多种疾病的发病机制中被突破。虽然动物模型可以重述这一疾病的某些方面, 但从小鼠和人类小肠中提取的 enteroids 的前体模型为测试各种治疗效果提供了一个平台。enteroid 模型的意义使研究人员能够将肠道干细胞培养并区分为所有上皮细胞亚型, 从幼稚或患病的动物和没有现有方法的人类。这种模?...

坏死性小肠结肠炎 (NEC) 是早产儿胃肠道疾病死亡的主要原因, 影响近 1 10 婴儿出生前29周妊娠1,2,3。有一半的婴儿与 NEC 进展到最严重的形式, 其中生存只有 10-30%4,5。在美国, 估计有 2-30亿美元/年用于治疗婴儿与 NEC6,7, 但在过去30年中, 存活率和治疗都没有改变。NEC 的发病特点是肠道损伤和黏膜愈合受损8,9,10,11, 然而, 信号通路导致恶化炎症反应和机制逆转炎症仍然不完全地被了解。
人类母乳的管理已被发现是唯一的保护策略, 针对 NEC 早产儿。我们以前已经表明, 母乳通过表皮生长因子受体 (EGFR) 信号通路11抑制肠道上皮中先天免疫受体 4 (TLR4), 从而防止 NEC 的发展。将母乳补充到一个实验 nec 公式中, 通过抑制 enterocyte 细胞凋亡和恢复 enterocyte 增殖的方式, 减弱了 nec 中所看到的炎症反应, 这种方法依赖于表皮生长因子 (EGF) 和 EGFR11。另一项研究表明, 母乳中的另一成分硝酸盐通过调节肠道灌注来促进其保护性, 与缺乏硝酸盐的婴儿配方奶粉相比, 这可能有助于提高 NEC 的频率。公式喂养婴儿12,13。母乳中含有的其他化合物包括人乳寡糖、l-精氨酸、谷氨酰胺和乳铁蛋白14,15,16, 17,18,19。母乳中的这些有益成分揭示了它在预防 nec 方面的必要性, 同时也强调了研究母乳如何调解对 nec 的保护所涉及的机制、信号通路和细胞效应的重要性。.
为了进一步研究 NEC 的小鼠模型中母乳的保护特性, 我们开发了一种新颖的、易于使用的技术, 用电动人乳泵从麻醉大坝中提取小鼠母乳的方法11,12.这项获取小鼠母乳的策略是有利的, 这不仅是因为人类母乳泵在母乳的采购方面是现成的, 而且还因为这种方法允许对特定种类的母乳进行分析。因此, 我们可以比较小鼠母乳与表达的人母乳的影响, 以及巴氏杀菌的人供奶从牛奶银行在物种特定的模型。这项技术允许研究母乳成分与他们对 NEC 预防的贡献。其他调查人员已经开发出母乳提取方法, 但是, 这些技术是手动的, 通常需要多个实验室成员20,21,22。这里提供了一个简单的技术, 可用于修改人的电动母乳泵从鼠标收集牛奶。这种技术也可以应用于其他物种。
为了充分审问与 NEC 有关的信号通路, 需要建立模型系统, 以评估已知受疾病影响的所有不同细胞类型。在这里, 我们讨论一个这样的模型系统-enteroids 和他们的建立从老鼠和人类小肠。人类肠道 enteroids (HIEs) 特别是提供了重要的承诺, 因为它们提供了一个创新的, 基因多样的前体人类模型, 以帮助研究在胃肠道发生的病理生理过程23. Enteroids 已被发现长期养殖, 可冷冻供以后使用23, 与人类肠道 Organoids (HIOs) 不同, 其培养方法来自诱导多潜能干细胞, Enteroids 是由干细胞产生的。在孤立的肠道墓穴中24。Enteroids 需要较少的维护, 可以快速感染25, 并且可以很容易地建立, 因为肠道墓穴比 HIOs23更加区分。因此, HIEs 提供了许多优势超过现有的技术, 因为它们可以被开发展示区域特定的成分和功能性质的人胃肠上皮23。使用 enteroids 是一个非常有效的选择, 当需要一个人的肠道模型, 坚持区域特定的限制和易用性。在这里, 我们展示了从小鼠和早产儿中分离和维持原发性干细胞源性小肠 enteroids 的技术。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM) with 4.5 g/L Glucose and L-Glutamine</td><td>Lonza</td><td>12-604F</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>26140-079</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Humulin N (Insulin)</td><td>Eli Lilly And Company</td><td>0002-8315</td><td></td></tr><tr><td>1x Dulbecco&#039;s Phosphate-Buffered Saline (DPBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15750-060</td><td></td></tr><tr><td>Amphotericin B</td><td>Gibco</td><td>15290-026</td><td></td></tr><tr><td>0.5 M Ethylenediaminetetraacetic acid (EDTA), pH 8.0</td><td>Invitrogen</td><td>15575-020</td><td></td></tr><tr><td>1x Advanced DMEM/F-12</td><td>Invitrogen</td><td>12634-028</td><td></td></tr><tr><td>200 mM L-Glutamine</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>1 M N-2-Hydroxyethylpiperazine-N-2-Ethane Sulfonic Acid (HEPES)</td><td>Sigma-Aldrich</td><td>H3537</td><td></td></tr><tr><td>N-Acetylcysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td></td></tr><tr><td>100x N-2 Supplement</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>50x B-27 Supplement Minus Vitamin A</td><td>Gibco</td><td>08-0085SA</td><td></td></tr><tr><td>100x ROCK Inhibitor Y-27632, Dihydrochloride</td><td>Sigma-Aldrich</td><td>Y0503</td><td></td></tr><tr><td>Recombinant Mouse Wnt3a Protein</td><td>R&amp;amp;D Systems</td><td>1324-WN</td><td></td></tr><tr><td>Murine Noggin</td><td>PeproTech</td><td>250-38</td><td></td></tr><tr><td>Recombinant Mouse R-Spondin 1</td><td>R&amp;amp;D Systems</td><td>3474-RS</td><td></td></tr><tr><td>Recombinant Murine Epidermal Growth Factor (EGF)</td><td>PeproTech</td><td>315-09</td><td></td></tr><tr><td>Matrigel Growth Factor Reduced Basement Membrane Matrix</td><td>Corning</td><td>356231</td><td></td></tr><tr><td>35 x 10 mm Cell Culture Petri Dish</td><td>Eppendorf</td><td>0030700112</td><td></td></tr><tr><td>24-Well Cell Culture Plate</td><td>Eppendorf</td><td>0030722116</td><td></td></tr><tr><td>48-Well Cell Culture Plate</td><td>Eppendorf</td><td>0030723112</td><td></td></tr><tr><td>8-Well Nunc Lab-Tek II Chamber Slide System</td><td>Thermo Scientific</td><td>154534</td><td></td></tr><tr><td>50 mL Conical Tube</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>100 &amp;mu;M Sterile Cell Strainer</td><td>Fisher Scientific</td><td>22-363-549</td><td></td></tr><tr><td>70 &amp;mu;M Sterile Cell Strainer</td><td>Fisher Scientific</td><td>22-363-548</td><td></td></tr><tr><td>1x Phosphate-Buffered Saline (PBS), pH 7.2</td><td>Invitrogen</td><td>20012-027</td><td></td></tr><tr><td>16% Paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Normal Donkey Serum (NDS)</td><td>Sigma-Aldrich</td><td>D9663</td><td></td></tr><tr><td>4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>Microscope Cover Glass</td><td>Fisher Scientific</td><td>12-544-D</td><td></td></tr><tr><td>Confocal Microscope Leica TCS SP8 X</td><td>Leica Microsystems</td><td>N/A</td><td></td></tr><tr><td>Photoshop CS6</td><td>Adobe Systems</td><td>N/A</td><td></td></tr><tr><td>18 G 1.5 Inch Needle</td><td>Becton Dickinson</td><td>305196</td><td></td></tr><tr><td>Isoflurane</td><td>Sigma-Aldrich</td><td>792632</td><td></td></tr><tr><td>Oxytocin</td><td>Sigma-Aldrich</td><td>O3251</td><td></td></tr><tr><td>Human Double Electric Breast Pump</td><td>Lansinoh</td><td>044677530163</td><td></td></tr><tr><td>5 mL Round Bottom Test Tube</td><td>Corning</td><td>352058</td><td></td></tr><tr><td>Rubber Stoppers</td><td>Frey Scientific</td><td>560761</td><td></td></tr><tr><td>Ki67 Antibody</td><td>Abcam</td><td>AB15580</td><td></td></tr><tr><td>Human Mki67 primer F: 5&#039;-GACCTCAAACTGGCTCCTAATC-3&#039; R: 5&#039;-GCTGCCAGATAGAGTCAGAAAG-3&#039;</td><td>Integrated DNA Technologies</td><td>N/A</td><td></td></tr></tbody></table>

breast milk enhances growth of enteroids: an ex vivo model of cell proliferation

人小肠 enteroids 来自于墓穴, 当生长在一个干细胞的利基包含所有的上皮细胞类型。建立人类 enteroid 体外培养系统的能力对于模拟肠道病理生理学和研究所涉及的特定细胞反应非常重要. 近年来, 来自老鼠和人类的 enteroids 正在被培养、传代, 并在世界各地的几个实验室中被储存起来供将来使用。这个 enteroid 平台可以用来测试各种治疗和药物的影响, 以及对不同的肠道细胞类型的影响。在此基础上, 建立了从新生小鼠和早肠中提取的原发性干细胞衍生小肠 enteroids 的方法。此外, 利用这种 enteroid 培养系统来检测特定品种母乳的效果。用改良的人乳泵可以有效地获得小鼠母乳, 并表达小鼠乳可以用于进一步的研究实验。我们现在展示了表达的小鼠, 人和供体母乳对新生小鼠或早产人小肠 enteroids 的生长和增殖的影响。

我们首先试图调查表达的人母乳或巴氏杀菌供乳是否对小肠 enteroids 有影响。事实上, 人类母乳和捐赠母乳增加了新生鼠的生长 (图 1A) 和过早的人类衍生 enteroids (图 1B)。
由于人类母乳增加小肠 enteroids 的生长, 我们接下来研究是否母乳对过早人类 enteroids 的增殖有影响。我们证明, 人...

Watch this Scientific Journal Video about Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation at JoVE.com

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

母乳促进 Enteroids 的生长: 一个前体细胞增殖模型

该协议描述了如何建立从新生鼠或早肠的 enteroid 培养系统以及从小鼠收集牛奶的有效方法。

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

breast-milk-enhances-growth-enteroids-an-ex-vivo-model-cell

Research

JoVE Journal

Medicine

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

19.4K 次观看.  Washington University School of Medicine. 该协议描述了如何建立从新生鼠或早肠的 enteroid 培养系统以及从小鼠收集牛奶的有效方法。

视频: Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

在球体哺乳动物细胞大规模扩张营养调控连续通

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

科研

生物工程

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

癌症研究

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

使用胎儿肠道有机体在体外重述吸吮到发胶过渡

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

发育生物学

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

通过微分试穿素生成马赛克乳腺有机体

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

医学

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

乳腺的发育和功能在小鼠模型的评价

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

生物学

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

乳腺辐照有机体的生长与表征

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

将3D多细胞乳房球体直接生物打印到内皮网络

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have recently emerged as a promising system to model gastrointestinal pathologies. However, currently utilized methods for enteroid manipulation either lack access to the apical surface of the epithelium (three-dimensional [3D]) or are time-consuming and resource-intensive (two-dimensional [2D] monolayers). These methods often require additional steps, such as microinjection, for the model to become physiologically translatable. Here, we describe a physiologically relevant and inexpensive protocol for studying NEC in vitro by reversing enteroid polarity, resulting in the apical surface facing outward (apical-out). An immunofluorescent staining protocol to examine enteroid barrier integrity and junctional protein expression following exposure to tumor necrosis factor-alpha (TNF-&#945;) or lipopolysaccharide (LPS) under normoxic or hypoxic conditions is also provided. The viability of 3D apical-out enteroids exposed to normoxic or hypoxic LPS or TNF-&#945; for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-&#945;, in combination with hypoxia, exhibited disruption of epithelial architecture, a loss of adherens junction protein expression, and a reduction in cell viability. This protocol describes a new apical-out NEC-in-a-dish model which presents a physiologically relevant and cost-effective platform to identify potential epithelial targets for NEC therapies and study the preterm intestinal response to therapeutics.

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

This protocol describes an apical-out necrotizing enterocolitis (NEC)-in-a-dish model utilizing small intestinal enteroids with reversed polarity, allowing access to the apical surface. We provide an immunofluorescent staining protocol to detect NEC-related epithelial disruption and a method to determine the viability of apical-out enteroids subjected to the NEC-in-a-dish protocol.

体外 坏死性小肠结肠炎的顶端出肠样模型

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have ...

Quantification of Proliferative and Dead Cells in Enteroids

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or &ldquo;enteroids&rdquo;. Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2&rsquo;-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

肠内增殖细胞和死细胞的定量

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. ...

母乳促进 Enteroids 的生长: 一个前体细胞增殖模型

摘要

探索更多视频

此视频中的章节

在球体哺乳动物细胞大规模扩张营养调控连续通

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型

使用胎儿肠道有机体在体外重述吸吮到发胶过渡

通过微分试穿素生成马赛克乳腺有机体

从整体组织和活检建立人类上皮Enteroids和Colonoids的

乳腺的发育和功能在小鼠模型的评价

乳腺辐照有机体的生长与表征

将3D多细胞乳房球体直接生物打印到内皮网络

体外坏死性小肠结肠炎的顶端出肠样模型

肠内增殖细胞和死细胞的定量

母乳促进 Enteroids 的生长: 一个前体细胞增殖模型

摘要

探索更多视频

此视频中的章节

在球体哺乳动物细胞大规模扩张营养调控连续通

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

使用胎儿肠道有机体在体外重述吸吮到发胶过渡

通过微分试穿素生成马赛克乳腺有机体

从整体组织和活检建立人类上皮Enteroids和Colonoids的

乳腺的发育和功能在小鼠模型的评价

乳腺辐照有机体的生长与表征

将3D多细胞乳房球体直接生物打印到内皮网络

体外 坏死性小肠结肠炎的顶端出肠样模型

肠内增殖细胞和死细胞的定量

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型

体外坏死性小肠结肠炎的顶端出肠样模型