Name: mouse- and human-derived primary gastric epithelial monolayer culture for the study of regeneration
Uploaded: 2018-05-07T13:30:00.000+00:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration at JoVE.com

这项工作得到 NIH (NIDDK) 5 R01 DK083402-07 补助金 (YZ) 的支持。

为了避免污染, 在无菌组织培养罩内全部执行协议。根据经批准的辛辛那提大学 irb 协议 (irb 协议号: 2015-4869), 从患者的袖状胃切除术中收集人体胃底组织。 所有老鼠的研究都得到了辛辛那提大学机构动物保育和使用委员会 (IACUC) 的批准, 它维持着美国实验室动物护理 (AAALAC) 设施的评估和认可协会。
1. 建立人源性3D 胃胃底 Organoids
注意: 此协议基于以前在本实验室中发布的研究8。
<ol><li>准备 3D organoid 的介质, 由50% 个 Wnt 条件和20% 个 spondin 条件的介质组成。<ol>
<li>制备 wnt 条件培养基, 培养 wnt 生长培养基中的 L 细胞 (Dulbecco 修饰的鹰培养基 (DMEM), 10% 胎牛血清 (血清), 1% 青霉素/链霉素) 7 天, 收获培养基, 并使用0.22 µm 过滤器进行过滤。</li>
		<li>准备 R-spondin 条件介质。在 r-spondin 生长培养基 (Dulbecco 修饰的鹰培养基 (DMEM), 10% 胎牛血清 (血清), 1% 青霉素/链霉素) 中生长一种改良的 HEK-293T r-spondin 分泌细胞系。2小时后附着, 将培养基从这些细胞转变为 OPTIMEM 生长培养基, 1% 青霉素/链霉素。将细胞培养7天。然后用0.22 µm 过滤器收割 spondin 条件介质和过滤器。 注意: 此协议以前已发布, 对于更详细的协议, 请参阅6、7、8。</li>
	</ol>
</li>
	<li>解冻生长因子减少, 苯酚无红基底膜基质为16小时的冰在4摄氏度。在一个大型塑料容器中收集在冷 Ca2 +/毫克2 +中的 DBPS 的组织。储存在冰上直到开始步骤1.4。 注: 组织是收集的病人进行袖状切除术。</li>
	<li>使用镊子, 在含有100毫升 Ca2 +/毫克2 +-游离 Dulbecco 磷酸盐缓冲盐水 (DPBS) 的无菌烧杯中大力冲洗组织。洗涤大约三十年代或直到残骸和血液被去除了。接下来, 使用无菌纱布, 从上皮中抹去粘液层。</li>
	<li>使用大钳, 牢牢抓住组织的肌肉层, 用力, 用大弯止血钳刮走上皮。将擦伤的上皮碎片收集成无菌的聚苯乙烯培养皿。</li>
	<li>用剃刀将上皮组织分成大约 2.0 x 2.0 毫米2大小的碎片, 以优化组织消化。用 ca2 +/毫克2 + -免费 DBPS 补充抗生素 (ca2 +/毫克2 + -免费 DPBS) 清洗组织碎片, 1% 青霉素/链霉素, 0.25 毫克/毫升两性霉素 B/10 毫克/毫升庆大霉素, 50 毫克/毫升卡那霉素), 直到洗的时候没有血。如有需要, 进行多次清洗。把洗涤用不育的纱布仔细过滤成一个废烧杯。</li>
	<li>收集洗涤片段成一个125毫升玻璃圆底烧瓶与25毫米搅拌棒和预热孵化介质 (基底介质补充2毫米 l-谷氨酰胺, 1% 青霉素/链霉素, 10 毫米 HEPES 缓冲, 1 毫克/毫升胶原酶 1, 2 毫克/毫升细胞培养级牛血清白蛋白)。用橡胶隔膜封住圆底烧瓶。</li>
	<li>将20克的脊椎针插入到隔膜中, 并将其连接到带橡胶冲洗的氧气罐中。要过滤掉任何污染物, 请在油管中插入纸巾以创建过滤器。打开低的氧气流出。插入 10-15 流出针进入隔膜, 以避免隔膜破裂。</li>
	<li>使用夹子将设置固定到一个环形支架上, 并将其放置在水浴中, 用搅拌板校准到37°c, 30-45 分钟。在组织孵化了15分钟后, 取出50µL 的孵化培养基, 并在视觉上检查游离腺体。如果腺体不致密或未与组织分离, 则额外留出 5-10 分钟。</li>
	<li>紧接孵化后, 加入50毫升预加热 DMEM/F12, 使用不育的血清吸管或直接倒入孵化混合物中孵化培养基。</li>
	<li>通过无菌纱布将腺体混合物过滤成四50毫升圆锥管。把滤液放在冰上15分钟, 让提取的腺体沉淀到锥形管的底部。注意不要扰乱已沉淀的腺体, 用血清学吸管去除和丢弃前40毫升的上清。并用重悬10毫升10毫升 DPBS 补充抗生素。</li>
	<li>将混合物均匀地分布到5毫升细胞培养试管中。离心机为5分钟在 65 x g 在4°c 和去除上清液用吸管仔细。用吸管或200µL 宽吸管尖并用重悬在解冻基底膜基质中的腺体颗粒。混合均匀。 注意: 在冰上执行这一步骤是非常重要的, 在混合时避免基底膜基质的聚合。另外, 通过抽样检查50µL 的腺体密度是很重要的。最佳密度为70% 融合。</li>
	<li>接下来, 用一个宽尖的吸管在50µL 基底膜基质中的腺体。避免在电镀时产生气泡。</li>
	<li>为使基底膜基质聚合, 孵育 10-15 分钟37摄氏度。如果镀在一个12井培养板, 添加1毫升的 hFGO 介质 (先进的 Dulbecco 的改良鹰 medium/F12 培养基补充 2mM l-谷氨酰胺, 1% 青霉素/链霉素, 0.25 毫克/毫升两性霉素 B/10 毫克/毫升庆大霉素, 50 毫克/毫升卡那霉素, 10 毫米 HEPES缓冲器, 1 毫米 n-Acteylcystine, 1 x N2, 1 x B27, 50% 个 Wnt 条件培养基, 20% R spondin 条件培养基补充 100 ng/毫升骨形成蛋白抑制剂, 1 nM 胃泌素, 50 ng/毫升表皮生长因子, 200 ng/毫升成纤维细胞生长因子 10, 10毫米烟酰胺, 和10µM Y-27632 岩石抑制剂) 每井。如果不使用12井培养板, 添加足够的介质淹没基底膜基质气泡。</li>
	<li>在 5% CO2加湿细胞培养孵化器中培养37°c 的细胞。每4-5 天更换一次媒体。</li>
</ol>2. 建立鼠源性3D 胃胃底 Organoids
注意: 此协议以前已发布6。在冰上解冻基底膜基质, 并在开始前准备所有试剂。
<ol><li>用研究机构批准的方法来牺牲老鼠。从鼠标中取出胃, 并根据以前发布的协议6进行解剖。20毫升冰冷 Ca2 +/毫克2 +-免费 DBPS。 注: C57BL/6 小鼠, 年龄为 8-10 周, 用于胃底胃 organoid 培养。在胃切除前, 通过吸入二氧化碳和人工颈椎脱位, 对小鼠进行安乐死。</li>
	<li>将肌肉层从胃和任何可见的血管剥离为以前发布的6。</li>
	<li>用不育的剃刀刀片从窦和前胃癌中分离出眼底。切开眼底使用小手术剪刀到片断大约 2.0 x 2.0 毫米. 使用镊子, 将碎片收集到含有 EDTA 孵化缓冲器的15毫升圆锥管 (Ca2 +/毫克2 +-免费 DPBS, 5mM EDTA), 并在4摄氏度孵化为 2 h 与温柔的鼓动。</li>
	<li>在无菌罩中, 在冰上留下圆锥管和碎片大约5分钟. 使用吸管去除尽可能多的孵化缓冲, 留下碎片。添加5毫升震动缓冲器 (1 克 D-山梨醇, 1.47 克蔗糖在100毫升 Ca2 +/毫克2 +-免费 DPBS) 和握手与一支力量为2分钟. 允许大的碎片定居和匆忙, 使用1000p 吸管, 删除尚未解决的小碎片在顶层。</li>
	<li>旋转被去除的片断为5分钟在4°c 在 65 x g。</li>
	<li>同时避免气泡, 去除上清, 并用重悬在基底膜基质和混合, 直到它是均匀的。</li>
	<li>使用宽尖端吸管将基底膜基质在50µL 每井, 然后孵育37摄氏度20分钟。</li>
	<li>添加足够的 mFGO 生长媒体 (先进的 Dulbecco 的改良鹰 medium/F12 培养基辅以 2mM l-谷氨酰胺, 1% 青霉素/链霉素, 10 毫米 HEPES 缓冲器, 1mM n Acteylcystine, 1 x N2, 1 x B27, 50% Wnt 条件的培养基, 20% R-spondin 条件培养基辅以 100 ng/毫升骨形态发生蛋白抑制剂, 10 nM 胃泌素, 50 ng/毫升表皮生长因子, 10 ng/毫升成纤维细胞生长因子 10, 10 µM Y-27632 岩石抑制剂) 淹没气泡。</li>
	<li>培养细胞在37°c, 5% CO2加湿细胞培养孵化器。每 4-5 天更改媒体。</li>
</ol>3. 2D 转移用胶原蛋白涂层
注意: 除非另有规定, 否则在无菌组织培养罩中执行所有步骤。在将 3D organoids 转移到单层之前, 开始胶原涂层24小时。保持鼠尾胶原蛋白在冰上, 并屏蔽光。胶原蛋白涂层板是良好的长达2周。如果不立即使用涂层板, 包装在 parafilm 和存储在4°c, 直到需要。
<ol><li>用20毫米醋酸稀释鼠尾胶原蛋白到50µg/毫升。</li>
	<li>用稀释后的胶原蛋白充分覆盖井表面。为一井在标准12井板材, 外套与大约1毫升胶原蛋白。在室温下以无菌罩过夜。</li>
	<li>清洗两次与1毫升 Ca2 +/毫克2 +-免费 DPBS 每井和离开发现2小时在室温下的无菌罩。</li>
</ol>4. 2D 转移用基底膜基质涂层
注意: 除非另有规定, 否则在无菌组织培养罩中执行所有步骤。在将 3D organoids 转移到单层之前, 开始基底膜基质涂层2小时。把地下室的膜基质放在冰上。涂层板应立即使用, 不能储存一段较长的时间。
<ol><li>在15毫升锥形管中稀释基底膜基质, 细胞培养级水的比例为1:10。在冰上稀释。</li>
	<li>用吸管将稀释基底膜基质的100µL 添加到标准12井板的每个井中。使用一个细胞刮刀, 均匀地分配稀释基底膜基质在整个井为均匀外套。</li>
	<li>孵育37°c 为1小时。</li>
	<li>在转移 3D organoids 前, 用吸管去除残余水分, 室温干燥1小时。</li>
</ol>5. 3D 米/hFGOs 到2D 胃上皮细胞膜的转移
<ol><li>3D 鼠或人源 FGOs 经过培养 6-7 天后, 开始向2D 单层转移 3D organoids。 注: 对于每一个单层所需的井, 建议使用300完整的 organoids 或从 300 organoids 中提取的单个细胞。在一个健壮的文化, 它有望获得大约 150 organoid/井内12井培养板。</li>
	<li>在转移前一定要涂上盘子。 注: 基底膜基质涂层板具有半透明的凝胶层, 但胶原涂层板不会有任何明显的涂层迹象。</li>
	<li>直接吹打1毫升无菌 Ca2 +/毫克2 +-自由 DBPS, 从板上取出3D 基底膜基质气泡到气泡中心。</li>
	<li>用吸管将基底膜基质和 organoid 混合物收集到细胞培养试管中。收集的比例为2基底膜基质气泡每管。离心机为5分钟在 40 x g 和4°c。</li>
	<li>使用真空系统, 去除上清和尽可能多的基底膜基质。并用重悬在4毫升的Ca2 +/毫克2 +-免费 DBPS, 删除上清, 并重复 2-3 次。对于整个 organoid 传输协议, 请继续执行步骤5.10。继续执行单单元暂停协议的步骤5.7。</li>
	<li>预热细胞脱离溶液至37摄氏度, 每试管加1毫升。用吸管轻轻地将管子或吹打混合。孵育10分钟, 在37摄氏度。</li>
	<li>将2毫升的 Ca2 +/毫克2 +-免费 DBPS 直接添加到每个测试管。</li>
	<li>使用26克针和注射器的混合物 2-3 倍。目视检查单个单元格。注射器 1-2 倍, 如果仍然有 organoids 或集群的细胞。</li>
	<li>离心机在 40 x g 5 分钟在4摄氏度。并用重悬 2D FGO 介质中的颗粒(2D 鼠标 FGO 生长介质: 高级 Dulbecco 改性鹰 medium/F12 培养基辅以 2mM l-谷氨酰胺, 10 毫米青霉素/链霉素, 10% 胎小牛血清, 10 毫米 HEPES 缓冲器, 1 x N2, 1 x B27,辅以 10 nM 胃泌素, 50 的表皮生长因子, 1 µM TGF β抑制剂, 10 µM Y-27632 岩石抑制剂。2D 人类 FGO 生长培养基:先进的 Dulbecco 改性鹰 medium/F12 培养基辅以 2mM l-谷氨酰胺, 1% 青霉素/链霉素, 10 毫米 HEPES 缓冲器, 10% 胎小牛血清, 10 毫米烟酰胺, 1 x N2, 1 x B27, 补充50毫克/毫升卡那霉素, 50 ng/毫升表皮生长因子, 和10µM Y-27632 岩石抑制剂, 1 µM TGF β抑制剂)。</li>
	<li>将泥沙在涂覆板上。孵育板在37°c。如果介质变黄, 则每隔4天或更早更换介质。单层需要大约4天的时间来形成。</li>
</ol>6. 用2D 胃上皮细胞单分子膜进行刮伤试验
<ol><li>使用剃刀刀片, 当文化达到100% 融合时, 划伤单层。</li>
	<li>室温下用3.7% 甲醛固定膜, 15 分钟。Permeabilize 0.5% 非离子洗涤剂/PBS, 室温20分钟。</li>
	<li>下块单膜与2% 驴血清1小时室温和孵化1:1000 稀释 h+, K+-atp 酶原抗体隔夜在4°c, 洗涤与0.1% 非离子洗涤剂/PBS 和孵化与1:100 稀释驴抗鼠488次抗体和 10μg/毫升赫斯特细胞核染色1小时室温。</li>
	<li>为确定2D 人源胃单层培养物中的表面黏液细胞, 用20微克/毫升荆豆刺猬 (UEAI) FITC 共轭染色细胞。共聚焦显微镜上的图像单膜。</li>
	<li>每隔几小时就拍下伤口的图像, 根据单层的质量和患者之间的变化, epithelialize 将会重 24-48 小时。</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histology+of+the+stomach+and+duodenum+in+Crohn's+disease.">Histology of the stomach and duodenum in Crohn's disease.</a> Am J Surg Pathol. 22 (4), 383-390 (1998).</li><li>Engevik, A. 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=The+Development+of+Spasmolytic+Polypeptide/TFF2-Expressing+Metaplasia+(SPEM)+During+Gastric+Repair+Is+Absent+in+the+Aged+Stomach.">The Development of Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) During Gastric Repair Is Absent in the Aged Stomach.</a> CMGH. , (2016).</li><li>Bertaux-Skeirik, 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=CD44+variant+isoform+9+emerges+in+response+to+injury+and+contributes+to+the+regeneration+of+the+gastric+epithelium.">CD44 variant isoform 9 emerges in response to injury and contributes to the regeneration of the gastric epithelium.</a> Journal of Pathology. 242 (4), 463-475 (2017).</li><li>Nam, K. T., 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=Mature+chief+cells+are+cryptic+progenitors+for+metaplasia+in+the+stomach.">Mature chief cells are cryptic progenitors for metaplasia in the stomach.</a> Gastroenterology. 139 (6), 2028-2037 (2010).</li></ol>

作者没有什么可透露的。

本议定书详细说明了建立人和老鼠的胃上皮膜, 可用于刮伤检测。该协议依赖于从原始人 (或小鼠) 组织中分离出的干细胞的概念, 是由 Schlaermann et al7首次发布的修改后的协议。特别是, 我们已经优化的协议, 以建立一个汇合和极化胃上皮单层, 表达的主要细胞血统, 可以发现在胃上皮内在体内。胃溃疡修复是一个复杂的过程, 涉及组织重新上皮, 再生和扩散<sup class="xref...

使用刮伤检测是研究修复和再生的常用方法1,2,3,4,5,6。建议的方法可用于专门研究胃再生和幽门螺杆菌的殖民化。过去, 胃癌细胞系被用来研究幽门螺杆菌 (幽门螺杆菌) 感染 1,2和直到最近的胃癌细胞株, 如 AGS 细胞被青睐1 ,2,3,4。胃癌细胞培养的一个局限性是它们未能重述胃上皮细胞的多样性。为了解决这一局限性, 这里展示了一个主要的人和小鼠3维胃底胃 organoids (FGOs) 的建立和转移的胃上皮单层的伤口愈合检测的基础上改进方法由 Schlaermann et al描述。7我们演示了从剪切后的3D 胃 FGOs 或 FGO 衍生的单细胞中提取的胃上皮细胞, 保留了一种极化的蜂窝状成分, 它与胃上皮的在体内有密切的关系。鉴于胃癌细胞系并没有证明这种技术所显示的细胞组成, 目前的协议比其他的擦伤伤口检测方法有优势。这种方法已被优化, 使单分子膜可以建立从整个 organoids 或从一个单一的细胞悬浮从分离 organoids 和电镀使用基底细胞膜基质或胶原涂层。该协议的总目标是建立一个 organoid 的胃上皮单层培养的新的伤口愈合试验。

<table><tbody><tr><td>Advanced Dulbecco&#039;s modified Eagle/F12 medium (basal media)</td><td>Life Technologies</td><td>12634-010</td><td></td></tr><tr><td>GlutaMAX (L-glutamine)</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Scientific</td><td>SV30010</td><td></td></tr><tr><td>Amphotericin B/Gentamicin</td><td>Thermo Scientific</td><td>R01510</td><td></td></tr><tr><td>Kanamycin</td><td>Thermo Fisher</td><td>RO1510</td><td></td></tr><tr><td>HEPES Buffer</td><td>Sigma Aldrich</td><td>H0887</td><td></td></tr><tr><td>n-Acetylcystine</td><td>Sigma Aldrich</td><td>A9165</td><td></td></tr><tr><td>N2</td><td>Life Technologies</td><td>17502-048</td><td></td></tr><tr><td>B27</td><td>Life Technologies</td><td>12587-010</td><td></td></tr><tr><td>BMP Inhibitor (Noggin)</td><td>Pepro Tech</td><td>250-38</td><td></td></tr><tr><td>Gastrin</td><td>Tocris</td><td>3006</td><td></td></tr><tr><td>EGF</td><td>Pepro Tech</td><td>315-09</td><td></td></tr><tr><td>FGF10</td><td>Pepro Tech</td><td>100-26</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma Aldrich</td><td>N0636</td><td></td></tr><tr><td>Y-27632 ROCK Inhibitor</td><td>Sigma Aldrich</td><td>Y0503</td><td></td></tr><tr><td>TGF-&amp;beta; Inhibitor</td><td>Tocris</td><td>2939</td><td></td></tr><tr><td>Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt; -free DPBS</td><td>Fisher</td><td>21-031CV</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>12634-010</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>FBS</td><td></td><td></td></tr><tr><td>OPTIMEM</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>0.22&amp;micro;M filter</td><td>Fisher</td><td>099-720-004</td><td></td></tr><tr><td>37 &amp;deg;C Water Bath</td><td></td><td></td><td></td></tr><tr><td>Collagenase Type 1</td><td>Worthington&amp;nbsp;</td><td>LS004214</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A9418</td><td></td></tr><tr><td>Kimwipes (tissue paper)</td><td>Fischer Scientific</td><td>06-666C</td><td></td></tr><tr><td>125 mL round bottom flask</td><td></td><td></td><td></td></tr><tr><td>1 in (25 mm) stir bar</td><td></td><td></td><td></td></tr><tr><td>Rubber Septa</td><td></td><td></td><td></td></tr><tr><td>Sterile Gauze</td><td></td><td></td><td></td></tr><tr><td>Stir Plate</td><td></td><td></td><td></td></tr><tr><td>Ring Stand</td><td></td><td></td><td></td></tr><tr><td>Ring Stand Clamps</td><td></td><td></td><td></td></tr><tr><td>Sterile petri dish</td><td></td><td></td><td></td></tr><tr><td>18 G needles of 1.5 in length</td><td>Thermo Fisher</td><td>305196</td><td></td></tr><tr><td>20 G spinal needles of 3.5 in length</td><td>Thermo Fisher</td><td>405182</td><td></td></tr><tr><td>12-well cell culture treated plate</td><td>Midwest Scientific</td><td>92012</td><td></td></tr><tr><td>Growth Factor Reduced, Phenol Red-free Matrigel&lt;sup&gt;TM &lt;/sup&gt;(Basement membrane matrix)</td><td>Fisher</td><td>CB-40230C</td><td></td></tr><tr><td>Sterile Razor Blades</td><td></td><td></td><td></td></tr><tr><td>Wide-tip tweezers</td><td></td><td></td><td></td></tr><tr><td>Curved Hemostatic Forceps</td><td></td><td></td><td></td></tr><tr><td>200 &amp;micro;L wide pipette tips</td><td>Thermo Fisher</td><td>02-707-134</td><td></td></tr><tr><td>5 mL cell culture test tubes</td><td>Fisher</td><td>14-956-3C</td><td></td></tr><tr><td>Oxygen Tank with rubber hosing</td><td></td><td></td><td></td></tr><tr><td>1 g D-Sorbitol</td><td></td><td></td><td></td></tr><tr><td>Sucrose</td><td>Fisher</td><td>SS-500</td><td></td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td></td></tr><tr><td>Dissecting Tray</td><td></td><td></td><td></td></tr><tr><td>Rocking Table</td><td></td><td></td><td></td></tr><tr><td>Rat Tail Collagen Type 1</td><td>Life Technologies</td><td>A10483-01</td><td></td></tr><tr><td>Cell culture grade glacial acetic acid</td><td>Fisher</td><td>A38-212</td><td></td></tr><tr><td>Cell culture grade water</td><td>Corning</td><td>25-055-CV</td><td></td></tr><tr><td>2-well chamber slide</td><td>Thermo Fisher</td><td>155380</td><td></td></tr><tr><td>12-well Transwell (polyester membrane inserts)</td><td>Corning</td><td>3460</td><td></td></tr><tr><td>Cell scraper</td><td>Corning</td><td>353086</td><td></td></tr><tr><td>Accutase (cell detachment solution)</td><td>Stemcell Technologies</td><td>7920</td><td></td></tr><tr><td>26&lt;sup&gt;3/8&lt;/sup&gt; G syringe</td><td>Thermo Fisher</td><td>309625</td><td></td></tr><tr><td>Razor blade</td><td></td><td></td><td></td></tr><tr><td>L Cells</td><td>L cells, a Wnt3a producing cell line, were received as a gift from Dr. Hans Clevers (Hubrecht Institute for Developmental Biology and Stem Cell Research, Netherlands).&amp;nbsp;&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>HEK-293T Rspondin secreting cells</td><td>A modified HEK-293T R-spondin secreting cell line was obtained from Dr. Jeff Whitsett (Section of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children&#039;s Hospital Medical Centre and The University of Cincinnati College of Medicine, Cincinnati, USA&amp;nbsp;).&amp;nbsp;&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>HK-ATPase Primary Antibody</td><td>Invitrogen</td><td>SC-374094</td><td></td></tr><tr><td>E-Cadherinn</td><td>SantaCruz</td><td>sc-59778</td><td></td></tr><tr><td>UEA1</td><td>Sigma Aldrich</td><td>L9006</td><td></td></tr><tr><td>Hoechst 33342</td><td>Thermo Fisher</td><td>H3570</td><td></td></tr><tr><td>Alexafluor 488 secondary antibody (Donkey anti-mouse 488 secondary antibody)</td><td>ThermoFisher Scientific</td><td>R37114</td><td></td></tr></tbody></table>

mouse- and human-derived primary gastric epithelial monolayer culture for the study of regeneration

体外对胃创面修复的研究通常涉及使用胃癌细胞系进行细胞增殖和迁移的刮伤试验。然而, 这种化验的一个关键限制是它们的细胞类型的同源分类。修复是一个复杂的过程, 需要几个细胞类型的相互作用。因此, 研究一种缺乏细胞亚型的文化, 是我们理解修复过程必须克服的一个问题。胃 organoid 模型可以缓解这个问题, 即细胞类型的异构收集密切反映的胃上皮或其他本机组织在体内。这里演示的是一本新颖的,体外刮伤试验, 从人或小鼠3维 organoids, 然后可以转移到胃上皮单层作为完整的 organoids 或作为一个单独的细胞悬浮分离organoids。该协议的目的是建立 organoid 的胃上皮膜, 可用于一个新的擦伤伤口检测系统, 以研究胃再生。

Organoids 是从人的主体/身体或鼠胃组织的眼底 (图 1A) 派生的。在胶原酶或 EDTA 消化的人或老鼠的胃组织分别后, 腺体埋入基底膜基质和培养 6-7 天 (图 1A)。图 1B演示了人源性胃 organoids (huFGOs) 的形成, 然后将其转移到基底膜基质涂层室幻灯片上的胃上皮层 (huGEM) 上。经过4天的文化, 汇合 huGEMs 被用于刮伤化验...

Watch this Scientific Journal Video about Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration at JoVE.com

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

在这里, 我们描述了建立和培养人和老鼠衍生的3维 (3D) 胃 organoids 的协议, 以及将 3D organoids 转移到2维单层的方法。本文还介绍了用胃上皮单层作为再生研究的一种新的擦伤创面检测方法。

小鼠和人源性胃上皮单层培养对再生的研究

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

mouse-human-derived-primary-gastric-epithelial-monolayer-culture-for

Research

JoVE Journal

Immunology and Infection

10.4K 次观看.  University of Cincinnati. 在这里, 我们描述了建立和培养人和老鼠衍生的3维 (3D) 胃 organoids 的协议, 以及将 3D organoids 转移到2维单层的方法。本文还介绍了用胃上皮单层作为再生研究的一种新的擦伤创面检测方法。

视频: Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

科研

癌症研究

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

类器官的模型传染病:人类的文化与小鼠胃组织体与幽门螺杆菌的显微注射

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

免疫与感染

Primary Cell Cultures to Study the Regeneration Potential of Murine M&#252;ller Glia after MicroRNA Treatment

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates such as fish, MG-driven regeneration occurs naturally; in mammals, however, stimulation with certain factors or genetic/epigenetic manipulation is required. Since MG comprise only 5% of the retinal cell population, there is a need for model systems that allow the study of this cell population exclusively. One of these model systems is primary MG cultures that are reproducible and can be used for a variety of applications, including molecule/factor screening and identification, testing of compounds or factors, cell monitoring, and/or functional tests. This model is used to study the potential of murine MG to convert into retinal neurons after supplementation or inhibition of microRNAs (miRNAs) via transfection of artificial miRNAs or their inhibitors. The use of MG-specific reporter mice in combination with immunofluorescent labeling and single-cell RNA sequencing (scRNA-seq) confirmed that 80%-90% of the cells found in these cultures are MG. Using this model, it was discovered that miRNAs can reprogram MG into retinal progenitor cells (RPCs), which subsequently differentiate into neuronal-like cells. The advantages of this technique are that miRNA candidates can be tested for their efficiency and outcome before their usage in in vivo applications.

M&#252;ller glia primary cultures obtained from mouse retinas represent a very robust and reliable tool to study the glial conversion into retinal progenitor cells after microRNA treatment. Single molecules or combinations can be tested before their subsequent application of in vivo approaches.

原代细胞培养物研究小鼠穆勒胶质细胞在微RNA处理后的再生潜力

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates ...

发育生物学

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to evade immune surveillance leading to the inhibition of CTL proliferation, survival, and effector function, and subsequently cancer persistence. Approximately 40% of gastric cancers express PD-L1, yet the response rate to immunotherapy is only 30%. We present the use of human-derived autologous gastric cancer organoid/immune cell co-culture as a preclinical model that may predict the efficacy of targeted therapies to improve the outcome of cancer patients. Although cancer organoid co-cultures with immune cells have been reported, this co-culture approach uses tumor antigen to pulse the antigen-presenting dendritic cells. Dendritic cells (DCs) are then cultured with the patient's CD8+ T cells to expand the cytolytic activity and proliferation of these T lymphocytes before co-culture. In addition, the differentiation and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in culture are investigated within this co-culture system. This organoid approach may be of broad interest and appropriate to predict the efficacy of therapy and patient outcome in other cancers, including pancreatic cancer.

The goals of the protocol are to use this approach to 1) understand the role of the immunosuppressive gastric tumor microenvironment and 2) predict the efficacy of patient response, thus increasing the survival rate of patients.

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to ...

医学

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

从小鼠和人类食管隔离肌纤维母细胞

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

生物学

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

小鼠小肠的混合原代培养拟肠激素分泌的研究和活细胞成像的肠内分泌细胞的供

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

来自肠道密码的穆林原结肠上皮单层的生成

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

从上消化道内窥镜活检生成人胃类器官

Establishment and Characterization of Patient-derived Gastric Organoids from Biopsies of Benign Gastric Body and Antral Epithelium

Gastric patient-derived organoids (PDOs) offer a unique tool for studying gastric biology and pathology. Consequently, these PDOs find increasing use in a wide array of research applications. However, a shortage of published approaches exists for producing gastric PDOs from single-cell digests while maintaining a standardized initial cell seeding density. In this protocol, the emphasis is on the initiation of gastric organoids from isolated single cells and the provision of a method for passaging organoids through fragmentation. Importantly, the protocol demonstrates that a standardized approach to the initial cell seeding density consistently yields gastric organoids from benign biopsy tissue and allows for standardized quantification of organoid growth. Finally, evidence supports the novel observation that gastric PDOs display varying rates of formation and growth based on whether the organoids originate from biopsies of the body or antral regions of the stomach. Specifically, it is revealed that the use of antral biopsy tissue for organoid initiation results in a greater number of organoids formed and more rapid organoid growth over a 20-day period when compared to organoids generated from biopsies of the gastric body. The protocol described herein offers investigators a timely and reproducible method for successfully generating and working with gastric PDOs.

作者聚焦:来自胃不同区域的患者来源的胃类器官的生成和比较 

Gastric patient-derived organoids find increasing use in research, yet formal protocols for generating human gastric organoids from single-cell digests with standardized seeding density are lacking. This protocol presents a detailed method for reliably creating gastric organoids from biopsy tissue obtained during upper endoscopy.

良性胃体和胃窦上皮活检患者来源的胃类器官的建立和表征

Gastric patient-derived organoids (PDOs) offer a unique tool for studying gastric biology and pathology. Consequently, these PDOs find increasing use in a ...