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>

<ol>
	<li>Nagy, T. A., 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=Helicobacter+pylori+regulates+cellular+migration+and+apoptosis+by+activation+of+phosphatidylinositol+3-kinase+signaling.">Helicobacter pylori regulates cellular migration and apoptosis by activation of phosphatidylinositol 3-kinase signaling.</a> J Infect Dis. 199 (5), 641-651 (2009).</li><li>Mnich, 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=Impact+of+Helicobacter+pylori+on+the+healing+process+of+the+gastric+barrier.">Impact of Helicobacter pylori on the healing process of the gastric barrier.</a> World J Gastroenterol. 22 (33), 7536-7558 (2016).</li><li>Zhang, Y., 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=Bm-TFF2,+a+toad+trefoil+factor,+promotes+cell+migration,+survival+and+wound+healing.">Bm-TFF2, a toad trefoil factor, promotes cell migration, survival and wound healing.</a> Biochem Biophys Res Commun. 398 (3), 559-564 (2010).</li><li>Crabtree, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cytokines+in+pathogenesis+of+Helicobacter+pylori-induced+mucosal+damage.">Role of cytokines in pathogenesis of Helicobacter pylori-induced mucosal damage.</a> Dig Dis Sci. 43 (Supplement), 46S-53S (1998).</li><li>Riahi, R., Yang, Y., Zhang, D. D., Wong, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+wound-healing+assays+for+probing+collective+cell+migration.">Advances in wound-healing assays for probing collective cell migration.</a> J Lab Autom. 17 (1), 59-65 (2012).</li><li>Zhang, M., Li, H., Ma, H., Qin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+microfluidic+strategy+for+cell+migration+assay+in+an+in+vitro+wound-healing+model.">A simple microfluidic strategy for cell migration assay in an in vitro wound-healing model.</a> Wound Repair Regen. 21 (6), 897-903 (2013).</li><li>Schlaermann, P., 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+novel+human+gastric+primary+cell+culture+system+for+modelling+Helicobacter+pylori+infection+in+vitro.">A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro.</a> Gut. 65 (2), 202-213 (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+plays+a+functional+role+in+Helicobacter+pylori-induced+epithelial+cell+proliferation.">CD44 plays a functional role in Helicobacter pylori-induced epithelial cell proliferation.</a> PLoS Pathog. 11 (2), e1004663 (2015).</li><li>Tarnawski, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+mechanisms+of+gastrointestinal+ulcer+healing.">Cellular and molecular mechanisms of gastrointestinal ulcer healing.</a> Dig Dis Sci. 50 (Suppl 1), S24-S33 (2005).</li><li>Wright, N. A., Pike, C., Elia, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+a+novel+epidermal+growth+factor-secreting+cell+lineage+by+mucosal+ulceration+in+human+gastrointestinal+stem+cells.">Induction of a novel epidermal growth factor-secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells.</a> Nature. 343, 82-85 (1990).</li><li>Wright, C. L., Riddell, R. 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&#39;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-&#946; Inhibitor</td>
			<td>Tocris</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>Ca2+/Mg2+ -free DPBS</td>
			<td>Fisher</td>
			<td>21-031CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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&#181;M filter</td>
			<td>Fisher</td>
			<td>099-720-004</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C Water Bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type 1</td>
			<td>Worthington&#160;</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 MatrigelTM (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 &#181;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>263/8 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).&#160;&#160;</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&#39;s Hospital Medical Centre and The University of Cincinnati College of Medicine, Cincinnati, USA&#160;).&#160;&#160;</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

10.3K 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

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

科研

免疫与感染

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

上皮损伤制作分析<em&gt;阿米巴</em&gt;感染

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

幽门螺杆菌感染与胃病理的小鼠模型

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

原发性鸡初囊-细胞培养模型研究传染性囊内病病毒发病机制

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

小鼠小肠上皮类器官与先天性淋巴细胞的共培养

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

小鼠和人源性胃上皮单层培养对再生的研究

摘要

探索更多视频

此视频中的章节