Name: isolation of salivary epithelial cells from human salivary glands for in vitro growth as salispheres or monolayers
Uploaded: 2019-07-15T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers at JoVE.com

我们要感谢詹姆斯·布里奇斯就培养原细胞的方法提供了重要指导。Matthew J. Beucler 得到了国家卫生训练学院 T32-ES007250 的支持。这项工作还得到了国家卫生研究院授予威廉·米勒的R01-AI121028和R21-DE026267的资助。

辛辛那提大学机构审查委员会(联邦范围的保障#00003152,IRB 协议 2016-4183) 对这些协议和研究进行了审查和批准。唾液组织通常在许多头部和颈部手术中被切除,并且通常不参与恶性过程。通常,从患者中取出后2-4小时内使用新切除的唾液腺组织(图1A)。
1. 试剂制备
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支气管上皮细胞生长培养基 (BEGM)
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<li>从制造商提供的套件中添加组件(?...

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唾液功能障碍代表了对Sjögren综合征患者以及接受唾液腺3、4、5附近癌症放射治疗的人的生活质量的关注。 16.治疗这些病人的一种建议疗法是在体外生长功能性唾液干细胞或器官,然后可以插入受损的唾液腺,以取代受影响的组织9。此外,唾液腺通常是人类病原体的持久性和传播的场所...

在哺乳动物中,唾液腺被组织成3对主要的唾液腺:小黄体、亚黄土和亚语言腺。也有一系列小腺位于舌头上,分散在整个口腔1。主要腺体负责产生的唾液的体积2。除了通过分泌因子提供抗菌保护外,唾液在咀嚼和润滑食物的过程中也非常重要,因为它通过食道2。因此,唾液腺功能障碍是一个医疗问题,患者谁不能产生足够的唾液更容易发展疾病,如蛀牙或口腔念珠菌病3。此外,唾液减少会导致饮食和消化食物更加困难,对生活质量有显著影响。
唾液腺功能障碍主要发生在患有Sjögren综合征(一种自身免疫性疾病)的患者中,或在接受辐照治疗3、4、5的头部和颈部癌症患者中。 6.由于自身免疫或放射治疗,腺体内受损的分泌性腺体不进行自我更新,导致患者生活在不可逆转的损害6。唾液腺的研究也引起了人们对病毒发病机制的关注或研究人员的关注。一些病原体,如人类巨细胞病毒(HCMV),在唾液腺内持续复制,然后允许通过唾液7、8将新生病毒传染给新的宿主。因此,开发健壮且可重复的唾液腺组织体外模型,以解决和理解各种医疗问题,是未满足的需求。
几个模型的鼠唾腺系统已被用作一个起点,了解和表征不同的上皮细胞,形成唾液腺9,10。人与鼠唾液腺11之间存在明显和重大的差异。最值得注意的是,人类的副体腺体相对于亚曼地腺体较大,而小鼠的亚曼底和花环在大小1、2、11上非常相似。虽然存在不朽的人类亚曼地状细胞系,但人们主要担心永生细胞不能准确维持主要组织的表型,二者担心永生细胞实际上并非来自唾液上皮本身12。由于这些原因,人们有兴趣和需要研究人类的主要唾液组织。

<table border="0" cellpadding="0" cellspacing="0" style="width:736px;" width="736">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">100 mm culture dishes</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:123px;">172931</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">15 mL conical tubes</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:123px;">339651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">50 mL conical tubes</td>
			<td style="width:132px;">Thermo Scientific</td>
			<td style="width:123px;">339653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Bronchial Epithelial Cell Growth Media</td>
			<td style="width:132px;">Lonza</td>
			<td style="width:123px;">CC-3171</td>
			<td>Add bullet kit as per manufacturer&#39;s instructions. Supplement with 20 mL of charcoal stripped serum.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Cell strainer 70 &#181;m nylon mesh</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:123px;">22-363-548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Charcoal stripped fetal bovine serum</td>
			<td style="width:132px;">Gibco</td>
			<td style="width:123px;">12676-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Collagenase type III</td>
			<td style="width:132px;">Worthington</td>
			<td style="width:123px;">LS004182</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Cryogenic Tube</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:123px;">5000-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Dispase</td>
			<td style="width:132px;">Cell Applications</td>
			<td style="width:123px;">07923</td>
			<td>Dissolve collagenase to make a 0.15% (w/v) stock. Filter sterilize then store at -20 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Dissecting scissors</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:123px;">08-940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Dulbecco phosphate buffered saline</td>
			<td style="width:132px;">Corning</td>
			<td style="width:123px;">55-031-PC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">General Chemicals</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">PathClear Basement membrane extract</td>
			<td style="width:132px;">Cultrex</td>
			<td style="width:123px;">3432-005-01</td>
			<td>Thaw at 4 &#176;C at least 24 hr prior to use. Always handle on ice.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Six-well culture dishes</td>
			<td style="width:132px;">Falcon</td>
			<td style="width:123px;">353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Surgical forceps</td>
			<td style="width:132px;">Fisher</td>
			<td style="width:123px;">22-079-742</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Trypsin-EDTA solution</td>
			<td style="width:132px;">Corning</td>
			<td style="width:123px;">25-052-CI</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of salivary epithelial cells from human salivary glands for in vitro growth as salispheres or monolayers

唾液腺是对人类疾病多方面感兴趣的研究人员非常感兴趣的场所。研究人员的目标之一是恢复因放射治疗或与Sjögren综合征相关的病理而受损的腺体的功能。研究人员的第二个目标是确定病毒在腺组织内复制的机制,然后它们能够获得对水平传播至关重要的唾液。这些目标突出表明,需要一种强大且易于获取的体外唾液腺模型,供对上述相关研究领域感兴趣的研究人员使用。在这里,我们讨论一个简单的协议,从人类唾液腺分离上皮细胞,并在体外繁殖它们。我们的协议可以进一步优化,以满足个别研究的需要。简单地说,唾液组织是机械和酶分离,以分离单个细胞或小簇细胞。上皮细胞的选择是在存在经过优化以促进上皮细胞生长的介质的情况下,电镀到基底膜基质上。这些生成的培养物可以作为三维簇,称为"盐层",或作为经处理的塑料组织培养皿的单层生长。该协议导致主要上皮细胞的异质种群的生长,在接受细胞衰老之前,可以繁殖5-8个通道(15-20个种群翻倍)。

在将细胞从消化组织电镀到BMM后两三天,细胞将很容易形成小簇,每个簇的大小将继续扩大至15-20个细胞(图2A)。通常可以看到细胞碎片和分离的死细胞,应通过吸气和补充新鲜介质来清除。细胞将继续增殖为盐层约3-10天,或只要BMM层保持完整。有时,由于BMM的部分分解,细胞将附着在底层塑料上,并开始增殖为单层。生长在BMM上的盐层在尺寸和结构复杂性方?...

Watch this Scientific Journal Video about Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers at JoVE.com

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

我们提出了一种分离和培养人类原发性唾液腺衍生上皮细胞的方法。这些细胞表现出与唾液上皮起源一致的基因表达模式,可以在从恩格尔布雷斯-霍尔姆-肖温肿瘤细胞中提取的基底膜基质上作为盐层生长,或作为治疗培养皿上的单层细胞生长。

从人类唾液腺中分离唾液表皮细胞,作为脂质层或单层体进行体外生长

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

生成主要细胞模型或器官状结构是回答生物学中许多当代问题的关键。例如，我们相当长一段时间一直对人类病毒用于促进唾液腺复制的机制感兴趣，因为它是水平传播的重要站点。本协议中描述的基于盐球的系统的发展是我们寻求回答这类问题的关键进展。这种技术使我们能够从各种不同的患者样本中生成原发细胞。细胞模型更让人想起体内类型的情况，因为细胞不像许多通常用于体外实验的细胞类型那样进行建模或转化。我们已经使用这个协议从小鼠原发性亚人类腺体生成类似的细胞，该协议可能对于具有类似细胞结构的其他器官有用。从患者那里切除了后两到四个小时内，在培养盘上，使用自动消毒的解剖剪刀和手术钳将唾液腺组织切碎成一到两毫米大小的细片。在切碎的组织中加入六毫升的 Dispase/胶原酶溶液。然后在37摄氏度下孵育约30分钟至1小时。用5毫升血清移液器移液15至20次，使组织中断。在37摄氏度下孵育30分钟到1小时。再重复移液和孵育步骤两到三次，或直到组织类似于泥浆，并很容易通过移液器开口。要用 BMM 覆盖油井，请缓慢移液 500 微升的隔夜解冻 BMM 到六孔板的每个孔中。缓慢旋转板，将 BMM 均匀分布到井上。然后在37摄氏度下孵育涂层板，在使用前至少15分钟，让BMM凝固。首先，将含有均质组织的 Dispase/胶原酶溶液转移到 15 毫升锥形管中。用六毫升 DPBS 清洗板壁一次，将剩余的细胞转移到锥形管中。然后通过70微米尼龙网状细胞过滤器，将均质过滤成新的50毫升锥形管，以去除未消化的组织。在500倍g下将细胞离心5分钟。吸上一道。然后将细胞颗粒重新在 10 毫升 1X RBC 解解缓冲液中。在37摄氏度下孵育5分钟。然后加入20至25毫升的DPBS，以中和RBC解液缓冲液，并最大限度地减少唾液细胞的解解。在500倍g下离心5分钟。白细胞颗粒表示所有RBC的完整解解。如果颗粒有红色，使用RBC解液缓冲液重复处理。吸升液，将颗粒重新在 BEGM 的一到两毫升中，然后将重新令人沮丧的细胞转移到 BMM 涂层孔上。在37摄氏度下孵育。一旦镀在BMM上，细胞将在两到三天的时间内形成球形结构或盐球。细胞可以保持为盐球约五到七天之前，BMM开始降解，允许细胞访问和粘附在塑料和成长为单层。首先，从油井中吸出介质，并在每口井中加入一毫升的 Dispase/科拉特纳塞溶液。在37摄氏度下孵育约15分钟，或直到BMM大部分溶解。然后将细胞转移到15毫升锥形管中，用1至3毫升的DPBS洗一次井，以获得剩余的细胞。在500倍g下离心5分钟。吸升液，将颗粒重新在两毫升的 Trypsin 中。在37摄氏度下再次孵育15分钟。接下来，加入管中任何含有10%血清的完整介质，以中和三辛。在500倍g下离心5分钟。吸上液去除残留介质、三辛和血清，然后在DPBS中重新喷洒细胞。在500倍g下离心5分钟。吸上一液，并在 BEGM 中重新暂停颗粒。为了保持颗粒作为球体，将重新暂停的细胞板到凝固的 BMM 涂层培养皿上。培养加湿培养箱中所有细胞在下培养前5至7天保持37摄氏度，二氧化碳含量为5%。每两到三天用新鲜的 BEGM 喂养。要将细胞作为单层扩散到塑料培养皿上，请从培养皿中吸出 BEGM 介质，用 DBPS 洗一次，以去除残留介质。加入两毫升的三辛，在37摄氏度下孵育15分钟，或直到细胞完全分离。使用含有10%血清的任何完整介质的四毫升中和 Trypsin。然后轻轻倾斜盘子，将细胞转移到15毫升锥形管。用大约五毫升的 DPBS 洗一次盘子，以收集剩余的细胞。在500倍g下离心5分钟。吸上一道。要去除残留介质，请重新在 DPBS 的 6 到 10 毫升中重新发送细胞。在500倍g下离心5分钟。吸上一道，在 BEGM 中重新暂停。然后将细胞板到经过处理的塑料组织培养皿上。培养加湿培养箱中的细胞在下培养前5至7天保持37摄氏度，二氧化碳含量为5%。每两到三天用新鲜的 BEGM 喂养。在该协议中，将消化的组织细胞电镀到BMM两到三天后，细胞很容易形成小簇，每个组的大小继续扩大，高达15至20个细胞。盐球细胞被镀在细胞培养处理后塑料上，并生长成单层，它们表现出与上皮起源细胞一致的形态。需要适当的无菌技术和护理，以避免细菌或真菌污染。任何抗生素或抗神秘药都无法拯救大量微生物的培养。我们正在回答有关病毒发病机制和传播的问题。但对于其他人，这提供了一种方法来研究人类唾液细胞为任何数量的应用，如组织再生疗法。对于研究人类巨细胞病毒的我们来说，这项技术使我们能够开始探索病毒利用的分子机制来感染并最终传染给新的宿主。本协议中使用的任何方法本质上都是危险的，但在处理人体组织和任何类型的化学试剂时应始终小心谨慎。

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Immunology and Infection

Unit 1

Tissues of the Human Body

SCIENTISTS IN ACTION

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

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

Application of a Mouse Ligated PeyerÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

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 ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

杂交<em&gt;原位</em&gt;和蚊虫唾液腺，卵巢，胚胎

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Method for the Measurement of Salivary Gland Function in Mice

小鼠涎腺功能的测定方法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...