这项工作得到了中国浙江省中医科技计划 （2017ZA055;2018ZA036），以及中国贵州省遵义市科技项目（遵义市科和扶 NS 〔2020） 第 18 号）给徐旭。作者感谢优佳（杭州）生物医药科技公司分子生物学实验室提供细胞培养培训。

根据浙江中医大学第一附属医院医学伦理委员会的指导方针（方案编号）。ChiMCTR2100005281，日期：2017-07-17）。
1. 组织采集
<ol><li>从成年女性的手术标本中收集新鲜的乳腺组织，将其收集到含有 10 mL 磷酸盐缓冲盐水 （PBS） 和 3% 青霉素/链霉素 （P/S） 的无菌管中。 注意：从手术中收集后 24 小时内，应根据以下详细信息处理乳腺组织。</li>
</ol>2. 组织预处理
<ol><li>用两对镊子从乳腺组织中取出脂肪组织，确保剩余的乳腺组织的重量为 ~1 克。</li>
	<li>用 75% 乙醇溶液 （5 mL） 冲洗乳腺组织 5 秒，然后用 20 mL 洗涤液（表 1）洗涤 2 x 5 分钟。</li>
</ol>3. 组织消化
<ol><li>将乳腺组织切成更小的碎片，用两个手术刀片将组织切碎 15 分钟以获得组织匀浆。将组织块转移到 50 mL 离心管中。</li>
	<li>在含有乳腺组织碎片的 50 mL 离心管中，将 10 mL 5.0 mg/mL 分散酶 + 5.0 mg/mL 胶原酶溶液、3 mL 0.25% 胰蛋白酶和 7 mL PBS 添加到总共 20 mL 消化液中。将试管置于 37 °C 的水浴中并孵育 1.5 小时;每 20 分钟摇动一次试管。</li>
	<li>通过注入 20 mL 中和溶液来终止消化过程。通过移液 ~15 次混合内容物。</li>
	<li>通过 100 μm 网状过滤器过滤混合物。以 156 × g 离心 5 分钟。</li>
	<li>去除上清液，重复沉淀与 20 mL 中和溶液的孵育。通过移液 15 次混合内容物，并以 156 × g 离心 5 分钟。</li>
	<li>小心去除上清液，并用 10 mL 初始培养基重悬细胞沉淀。将细胞悬液接种在 100 mm 细胞培养皿中。</li>
	<li>在 37 °C 的 5% CO2 培养箱中培养细胞。 每三天用新鲜的上皮细胞培养基替换原始培养基。检查细胞并每 2 天刷新一次培养基。</li>
</ol>4. 细胞传代
<ol><li>当细胞达到 80%-90% 汇合时，从培养箱中取出 100 mm 培养皿，丢弃用过的培养基，并用 2 mL PBS 冲洗培养皿 2 次。去除 PBS，然后将 2 mL 0.05% 胰蛋白酶添加到每个 100 mm 培养皿中。 注意：旋转培养皿以确保胰蛋白酶溶液与培养皿底部充分接触。</li>
	<li>将 100 mm 培养皿置于 37 °C 培养箱中 ~7 分钟以进行消化过程。</li>
	<li>使用 40 倍物镜用显微镜检查细胞，以确保大多数细胞已从培养皿底部解离。</li>
	<li>加入 8 mL 中和溶液终止消化过程，并将细胞转移到 15 mL 试管中。通过上下吹打 10-15 次来混合细胞。将细胞以 156 × g 离心 5 分钟。</li>
	<li>小心去除上清液，用 10 mL 上皮细胞培养基重悬细胞，并计数细胞数。</li>
	<li>在 100 mm 培养皿的 10 mL 上皮细胞培养基中加入 1 ×10 6 个细胞。</li>
	<li>刷新上皮细胞培养基并每 2 天观察一次细胞。</li>
</ol>5. 细胞冻存
<ol><li>重复步骤 4.1-4.4。</li>
	<li>小心去除上清液，并将细胞重悬于 2 mL 细胞冻存液中。</li>
	<li>将 1 mL 含有细胞的冻存溶液转移到每个冻存管中。</li>
	<li>记录冻存细胞的名称、传代编号和日期。</li>
	<li>将样品瓶置于 -80 °C 的受控速率冷冻容器中过夜。</li>
	<li>从 -80 °C 冰箱中取出冻存管，并迅速将其转移到液氮中进行长期储存。</li>
</ol>

从人体组织中分离乳腺上皮细胞的方案

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作者声明，本文的发布不存在利益冲突。

HMEC 对于保持乳腺组织的解剖和功能完整性至关重要，它们在科学调查、临床实施和相关领域中非常有用15。原代上皮细胞是一种传代受限且寿命较短的特殊细胞。然而，HMEC 的增长受到技术限制的阻碍，从而阻碍了乳腺癌和其他与乳腺相关的炎症性疾病的研究进展16。
因此，迫切需要开发一种稳定、可行且高效的方法来从新鲜乳腺组织中获...

乳腺癌是全球女性诊断出的主要癌症类型，也是癌症死亡的主要原因1。乳腺癌的发病机制很复杂，涉及遗传、环境和生活方式等多种因素。HMEC 是活性产奶细胞，是乳腺组织最重要的成分之一，很可能是参与乳腺癌致癌的原始细胞。因此，HMEC 在乳腺癌研究中受到了研究人员的最大关注2。此外，原代细胞能够提供复杂细胞过程的生物学相关特征，因为它们保留了遗传稳定性、正常形态和更完整的基本细胞功能，而永生化细胞系无法实现3。因此，原代 HMEC 的分离和培养是研究大多数乳腺相关疾病（如乳腺癌和乳腺炎症性疾病）的重要步骤。
目前，已经建立了一个稳定且可重复的系统，用于分离、培养和鉴定大鼠、奶牛、猪和山羊的乳腺上皮细胞 4,5,6,7。然而，由于复杂的微环境和低细胞产量，原代 HMEC 的分离和培养具有挑战性。几十年来，科学家们一直在寻找分离和培养 HMEC 的最有效方法，尽管 HMEC 培养系统是在近 20 年前建立的。例如，Hammond 等人开发了一种无血清培养基，其中 HMEC 可有效生长8。最近，Zubeldia-Plazaola 等人使用快/慢酶消化程序结合顺序过滤或差速离心步骤测试了四种不同的分离方法，以获得 HMEC9。他们发现，慢速消化法与差速离心法是从新鲜乳腺组织中分离 HMEC 的最有效方法。然而，这种分离方法需要大块组织 （40-75 g） 并使用大量的消化酶。他们的程序很复杂（至少需要 3 次不同的离心以获得不同的细胞组分），并且很耗时。因此，仍然需要一种简单快速的方法，从少量乳腺组织中有效地获得 HMEC 群体，用于研究和临床应用9。
我们之前的研究表明，在初始培养基中加入 Rho 相关激酶 （ROCK） 抑制剂 Y-27632 可以简化分离人皮肤表皮细胞的过程10，这也已成功用于分离牙龈上皮细胞11。此外，Zubeldia-Plazaola 小组和 Jin 小组进行的早期研究表明，Y-27632 具有刺激来源于乳腺组织的原代上皮细胞快速和无限体外生长的能力 9,12。本研究旨在测试使用 Y-27632 是否会简化 HMEC 的分离和培养，我们成功建立了一种简单易行的方法，从小块 （1 g） 乳腺组织中分离 HMEC。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin</td>
			<td>Basalmedia</td>
			<td>K431010</td>
			<td>For HMECs&#160; dissociation</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge Tubes</td>
			<td>NEST</td>
			<td>081722CK01</td>
			<td>For cell digestion</td>
		</tr>
		<tr>
			<td>100 &#181;m mesh filter</td>
			<td>Solarbio</td>
			<td>431752</td>
			<td>For HMECs filtration</td>
		</tr>
		<tr>
			<td>100 mm Cell Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>solarbio</td>
			<td>P1110-100ml</td>
			<td>For immunofluorescence staining to check differentiation marker of HMECs</td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>Corning</td>
			<td>430829</td>
			<td>For cell centrifugation</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Solarbio</td>
			<td>431752</td>
			<td>Cell filtration</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5404HN133048</td>
			<td>Cell centrifuge</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>42820906</td>
			<td>For cell incubation</td>
		</tr>
		<tr>
			<td>Collagenase Type I</td>
			<td>Merck</td>
			<td>SKU:SCR103</td>
			<td>For HMECs isolation</td>
		</tr>
		<tr>
			<td>Dispase&#160;</td>
			<td>Solarbio</td>
			<td>CAS:42613-33-2</td>
			<td>For HMECs isolation</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>8122622</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>2556132P</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Phosphate buffered solution</td>
			<td>Tecono</td>
			<td>20201033</td>
			<td>Washing solution</td>
		</tr>
		<tr>
			<td>rabbit anti CK7</td>
			<td>abcam</td>
			<td>ab68459</td>
			<td>For immunofluorescence staining to check differentiation marker of HMECs</td>
		</tr>
		<tr>
			<td>rabbit anti GATA3</td>
			<td>abcam</td>
			<td>ab199428</td>
			<td>For immunofluorescence staining to check differentiation marker of HMECs</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Solarbio</td>
			<td>IY0040</td>
			<td>ROCK inhibitor</td>
		</tr>
	</tbody>
</table>

isolation and culture of primary human mammary epithelial cells

乳腺是乳房的基本结构，在生殖中起着至关重要的作用。人乳腺上皮细胞 （HMEC） 是乳腺癌和其他乳腺相关炎症性疾病的起源细胞，引起了相当大的关注。然而，由于其高度分化、角化性质和较短的寿命，出于研究目的在 体外 分离和培养原代 HMEC 一直具有挑战性。因此，开发一种简单有效的分离和培养 HMEC 的方法对于乳腺生物学和乳腺相关疾病的研究具有重要的科学价值。在这项研究中，我们成功地从少量乳腺组织中分离出原代 HMECs，方法是用酶混合物消化，并在含有 Rho 相关激酶 （ROCK） 抑制剂 Y-27632 的 5% 胎牛血清-DMEM 中进行初始培养，然后在无血清角质形成细胞培养基中扩增。这种方法选择性地促进上皮细胞的生长，从而获得优化的细胞产量。这种方法的简单性和便利性使其适用于实验室和临床研究，这应该为这些重要的研究领域提供有价值的见解。

Breast cancer is the primary type of cancer diagnosed in women globally and is the primary cause of death from cancer1. The pathogenesis of breast cancer is complex, involving multiple factors such as genetics, environment, and lifestyle. HMECs, active milk-producing cells, are one of the most important components of mammary tissue and likely are the original cells involved in breast cancer carcinogenesis. Therefore, HMECs have received the most attention from researchers for the study of breast cancer2. Furthermore, primary cells have the ability to provide a biologically relevant characterization of complex cellular processes due to their retention of genetic stability, normal morphology, and a more complete set of basic cellular functions that cannot be achieved with immortalized cell lines3. Hence, the isolation and culture of primary HMECs is an essential step for the study of most breast-related diseases such as breast cancer and breast inflammatory diseases.
At present, a stable and reproducible system for the isolation, culture, and identification of mammary epithelial cells from rats, cows, pigs, and goats has been established4,5,6,7. However, the isolation and culture of primary HMECs are challenging due to the complex microenvironment and the low yield of cells. For decades, scientists have been searching for the most effective method to isolate and cultivate HMECs although a culture system for HMECs was established nearly 20 years ago. For example, Hammond et al. developed a serum-free culture medium in which HMECs grew efficiently8. Recently, Zubeldia-Plazaola et al. tested four different isolation methods using fast/slow enzyme digestion procedures combined with sequential filtration or differential centrifugation steps to obtain HMECs9. They found that the slow digestion method together with differential centrifugation is the most efficient method to isolate HMECs from fresh breast tissue. However, that isolation method requires large pieces of tissue (40-75 g) and uses larger amounts of digestion enzymes. Their procedure is complicated (at least three different centrifugations to obtain different cell fractions), as well as time-consuming. Therefore, a simple and quick method is still needed to efficiently obtain populations of HMECs from small amounts of mammary tissue for research and clinical applications9.
Our previous studies showed that adding the Rho-associated kinase (ROCK) inhibitor Y-27632 into the initial culture medium can simplify the process of isolating human skin epidermal cells10, which has also been successfully used for the isolation of gingival epithelial cells11. Additionally, earlier research conducted by Zubeldia-Plazaola&#39;s group and Jin&#39;s group has indicated that Y-27632 has the ability to stimulate quick and unlimited in vitro growth of primary epithelial cells derived from mammary tissue9,12. The present study aimed to test whether using Y-27632 would simplify the isolation and culture of HMECs and we successfully established a simple and easily performed method to isolate HMECs from small pieces (1 g) of mammary tissue.

作者聚焦：东西方医学结合治疗肉芽肿性乳腺炎

原代人乳腺上皮细胞的分离和培养

图 1 显示了该过程的示意图。该方案涉及使用酶的组合，即分散酶、胶原酶和胰蛋白酶。这种组合用于从其下方的成纤维细胞层分离上皮片，然后利用胰蛋白酶将乳腺上皮细胞解离成悬浮液。此外，在初始培养基中加入 Y-27632 可有效促进上皮细胞的生长。因此，该方法可产生大量 HMEC，平均只需 10 天即可满足传代要求。
为了测试 Y-27632 在分离后细胞生?...

Read this Scientific Article Protocol about Isolation and Culture of Primary Human Mammary Epithelial Cells at JoVE.com

本研究报告了一种更简单、省时且经济的方案，可从少量乳腺组织中有效地分离和生长原代人乳腺上皮细胞 （HMEC）。该方案适用于快速生产用于实验室和临床应用的初级 HMEC。

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...

我们的研究重点是阐明难治性肉芽肿性乳腺炎的治疗途径。利用传统中西医方法同时结合的综合方法，将揭示驱动疾病及其并发症的复杂致病机制，重点是努力缓解这些问题并改善患者的预后。我们的研究报告了一种更简单、省时且经济的方案，用于从少量乳腺组织中有效分离和生长原代人乳腺上皮细胞 （HMEC）。该方案适用于快速生产用于实验室和临床应用的初级 HMEC。在这项研究的基础上，我们的目标是开发肉芽肿性乳腺炎乳腺上皮细胞模型，以加深我们对病因和经过验证的治疗策略的理解。

isolation-and-culture-of-primary-human-mammary-epithelial-cells

Research

JoVE Journal

Biology

Isolation and Culture of Primary Human Mammary Epithelial Cells

671 次观看.  The First Clinical Medical College of Zhejiang Chinese Medical University. 我们的研究重点是阐明难治性肉芽肿性乳腺炎的治疗途径。利用传统中西医方法同时结合的综合方法，将揭示驱动疾病及其并发症的复杂致病机制，重点是努力缓解这些问题并改善患者的预后。我们的研究报告了一种更简单、省时且经济的方案，用于从少量乳腺组织中有效分离和生长原代人乳腺上皮细胞 （HMEC）。该方案适用于快速生产用于实验室和临床应用的初级 HMEC...

视频: 作者聚焦：东西方医学结合治疗肉芽肿性乳腺炎

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.