CN113502259A - 一种毛囊干细胞的分离培养方法 - Google Patents
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Abstract
本发明属于干细胞领域，具体涉及一种毛囊干细胞的分离培养方法。该毛囊干细胞的分离方法包括：S1、进行毛囊的显微剥离得到毛囊；S2、在完全培养基中培养毛囊，经过纯化后得到毛囊干细胞；所述完全培养基包括基础培养基、表皮生长因子、胰岛素、氢化可的松、L‑谷酰胺、维生素A、维生素D2、转铁蛋白、亚油酸、牛血清白蛋白和胎牛血清。本发明中制备毛囊干细胞所需要的皮肤材料少，只用几根毛囊甚至一根即可；开始时细胞在其原来的微环境中生长，对细胞损伤小，克隆形成效率高；且能够稳定传代，可以获得足量毛囊干细胞。
Description
技术领域
本发明属于干细胞领域，具体涉及一种毛囊干细胞的分离培养方法。
背景技术
毛囊作为皮肤的附属物，其外根部的隆突部中存在有毛囊干细胞。毛囊干细胞是成体干细胞的一种，具有较强的多向分化潜能。小鼠体内实验证明，它能形成完整的表皮、皮脂腺和毛囊。相对胚胎干细胞和目前新兴起的诱导多能性干细胞来说，由于哺乳动物几乎所有体表都覆盖有毛囊，毛囊干细胞更容易从自身获得，不受伦理因素的制约，避免了异体干细胞移植带来的免疫排斥，因此具有很大的临床应用前景与价值。
如何获得足量毛囊干细胞是毛囊干细胞移植过程中重要的问题之一。传统的毛囊干细胞分离方法主要是基于流式细胞仪的分选方法，但是这种基于流式细胞仪分选的方法有很大的缺点，即需要大量皮肤用于制备单细胞悬液，这对于大面积皮肤损伤(烧伤或烫伤等)的患者来说是不可能的。此外，分选过程中，单个细胞在体外时间长，处于悬浮状态，细胞容易发生凋亡，细胞经过流式细胞仪时容易受到机械损伤，这些都导致分选到的毛囊干细胞不易成活，克隆形成效率极低。因此，如何利用尽可能少的皮肤组织高效获得足量的毛囊干细胞用于干细胞移植，是当前亟待解决的难题。
发明内容
本发明的目的在于提供一种新的有效的分离毛囊干细胞的方法。
本发明通过显微剥离技术在体视镜下剥离出毛囊，利用器官培养的方法，将毛囊用完全培养基进行培养，毛囊贴壁后，毛囊干细胞从隆突部长出并形成克隆，此细胞的克隆形成效率高，易纯化，纯度几乎达到100％，能长期传代并保持特性不变。由于开始培养时毛囊干细胞在其原来的微环境中生长，没有受到损伤，克服了传统的流式细胞仪分选方法对毛囊干细胞造成严重机械损伤而导致克隆形成效率极低的缺点，并且培养基中添加了有利于毛囊干细胞稳定增殖的因子，只需要很小的皮肤组织，就可以获得可以长期使用的足量的毛囊干细胞。
具体的，本发明的技术方案如下：
本发明第一个方面公开了一种毛囊干细胞的分离方法，包括：
S1、进行毛囊的显微剥离得到毛囊；
S2、在完全培养基中培养毛囊，经过纯化后得到毛囊干细胞；所述完全培养基包括基础培养基、表皮生长因子、胰岛素、氢化可的松、L-谷酰胺、维生素A、维生素D2、转铁蛋白、亚油酸、牛血清白蛋白和胎牛血清。
优选的，所述S1包括：取毛囊处皮肤，消毒后在体视镜下用镊子夹住毛囊靠近表皮的峡部，向真皮方向用力撕出毛囊；用针头固定住毛囊，用解剖刀小心划破真皮鞘，并将毛囊剥离出来，切断真皮乳头和真皮鞘间的连接。
在本发明的一些具体实施例中，取一小块人毛囊处皮肤，用75％酒精消毒3-5分钟；在体视镜下，用镊子夹住毛囊靠近表皮的峡部，向真皮方向用力撕出毛囊；用针头固定住毛囊，用解剖刀小心划破真皮鞘，并将毛囊剥离出来，切断真皮乳头和真皮鞘间的连接。
优选的，所述完全培养基包括基础培养基、5-20ng/mL的表皮生长因子、2-10μg/mL的胰岛素、0.5-4μg/mL的氢化可的松、2-9mmol/L的L-谷酰胺、0.005-0.3μg/mL的维生素A、0.5-3μg/mL的维生素D2、0.5-3μg/mL转铁蛋白、50-200ng/mL的亚油酸、10-40μg/mL的牛血清白蛋白和10-25％的胎牛血清。
更优选的，所述完全培养基还包括青霉素钠和硫酸链霉素。
在本发明的一些具体实施例中，所述完全培养基的配制方法为向T4培养基(Gibco)中添加以下因子，各种因子的最终浓度为：10纳克/毫升的表皮生长因子，5微克/毫升的胰岛素，1微克/毫升的氢化可的松，5.8毫摩尔/升的L-谷酰胺，0.115微克/毫升的维生素A，1微克/毫升的维生素D2，1微克/毫升的转铁蛋白，100纳克/毫升的亚油酸，20微克/毫升的牛血清白蛋白，200单位/毫升的青霉素200单位/毫升的硫酸链霉素。添加因子后培养基中加体积百分含量为15％的胎牛血清，即为完全培养基。
优选的，将S1中得到的毛囊转入培养容器中，加入完全培养基培养3-7天。
优选的，所述纯化步骤包括：
在室温的条件下，用消化液消化至成纤维细胞脱落，将成纤维细胞消化下来收集备用，再向培养板中加消化液，转到37℃培养箱继续消化3-5分钟，用缓冲液收集消化下来的毛囊干细胞。
优选的，在S2之后还包括步骤S3：将得到的毛囊干细胞进行传代。
更优选的，在S3中，将毛囊干细胞和成纤维细胞共培养，加入完全培养基后进行培养，每2-3天更换培养液，待毛囊干细胞汇合度达到80-90％进行传代。
在本发明的一些优选实施例中，将单根毛囊生长出的毛囊干细胞和成纤维细胞按照1:1的比例接种到培养皿中，加入完全培养基后放入的培养箱中进行培养，2-3天更换培养液，大约2周后细胞长满，可继续传代或在液氮中冻存备用。
优选的，所述毛囊干细胞为人毛囊干细胞。
本发明第二个方面公开了上述方法得到的毛囊干细胞。
本发明第三个方面公开了一种用于皮肤修复的产品，所述产品包括上述的毛囊干细胞。
本发明第四个方面公开了上述的毛囊干细胞在医学领域中的应用；优选的，所述毛囊干细胞在皮肤修复领域中的应用。
在符合本领域常识的基础上，上述各优选条件，可任意组合，而不超出本发明的构思与保护范围。
本发明相对于现有技术具有如下的显著优点及效果：本发明中制备毛囊干细胞所需要的皮肤材料少，只用几根毛囊甚至一根即可；开始时细胞在其原来的微环境中生长，对细胞损伤小，克隆形成效率高；且能够稳定传代，可以获得足量毛囊干细胞。
附图说明
图1为人头皮的显微镜下放大照片(10×)；
图2为人毛囊分离后培养，得到的毛囊干细胞培养显微镜下放大照片(A图为100×，B图为200×)。
具体实施方式
下面结合附图和实施例对本发明的技术方案进行详细描述，但并不因此将本发明限制在所述的实施例范围之中。
下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。本发明所用试剂和原料均市售可得。
实施例1
本实施例公开了一种毛囊干细胞的分离培养方法，包括：
一、毛囊的显微剥离
取一小块人毛囊处皮肤(如图1所示)，用75％酒精消毒3-5分钟；在体视镜下，用镊子夹住毛囊靠近表皮的峡部，向真皮方向用力撕出毛囊；用针头固定住毛囊，用解剖刀小心划破真皮鞘，并将毛囊剥离出来，切断真皮乳头和真皮鞘间的连接。
二、毛囊干细胞的分离
1.向T4培养基(Gibco)中添加以下因子，各种因子的最终浓度为：10纳克/毫升的表皮生长因子，5微克/毫升的胰岛素，1微克/毫升的氢化可的松，5.8毫摩尔/升的L-谷酰胺，0.115微克/毫升的维生素A，1微克/毫升的维生素D2，I微克/毫升的转铁蛋白，100纳克/毫升的亚油酸，20微克/毫升的牛血清白蛋白，200单位/毫升的青霉素，200单位/毫升的硫酸链霉素。添加因子后培养基中加体积百分含量为15％的胎牛血清，即为完全培养基。
2.将毛囊转入6孔培养板，加入2mL完全培养基进行培养。毛囊一般在第3-7天贴壁，然后毛囊干细胞会从隆突部爬出，如图2所示，细胞边缘明亮，紧密排列，为典型角质细胞特征。
三、毛囊干细胞的纯化
由于毛囊干细胞具有贴壁紧的特点而成纤维细吧贴壁能力较弱，因此在室温25℃左右，用消化液(0.1％的胰酶和0.008％的EDTA的磷酸缓冲液)消化时，成纤维细胞会被消化下来，而毛囊干细胞克隆仍紧贴于培养板上。消化后，将成纤维细胞离心富集，用磷酸缓冲液收集，收集后可以传代或冻存，用于后续使用。再向培养板中加消化液，转到37℃培养箱继续消化3-5分钟，用磷酸缓冲液收集消化下来的毛囊干细胞。
三、毛囊干细胞的传代与培养
由于成纤维细胞可为干细胞提供与体内相似的模拟环境，有利于干细胞的生长，因此我们将单根毛囊生长出的毛囊干细胞和成纤维细胞按照1:1的比例接种到10cm培养皿中共培养，加入完全培养基后放入的培养箱中进行培养，37℃、5％CO2、100％湿度的培养箱中培养，2-3天更换培养液，大约2周后细胞长满，细胞消化后可继续传代或在液氮中冻存备用,纯化后的毛囊干细胞可以长期稳定传代并保持细胞和克隆形态不变。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (10)
1.一种毛囊干细胞的分离方法，其特征在于，包括：
S1、进行毛囊的显微剥离得到毛囊；
S2、在完全培养基中培养毛囊，经过纯化后得到毛囊干细胞；所述完全培养基包括基础培养基、表皮生长因子、胰岛素、氢化可的松、L-谷酰胺、维生素A、维生素D2、转铁蛋白、亚油酸、牛血清白蛋白和胎牛血清。
2.根据权利要求1所述的方法，其特征在于，所述S1包括：取毛囊处皮肤，消毒后在体视镜下用镊子夹住毛囊靠近表皮的峡部，向真皮方向用力撕出毛囊；用针头固定住毛囊，用解剖刀小心划破真皮鞘，并将毛囊剥离出来，切断真皮乳头和真皮鞘间的连接。
3.根据权利要求1所述的方法，其特征在于，所述完全培养基包括基础培养基、5-20ng/mL的表皮生长因子、2-10μg/mL的胰岛素、0.5-4μg/mL的氢化可的松、2-9mmol/L的L-谷酰胺、0.005-0.3μg/mL的维生素A、0.5-3μg/mL的维生素D2、0.5-3μg/mL转铁蛋白、50-200ng/mL的亚油酸、10-40μg/mL的牛血清白蛋白和10-25％的胎牛血清。
4.根据权利要求1所述的方法，其特征在于，将S1中得到的毛囊转入培养容器中，加入完全培养基培养3-7天。
5.根据权利要求1所述的方法，其特征在于，所述纯化步骤包括：
在室温的条件下，用消化液消化至成纤维细胞脱落，将成纤维细胞消化下来收集备用，再向培养板中加消化液，转到37℃培养箱继续消化3-5分钟，用缓冲液收集消化下来的毛囊干细胞。
6.根据权利要求1所述的方法，其特征在于，在S2之后还包括步骤S3：将得到的毛囊干细胞进行传代；
优选的，在S3中，将毛囊干细胞和成纤维细胞共培养，加入完全培养基后进行培养，每2-3天更换培养液，待毛囊干细胞汇合度达到80-90％进行传代。
7.根据权利要求1所述的方法，其特征在于，所述毛囊干细胞为人毛囊干细胞。
8.根据权利要求1-6任一项所述方法得到的毛囊干细胞。
9.一种用于皮肤修复的产品，其特征在于，所述产品包括权利要求8所述的毛囊干细胞。
10.权利要求8所述的毛囊干细胞在医学领域中的应用；优选的，所述毛囊干细胞在皮肤修复领域中的应用。
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114350596A (zh) * | 2022-02-17 | 2022-04-15 | 浙江卫未生物医药科技有限公司 | 一种毛囊外根鞘细胞培养基 |
| CN115011545A (zh) * | 2022-06-06 | 2022-09-06 | 北京熙朵医疗美容门诊部有限公司 | 一种毛囊干细胞的培养方法 |
| CN115197898A (zh) * | 2022-07-06 | 2022-10-18 | 北京晶莱华科生物技术有限公司 | 一种毛囊干细胞的制备方法 |
| CN116254222A (zh) * | 2023-01-16 | 2023-06-13 | 吴皖 | 一种机械分离毛囊干细胞的方法 |
| CN117646052A (zh) * | 2023-12-04 | 2024-03-05 | 安徽科门生物科技有限公司 | 一种用于白发转黑发的干细胞活性因子制剂提取制备方法 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101045916A (zh) * | 2007-03-13 | 2007-10-03 | 天津市天津医院 | 人发来源的毛囊干细胞及其扩增制备方法 |
| KR20080071656A (ko) * | 2007-01-31 | 2008-08-05 | 주식회사 에버브릿지 | 배양된 모낭구성세포를 이용한 생인공피부 제조방법 |
| US20110086007A1 (en) * | 2007-09-21 | 2011-04-14 | Aderans Research Institute, Inc. | Epidermal stimulation to enhance hair follicle formation |
| CN102344906A (zh) * | 2010-07-21 | 2012-02-08 | 中国科学院动物研究所 | 一种毛囊干细胞的分离培养方法 |
-
2021
- 2021-07-12 CN CN202110784466.5A patent/CN113502259A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20080071656A (ko) * | 2007-01-31 | 2008-08-05 | 주식회사 에버브릿지 | 배양된 모낭구성세포를 이용한 생인공피부 제조방법 |
| CN101045916A (zh) * | 2007-03-13 | 2007-10-03 | 天津市天津医院 | 人发来源的毛囊干细胞及其扩增制备方法 |
| US20110086007A1 (en) * | 2007-09-21 | 2011-04-14 | Aderans Research Institute, Inc. | Epidermal stimulation to enhance hair follicle formation |
| CN102344906A (zh) * | 2010-07-21 | 2012-02-08 | 中国科学院动物研究所 | 一种毛囊干细胞的分离培养方法 |
Non-Patent Citations (3)
| Title |
|---|
| 刘海波: "《生物医药及高性能医疗器械 中国制造2025大众读本》", 30 April 2018, pages: 239 * |
| 张菊芳: "《毛发整形美容学》", 28 February 2013, pages: 327 * |
| 张菊芳: "《高密式毛发移植》", 31 May 2011, pages: 21 - 23 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114350596A (zh) * | 2022-02-17 | 2022-04-15 | 浙江卫未生物医药科技有限公司 | 一种毛囊外根鞘细胞培养基 |
| CN114350596B (zh) * | 2022-02-17 | 2023-11-10 | 浙江卫未生物医药科技有限公司 | 一种毛囊外根鞘细胞培养基 |
| CN115011545A (zh) * | 2022-06-06 | 2022-09-06 | 北京熙朵医疗美容门诊部有限公司 | 一种毛囊干细胞的培养方法 |
| CN115197898A (zh) * | 2022-07-06 | 2022-10-18 | 北京晶莱华科生物技术有限公司 | 一种毛囊干细胞的制备方法 |
| CN116254222A (zh) * | 2023-01-16 | 2023-06-13 | 吴皖 | 一种机械分离毛囊干细胞的方法 |
| CN117646052A (zh) * | 2023-12-04 | 2024-03-05 | 安徽科门生物科技有限公司 | 一种用于白发转黑发的干细胞活性因子制剂提取制备方法 |
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