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
Summary
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。
Abstract
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)。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 100 mm culture dishes | Thermo Scientific | 172931 | |
| 15 mL conical tubes | Thermo Scientific | 339651 | |
| 50 mL conical tubes | Thermo Scientific | 339653 | |
| Bronchial Epithelial Cell Growth Media | Lonza | CC-3171 | Add bullet kit as per manufacturer's instructions. Supplement with 20 mL of charcoal stripped serum. |
| Cell strainer 70 µm nylon mesh | Fisher | 22-363-548 | |
| Charcoal stripped fetal bovine serum | Gibco | 12676-029 | |
| Collagenase type III | Worthington | LS004182 | Store at 4 °C. |
| Cryogenic Tube | Fisher | 5000-0020 | |
| Dispase | Cell Applications | 07923 | Dissolve collagenase to make a 0.15% (w/v) stock. Filter sterilize then store at -20 °C. |
| Dissecting scissors | Fisher | 08-940 | |
| Dulbecco phosphate buffered saline | Corning | 55-031-PC | |
| General Chemicals | Sigma | ||
| PathClear Basement membrane extract | Cultrex | 3432-005-01 | Thaw at 4 °C at least 24 hr prior to use. Always handle on ice. |
| Six-well culture dishes | Falcon | 353046 | |
| Surgical forceps | Fisher | 22-079-742 | |
| Trypsin-EDTA solution | Corning | 25-052-CI |
Referenzen
- Kessler, A. T., Bhatt, A. A. Review of the Major and Minor Salivary Glands, Part 1: Anatomy, Infectious, and Inflammatory Processes. Journal of Clinical Imaging Science. 8, 47 (2018).
- Holmberg, K. V., Hoffman, M. P., Ligtenberg, A. J. M., Veerman, E. C. I. Anatomy, Biogenesis and Regeneration of Salivary Glands. Monographs in Oral Science. 24, 1-13 (2014).
- Vissink, A., et al. Prevention and Treatment of the Consequences of Head and Neck Radiotherapy. Critical Reviews in Oral Biology & Medicine. 14, 213-225 (2003).
- Gualtierotti, R., Marzano, A., Spadari, F., Cugno, M. Main Oral Manifestations in Immune-Mediated and Inflammatory Rheumatic Diseases. Journal of Clinical Medicine. 8, 21 (2018).
- Tsukamoto, M., Suzuki, K., Takeuchi, T. Ten-year observation of patients with primary Sjögren’s syndrome: Initial presenting characteristics and the associated outcomes. International Journal of Rheumatology. Dis. , (2018).
- Dirix, P., Nuyts, S., Van den Bogaert, W. Radiation-induced xerostomia in patients with head and neck cancer: A literature review. Cancer. 107, 2525-2534 (2006).
- Morrison, K. M., et al. Development of a primary human cell model for the study of human cytomegalovirus replication and spread within salivary epithelium. Journal of Virology. , (2018).
- Pomeroy, C., Englund, J. A. Cytomegalovirus: epidemiology and infection control. American Journal of Infection Control. 15, 107-119 (1987).
- Maimets, M., et al. Long-Term In Vitro Expansion of Salivary Gland Stem Cells Driven by Wnt Signals. Stem Cell Reports. 6, 150-162 (2016).
- Varghese, J. J., et al. Salivary gland cell aggregates are derived from self-organization of acinar lineage cells. Archives of Oral Biology. 97, 122-130 (2019).
- Maruyama, C., Monroe, M., Hunt, J., Buchmann, L., Baker, O. Comparing human and mouse salivary glands: A practice guide for salivary researchers. Oral Diseases. , (2018).
- Lin, L. C., et al. Cross-contamination of the human salivary gland HSG cell line with HeLa cells: A STR analysis study. Oral Diseases. 24, 1477-1483 (2018).
- Tran, S. D., et al. Primary Culture of Polarized Human Salivary Epithelial Cells for Use in Developing an Artificial Salivary Gland. Tissue Engineering. 11, 172-181 (2005).
- Pringle, S., et al. Isolation of Mouse Salivary Gland Stem Cells. Journal of Visualized Experiments. , (2011).
- Nam, H., et al. Characterization of Primary Epithelial Cells Derived from Human Salivary Gland Contributing to in vivo Formation of Acini-like Structures. Molecules and Cells. 41, 515-522 (2018).
- Vissink, A., Jansma, J., Spijkervet, F. K. L., Burlage, F. R., Coppes, R. P. Oral Sequelae of Head and Neck Radiotherapy. Critical Reviews in Oral Biology & Medicine. 14, 199-212 (2003).
- Cannon, M. J., et al. Repeated measures study of weekly and daily cytomegalovirus shedding patterns in saliva and urine of healthy cytomegalovirus-seropositive children. BMC Infect. Dis. 14, (2014).
- Pringle, S., et al. Human Salivary Gland Stem Cells Functionally Restore Radiation Damaged Salivary Glands: Hyposalivation Cell Therapy. STEM CELLS. 34, 640-652 (2016).
- Maria, O. M., Maria, O., Liu, Y., Komarova, S. V., Tran, S. D. Matrigel improves functional properties of human submandibular salivary gland cell line. International Journal of Biochemistry & Cell Biology. 43, 622-631 (2011).
- Srinivasan, P. P., et al. Primary Salivary Human Stem/Progenitor Cells Undergo Microenvironment-Driven Acinar-Like Differentiation in Hyaluronate Hydrogel Culture: Differentiation of Salivary Stem/Progenitor Cells. STEM CELLS Translational Medicine. 6, 110-120 (2017).
- Foraida, Z. I., et al. Elastin-PLGA hybrid electrospun nanofiber scaffolds for salivary epithelial cell self-organization and polarization. Acta Biomaterialia. 62, 116-127 (2017).
