奇托桑膜上人类牙周韧带细胞球体的形成
1Department of Periodontology, School & Hospital of Stomatology,Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, 2Department of Periodontology,Dental Diseases Prevention & Treatment Center of Jiading District, 3College of Materials Science and Engineering,Tongji University
Summary
在这里,我们提出通过甲骨膜培养人类牙周韧带(PDL)细胞球体的方案。三维 (3D) 细胞球体培养提供了传统组织培养聚苯乙烯 (TCPS) 培养系统的替代方案。
Abstract
牙周韧带(PDL)细胞对牙周组织再生大有希望。按照惯例,PDL细胞在二维(2D)基材上培养,如组织培养聚苯乙烯(TCPS)。然而,在体外培养过程中观察到PDL细胞的特征变化。这种现象可能是因为2DTCPS不同于体内三维(3D)微环境。与在2D基质上培养的细胞相比,在3D微环境中生长的细胞与体内细胞的相似性更大。因此,3D细胞培养模型为传统的2D单层细胞培养提供了一个有前途的替代方法。为了改进传统的PDL细胞培养模型,我们最近开发了一种3D细胞培养方法,该方法基于基托桑薄膜上PDL细胞的球形形成。在这里,我们提出了基于甲体膜的详细细胞球体培养方案。PDL细胞球体的3D培养系统克服了与传统的2D单层细胞培养相关的一些限制,因此可能适合生产PDL细胞,具有增强的疗效,为未来牙周组织再生。
Introduction
牙周炎,主要由牙菌斑1初始化,其特征是牙周韧带(PDL)、腹腔骨和水泥等牙周组织损伤。目前对牙周炎的治疗通常成功地阻止了活动性疾病的进展,但丢失的牙周组织的再生仍然是一个临床挑战。最近,在细胞为基础的牙周组织再生方法方面取得了重要进展,克服了目前治疗2、3、4的缺点。
我们以前的系统审查显示,PDL细胞显示出巨大的潜力,牙周再生5。按照惯例,PDL细胞在二维(2D)基材上培养,如组织培养聚苯乙烯(TCPS)。然而,在体外培养6中观察到PDL细胞的特征变化。这种现象可能是因为2DTCPS不同于体内三维(3D)微环境7。与在2D基质上培养的细胞相比,在3D微环境中生长的细胞与体内细胞8的相似性更大。因此,3D细胞培养模型为传统的2D单层细胞培养提供了一个有前途的替代方法。
传统的 3D 培养方法是将细胞封装在 3D 生物材料中。与封装在3D生物材料中的细胞相比,细胞球体更模仿体内的情况,因为球体是无异物生长的细胞的聚合体9、10、1112.据报道,细胞球体通过保存细胞外基质(ECM)成分,包括纤维素和拉米宁13,促进MSC生物活性。为了改进传统的PDL细胞培养模型,我们最近开发了一种3DPDL细胞培养方法,该方法基于基托桑薄膜14上的PDL细胞球体形成。球形形成增加了PDL细胞14的自我更新和成骨分化能力。在这里,我们提出了详细的PDL细胞球体培养方案基于甲酮薄膜。PDL细胞球体的3D培养系统克服了传统TCPS细胞培养的一些缺陷,因此适合生产PDL细胞,具有增强的疗效,为未来牙周组织再生。
Protocol
该研究方案经同济大学口腔医学院和医院伦理委员会批准。所有患者均提供书面知情同意。 1. PDL细胞隔离 使增殖培养基培养为PDL细胞:β-MEM培养基,辅以10S和100U/mL笔/链球菌。 准备一个装有冰的容器,以转移孤立的第三摩尔。 使用高压灭菌器对手术器械进行消毒。 在同济大学口腔医学院口腔医院牙科诊所提取正常人从第三种摩尔(18-28岁)中提取第…
Representative Results
利用本方案,成功形成可行的PDL细胞球体。图1显示,悬浮细胞或球体代替附着细胞主要在甲酮薄膜上观察到。对于0.5 x 104细胞/cm2的种子密度,在第1天和第3天偶尔发现附着的PDL细胞,很少观察到PDL细胞球体。相反,对于3 x 104和6 x 104细胞/cm2的种子密度,从第1天起就发现了不同大小的PDL细胞球体。PDL细胞球形形成从3天…
Discussion
本研究引入了一种三维细胞培养系统,以克服与传统的2D单层细胞培养相关的一些限制。根据该协议,PDL细胞球体通过在甲酮薄膜上培养细胞而成功形成。我们先前的研究报告说,球形的形成增加了PDL细胞14的自我更新和成骨分化能力。PDL细胞球体不用酶从TCPS中采集细胞,只需将介质移液几倍14,就可以从甲酮薄膜中收获。因此,ECM 和细胞间结可以很好地保存。
<p cla…Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究由中国国家自然科学基金委员会(国家自然科学基金委员会81700978)、中央高校基础研究基金(1504219050)、上海自然科学基金(17ZR1432800)和上海医学探索项目()共同发起。17411972600)。
Materials
| α-MEM | Gibco | 11900-073 | |
| acetic acid | Sigma-Aldrich | 64197 | |
| Cell culture flask 25 cm2 | Corning | 430639 | |
| Cell culture flask 75 cm2 | Corning | 430641 | |
| Chitosan | Heppe Medical Chitosan GmbH | / | molecular weight 500 kDa, degree of deacetylation 85% |
| FCS | Gibco | 26140-079 | |
| Live/Dead Viability/Cytotoxicity Kit | Molecular Probes | L3224 | |
| NaOH | Sigma-Aldrich | 1310732 | |
| PBS | KeyGen Biotech | KGB5001 | |
| pen/strep | Gibco | 15140-122 | |
| Trypsin/EDTA | KeyGen Biotech | KGM25200 | |
| 15 mL conical centrifuge tube | Corning | 430790 | |
| 24-well plate | Corning | 3524 |
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Cite This Article
Yan, X., Ran, X., Xia, S., Yang, Y., Zhou, M., Yuan, C., Luo, L. Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films. J. Vis. Exp. (148), e59855, doi:10.3791/59855 (2019).