Name: formation of human periodontal ligament cell spheroids on chitosan films
Uploaded: 2019-06-19T15:00:00
Description: Watch this Scientific Journal Video about Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films at JoVE.com

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)。

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
<ol><li>使增殖培养基培养为PDL细胞:β-MEM培养基,辅以10%FCS和100U/mL笔/链球菌。</li>
	<li>准备一个装有冰的容器,以转移孤立的第三摩尔。</li>
	<li>使用高压灭菌器对手术器械进行消毒。</li>
	<li>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...

<ol>
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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。
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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。

<table border="0" cellpadding="0" cellspacing="0" style="width:718px;" width="718">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">&#945;-MEM</td>
			<td style="width:158px;">Gibco</td>
			<td style="width:166px;">11900-073</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">acetic acid&#160;</td>
			<td style="width:158px;">Sigma-Aldrich</td>
			<td style="width:166px;">64197</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell culture flask 25 cm2</td>
			<td style="width:158px;">Corning</td>
			<td style="width:166px;">430639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell culture flask 75 cm2</td>
			<td style="width:158px;">Corning</td>
			<td style="width:166px;">430641</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:223px;">Chitosan</td>
			<td style="width:158px;">Heppe Medical&#160;Chitosan GmbH</td>
			<td style="width:166px;">/</td>
			<td>molecular weight 500 kDa, degree of deacetylation 85%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">FCS</td>
			<td style="width:158px;">Gibco</td>
			<td style="width:166px;">26140-079</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td style="width:158px;">Molecular&#160;Probes</td>
			<td style="width:166px;">L3224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">NaOH</td>
			<td style="width:158px;">Sigma-Aldrich</td>
			<td style="width:166px;">1310732</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">PBS</td>
			<td>KeyGen Biotech&#160;</td>
			<td style="width:166px;">KGB5001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">pen/strep</td>
			<td style="width:158px;">Gibco</td>
			<td style="width:166px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Trypsin/EDTA&#160;</td>
			<td>KeyGen Biotech&#160;</td>
			<td style="width:166px;">KGM25200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">15 mL conical centrifuge tube</td>
			<td style="width:158px;">Corning</td>
			<td style="width:166px;">430790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">24-well plate</td>
			<td style="width:158px;">Corning</td>
			<td style="width:166px;">3524</td>
			<td></td>
		</tr>
	</tbody>
</table>

formation of human periodontal ligament cell spheroids on chitosan films

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。

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...

Watch this Scientific Journal Video about Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films at JoVE.com

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

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。

奇托桑膜上人类牙周韧带细胞球体的形成

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) ...

这是一个详细的协议，通过切托桑薄膜培养周周韧带细胞球体。与传统培养系统相比，球状培养系统产生具有增强自我更新和骨源分化能力的周周韧带细胞。首先，按照手稿中指示，准备 1% 的重量到体积的气当液溶液。将 0.5 毫升甲氨基齐桑溶液加入 24 孔组织培养聚苯乙烯板的每个孔中。在60摄氏度的烤箱中干燥盘子24小时，形成气膜。要中和甲酸山薄膜，在24井板的每个井中加入0.5毫升0.5普通氢氧化钠，并在室温下孵育两小时。然后，用双蒸馏水洗三次气精膜。在室温下，在70%酒精中对齐藤桑涂层的24孔板进行消毒。用 PBS 冲洗盘子三次，然后在紫外线下过夜。在细胞播种之前，预热增殖介质、PBS溶液和尝试性EDTA溶液达到37摄氏度。从细胞培养瓶中丢弃上流液，用10毫升PBS洗涤汇合期周韧带或PDL细胞。丢弃 PBS，在烧瓶中加入一毫升的尝试素 EDTA 溶液。在37摄氏度下孵育烧瓶3分钟，然后加入三毫升增殖介质，以终止三辛消化。将细胞悬浮液转移到15毫升锥形管中，然后在300倍g下将其离心5分钟。丢弃上一级，在增殖介质的500微升中重新悬浮细胞。用血细胞计计算细胞数，并在每平方厘米5、10、30和6万个细胞的盘上播种。在5%的二氧化碳和加湿空气中培养孵化器中的PDL细胞，每周两次改变培养基。通过逆相对比显微镜每天观察细胞形态五天。使用该协议成功形成可行的PDL细胞球体。在1天和3天，很少观察到种子密度较低的球体。但在两个更高的密度，各种大小的球体存在从第一天。最佳播种密度为每厘米平方3万个细胞，因为它产生均匀的球状体。经过五天的培养，观察到所有播种密度的球体更大。可行性测定表明，大多数PDL细胞在1天、第3天和6天存活，但死亡细胞的数量在6天增加。该协议的一个限制是球体形成后细胞的增殖减少。因此，需要进一步研究，以促进球状形成后细胞的增殖。

Research

JoVE Journal

Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

一种新的文化模式的人多能干明胶胎盘条件培养基细胞增殖

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

HUVEC 管形成测定评估天然产物对血管生成的影响

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

通过转录因子的强制表达，人类纤维细胞的造血再编程

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...