PURPOSE Periodontal ligament (PDL) cell cultures are classically maintained in serum-containing media. However, unwanted side-effects of these conditions on cellular and molecular characteristics demand a serum-free alternative. Even though these limitations are well known and efforts for the development of adequate serum-free alternatives have been made, these approaches for replacement remained unsuccessful so far. This study aimed at developing a well-defined, serum-free formulation supporting both isolation from tissue samples and efficient expansion of PDL cells. Here, of particular focus was the perpetuation of tissue-characteristic markers detectable in primary tissues and of stemness features. BASIC PROCEDURES Primary PDL cell cultures from generally healthy human donors (n = 3) were maintained in basal media N2B27 and E6 together with different concentrations of growth and attachment factors. Cell proliferation was recorded via microscopy and WST assay. Gene expression of RUNX2, Periostin, ALP, CD73, CD90, CD105, CD45, SOX10 and SOX2 was compared to primary PDL explants via qRT-PCR. Immunocytochemistry was performed for anti-CD105, SSEA-3, CD271, HNK1. Serum-containing sDMEM medium served as control. MAIN FINDINGS N2B27 medium substituted with 25 ng/mL EGF, 25 ng/mL IGF1, 0.5 mg/mL Fetuin plus gelatine coating (designated N2B27-PDLsf) emerged as potent serum-free formulation ensuring adequate culture isolation and expansion. Here, PDL primary tissue signature markers RUNX2 and Periostin remained stable in N2B27-PDLsf compared to controls (229.0-fold ±101.0 and 83.2-fold ±9.6 increase). Additionally, stemness markers ALP and CD105 were significantly upregulated on transcriptional, and CD105 and SOX2 on protein level. PRINCIPAL CONCLUSIONS This investigation identified a novel serum-free medium for the isolation, and expansion of primary human PDL cells with constantly high proliferation rates. Here, purity and stemness properties are maintained. Thus, N2B27-PDLsf represents a valid replacement for serum-containing media in PDL cultures.

中文翻译：

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一种新型无血清培养基，用于分离，扩增和维持人牙周膜韧带细胞的干性和组织特异性标志物。

目的牙周膜（PDL）细胞培养物通常在含血清的培养基中维持。然而，这些条件对细胞和分子特性的不良副作用需要无血清替代品。尽管这些局限性是众所周知的，并且已经为开发足够的无血清替代品做出了努力，但是到目前为止，这些替代方法仍然没有成功。这项研究旨在开发一种定义明确，无血清的配方，该配方支持从组织样品中分离和有效扩增PDL细胞。在此，特别关注的是永久性可在原发组织中检测到的组织特征标记和茎特征。基本程序将来自一般健康人类供体（n = 3）的原代PDL细胞培养物与不同浓度的生长和附着因子一起保存在基础培养基N2B27和E6中。通过显微镜和WST测定记录细胞增殖。通过qRT-PCR将RUNX2，Periostin，ALP，CD73，CD90，CD105，CD45，SOX10和SOX2的基因表达与主要PDL外植体进行了比较。对抗CD105，SSEA-3，CD271，HNK1进行了免疫细胞化学分析。含血清的sDMEM培养基用作对照。主要发现出现了用25 ng / mL EGF，25 ng / mL IGF1、0.5 mg / mL Fetuin加明胶涂层（命名为N2B27-PDLsf）替代的N2B27培养基，该制剂为有效的无血清制剂，可确保适当的培养物分离和扩增。这里，与对照组相比，PDL主要组织标志物标记RUNX2和骨膜素在N2B27-PDLsf中保持稳定（增加229.0倍±101.0和83.2倍±9.6）。此外，茎标记ALP和CD105在转录上显着上调，而CD105和SOX2在蛋白质水平上显着上调。主要结论这项研究确定了一种新型的无血清培养基，用于分离和扩增具有持续高增殖率的原代人PDL细胞。在此，保持纯度和茎干特性。因此，N2B27-PDLsf代表了PDL培养物中含血清培养基的有效替代品。主要结论这项研究确定了一种新型的无血清培养基，用于分离和扩增具有持续高增殖率的原代人PDL细胞。在此，保持纯度和茎性。因此，N2B27-PDLsf代表了PDL培养物中含血清培养基的有效替代品。主要结论这项研究确定了一种新型的无血清培养基，用于分离和扩增具有持续高增殖率的原代人PDL细胞。在此，保持纯度和茎干特性。因此，N2B27-PDLsf代表了PDL培养物中含血清培养基的有效替代品。