Isolation and culture of human primary osteoblasts: Comparing the effects of differences in method details on osteoblast characteristics.
Isolation and culture of human primary osteoblasts: Comparing the effects of differences in method details on osteoblast characteristics.
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DOI：
10.1016/j.gendis.2023.03.024
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发表时间：
2024-03
期刊：
影响因子：
6.8
通讯作者：
Guo, Yan
中科院分区：
文献类型：
--
作者：
Chen, Fei;Zhang, Yan;Peng, Pai;Huang, Xiao-Ting;Qiu, Zi-Han;Liu, Bao-Cheng;Yang, Tie-Lin;Yang, Bo;Guo, Yan
成骨细胞在维持人体骨稳态中是必不可少的。1成骨细胞的异常形成和分化能力受损是导致骨相关疾病的关键因素，提示对成骨细胞的深入研究可以为这些疾病提供新的治疗策略。2人原代成骨细胞是骨相关研究中珍贵的实验模型。3然而，人原代成骨细胞的分离培养仍存在一定的困难和局限性。在这里，我们改进了一种分离和培养人原代成骨细胞的方法，通过骨组织取样器取松质骨，用胰酶预消化，这使得该过程更容易，更便宜，更有效。此外，我们还比较了分离和培养方法的细微变化对人原代成骨细胞表型的影响，以及微植块培养成骨细胞（MEC-OBs）和植块培养成骨细胞（EC-OBs）在形态、增殖和分化方面的差异。我们的研究结果表明，尽管MEC-OBs和EC-OBs都具有突出的成骨表型，但它们仍然有自己的特点。本研究对人原代成骨细胞的分离培养方法进行了改进，在补充资料中详细描述了该方法的具体操作步骤和优点，并在补充资料中提供了该方法的操作视频。在培养过程中，我们在显微镜下连续观察不同培养时间的人原代成骨细胞的形态变化（图1A）。收集消化的细胞和能够通过具有70 μm孔的细胞过滤器的微外植体并进行培养。当培养4-6天时，几个细胞生长出来并附着在培养皿的底部。随后，细胞突起逐渐延伸，细胞变得更长更大，形成梭形、三角形或多边形的成骨细胞形态（图1A）。随着细胞继续增殖，大约30天后，细胞完全融合。在诱导前后对MEC-OBs和EC-OBs进行HE染色，进一步观察其形态学变化。在诱导前，MEC-OB和EC-OB主要为三角形和多边形，苏木精染色的紫色核为圆形或椭圆形（图1 B）。诱导后，随着生长密度的增加，MEC-OBs和EC-OBs变成长梭形（图1 B）。比较这两种类型的细胞，我们发现，MEC-OBs的细胞大小和形态更均匀，而EC-OBs的细胞大小和形态表现出多态性。这意味着MEC-OBs的分化阶段可能更同步，MEC-OBs更同质，而EC-OBs之间的分化阶段可能有更大的跨度。
Osteoblasts are essential in the maintenance of human bone homeostasis. 1 The abnormal formation and impaired differentiation ability of osteoblasts are pivotal factors leading to bone-related diseases, suggesting that an in-depth study of osteoblasts can provide novel treatment strategies for these diseases. 2 Human primary osteoblasts are precious experimental models in bone-related research. 3 However, the isolation and culture of human primary osteoblasts still have some difficulties and limitations. Here, we refined a method to isolate and culture human primary osteoblasts by taking cancellous bone through a bone tissue sampler and pre-digesting it with pancreatin, which makes the process easier, cheaper, and more efficient. Furthermore, we compared the effects of the subtle changes in isolation and culture methods on the phenotype of human primary osteoblasts and examined the differences in morphology, proliferation, and differentiation between micro-explants cultured osteoblasts (MEC-OBs) and explants cultured osteoblasts (EC-OBs). Our results showed that although both MEC-OBs and EC-OBs have prominent osteoblastic phenotypes, they still have their own characteristics. Collectively, this improved method could reduce the difficulty and provide benefits for the research and application of human primary osteoblasts, such as human bone-related organoid culture and medical treatment of bone-related diseases.In our study, we improved the isolation and culture method of human primary osteoblasts, the specific operation steps and advantages of the method were described in detail in the supplementary data, and we also provided the operation video of this method in the supplementary material. During the culture process, we continuously observed the morphological changes of human primary osteoblasts under the microscope at different culture times (Fig. 1 A). Digested cells and micro-explants that can pass through the cell strainer with 70 μm pores were collected and cultured. When cultured for 4–6 days, several cells grew out and attached to the bottom of the culture dish. Subsequently, the cell processes gradually extended, and the cells became longer and larger, forming the spindle, triangle, or polygonal osteoblast morphology (Fig. 1 A). As the cells continued to proliferate, about 30 days later, the cells fully converged. Then hematoxylin and eosin (HE) staining was performed on MEC-OBs and EC-OBs before and after induction to further observe their morphology. Before induction, MEC-OBs and EC-OBs were mainly triangular and polygonal, and the nuclei-stained purple by hematoxylin was round or oval (Fig. 1 B). After induction, MEC-OBs and EC-OBs became long fusiform as the increase in growth density (Fig. 1 B). Comparing these two types of cells, we found that MEC-OBs were more uniform in cell size and morphology, while EC-OBs showed polymorphism in cell size and morphology. This implies that the differentiation stage of MEC-OBs may be more synchronized and MEC-OBs are more homogeneous, while the differentiation stage between EC-OBs may have a larger span.
影响因子：
13.6
作者：
Park Y;Cheong E;Kwak JG;Carpenter R;Shim JH;Lee J
通讯作者：
Lee J