Miniaturization of an Anoikis assay using non-adhesive micromolded hydrogels

Anoikis is a specific form of apoptosis resulting from the loss of cellular attachment to extracellular matrix or other cells. Challenges in simulating these conditions in vitro make it difficult to generate a controlled, efficient assay to study anoikis. We developed a microscale method for analysis and quantification of anoikis using micromolded, non-adhesive hydrogels. These hydrogels allow for isolation and observation of single, unattached cells in an ordered array, and controlled distribution. Cell distributions resulting from multiple seeding densities were compared to a mathematical probability model. Normal human fibroblasts, human umbilical vein endothelial cells, and Mandin-Darby canine kidney epithelial cells were seeded at low densities of approximately one cell/well. Because the hydrogel is made of non-adhesive agarose, attachment was negligible. Survival was monitored using fluorescent microscopy, and quantified by image analysis. The attachment and proliferative potential of cells after being held in a non-adherent environment was assessed with a companion attachment assay. The data from both methods revealed that cells were able to survive much longer than expected without attachment. When tested with H35 rat hepatoma cells we showed that single cancer cells could grow into three-dimensional spheroids, demonstrating the utility of this method in understanding the role of anoikis in cancer.     

In vivo therapeutic applications of cell spheroids

Spheroids are increasingly being employed to answer a wide range of clinical and biomedical inquiries ranging from pharmacology to disease pathophysiology, with the ultimate goal of using spheroids for tissue engineering and regeneration. When compared to traditional two-dimensional cell culture, spheroids have the advantage of better replicating the 3D extracellular microenvironment and its associated growth factors and signaling cascades. As knowledge about the preparation and maintenance of spheroids has improved, there has been a plethora of translational experiments investigating in vivo implantation of spheroids into various animal models studying tissue regeneration.We review methods for spheroid delivery and how they have been utilized in tissue engineering experiments. We break down efforts in this field by organ systems, discussing applications of spheroids to various animal models of disease processes and their potential clinical implications. These breakthroughs have been made possible by advancements in spheroid formation, in vivo delivery and assessment. There is unexplored potential and room for further research and development in spheroid-based tissue engineering approaches. Regenerative medicine and other clinical applications ensure this exciting area of research remains relevant for patient care.     

HistoGel预包埋法在微小组织石蜡切片中的应用

目的：探讨微小组织HistoGel预包埋的石蜡切片制作方法和优势。方法：选择拟胚体(embryoid body, EB)为微小组织,将人 类胚胎干细胞(human Embryonic Stem Cell, hESC)切割成小块,用拟胚体培养基在低贴附培养皿中悬浮培养形成拟胚体。收集拟 胚体,4 %的多聚甲醛固定。将HistoGel 加热到60 ℃熔解,离心去除拟胚体中的固定液,把液态的HistoGel 加到拟胚体上,调整拟 胚体的相对位置使其相对集中,待胶冷却凝固,将含有拟胚体的胶块取出,进行常规的石蜡切片操作,包括脱水、透明、浸蜡、包 埋、切片和苏木素- 伊红(hematoxylin-eosin, HE)染色。显微镜下观察拟胚体的形态,并用拟胚体的形态完好度来评判这种方法。结 果：HistoGel 仅在60 ℃便可熔解,室温可以冷却凝固,含有拟胚体的胶块在整个操作过程中很方便。展片过程中,石蜡和HistoGel 能够保持平整。HE 染色的结果可以看出,拟胚体内部结构完好,细胞核和细胞质清晰可辨。结论：HistoGel可以作为一种微量细 胞组织的预包埋胶制作石蜡切片,而且相比琼脂预包埋,HistoGel 因其操作更加方便和特有的物理特性显示出更多的优点。