Abu-Yousif Adnan O, Rizvi Imran, Evans Conor L, Celli Jonathan P, Hasan Tayyaba
Wellman Center for Photomedicine Massachusetts General Hospital, Harvard Medical School.
J Vis Exp. 2009 Dec 17(34):1692. doi: 10.3791/1692.
Increasing evidence suggests that culturing cancer cells in three dimensions more accurately recapitulates the complexity of tumor biology. Many of these models utilize reconstituted basement membrane derived from animals which contain a variable amount of growth factors and cytokines that can influence the growth of these cell culture models. Here, we describe in detail the preparation and use of PuraMatrix, a commercially available self assembling peptide gel that is devoid of animal-derived material and pathogens to encapsulate and propagate the ovarian cancer cell line, OVCAR-5. We begin by describing how to prepare the PuraMatrix prior to use. Next, we demonstrate how to properly mix the PuraMatrix and cell suspension to encapsulate the cells in the hydrogel. Upon the addition of cell culture media or injection into a physiological environment, the peptide component of PuraMatrix rapidly self assembles into a 3D hydrogel that exhibits a nanometer scale fibrous structure with an average pore size of 5-200 nm(1). In addition, we demonstrate how to propagate cultures grown in encapsulated PuraMatrix. When encapsulated in PuraMatrix, OVCAR-5 cells assemble into three dimensional acinar structures that more closely resemble the morphology of micrometastatic nodules observed in the clinic than monolayer in vitro models. Using confocal microscopy we illustrate the appearance of representative OVCAR-5 cells encapsulated in PuraMatrix on day 1, 3, 5, and 7 post plating. The use of PuraMatrix to culture cancer cells should improve our understanding of the disease and allow us to assess treatment response in more clinically predictive model systems.
越来越多的证据表明，在三维空间中培养癌细胞能更准确地重现肿瘤生物学的复杂性。许多此类模型利用源自动物的重组基底膜，其中含有数量可变的生长因子和细胞因子，这些物质会影响这些细胞培养模型的生长。在此，我们详细描述PuraMatrix的制备和使用方法，PuraMatrix是一种市售的自组装肽凝胶，不含动物源材料和病原体，用于包封和培养卵巢癌细胞系OVCAR-5。我们首先描述使用前如何制备PuraMatrix。接下来，我们展示如何正确混合PuraMatrix和细胞悬液，以便将细胞包封在水凝胶中。加入细胞培养基或注入生理环境后，PuraMatrix的肽成分会迅速自组装成三维水凝胶，呈现出纳米级纤维结构，平均孔径为5-200纳米(1)。此外，我们展示如何传代培养在包封的PuraMatrix中生长的细胞。当包封在PuraMatrix中时，OVCAR-5细胞会组装成三维腺泡结构，与临床观察到的微转移结节形态相比，比单层体外模型更相似。我们使用共聚焦显微镜展示接种后第1、3、5和7天包封在PuraMatrix中的代表性OVCAR-5细胞的外观。使用PuraMatrix培养癌细胞应能增进我们对该疾病的了解，并使我们能够在更具临床预测性的模型系统中评估治疗反应。
