大部分与癌症相关的体外研究都是在细胞单层或球体上进行。尽管这种方法已经取得了重要的发现，但在复制肿瘤发展的不同阶段方面仍然效果不佳。新型的三维（3D）系统和其制备技术的出现将有助于改善这一领域，提供更贴近生理的，能够高通量地研究肿瘤发展和治疗的体外系统。在这里，我们描述了一种基于海藻酸盐的3D模型的制备方法，该模型能够复制结直肠癌的早期阶段，并追踪肿瘤发展的两个主要生物标志物：CD44和HIF-1α。我们优化了制备过程，以获得具有控制大小和刚度的海藻酸盐微珠，模拟结直肠癌的早期阶段。人结直肠HCT-116癌细胞被包埋进去，初始细胞数受到控制，并将细胞存活率和蛋白质表达与当前的金标准（细胞单层和球体）进行了比较。我们的结果表明，包埋的HCT-116表现出高存活率，干细胞样细胞群体的增加（CD44表达增加）和缺氧区域的减少（较低的HIF-1α表达），与球体培养相比。总之，我们展示了我们的生物制造系统是一种高度可复制和易于获取的替代方法，用于研究细胞行为，能够更好地模拟结直肠癌的早期阶段，与其他体外模型相比。使用生物制造的体外模型将提高结果的可转化性，尤其是在测试治疗干预策略时。知识共享署名许可。

The majority of cancer-relatedin vitrostudies are conducted on cell monolayers or spheroids. Although this approach has led to key discoveries, it still has a poor outcome in recapitulating the different stages of tumor development. The advent of novel three-dimensional (3D) systems and technological methods for their fabrication is set to improve the field, offering a more physiologically relevant and high throughputin vitrosystem for the study of tumor development and treatment. Here we describe the fabrication of alginate-based 3D models that recapitulate the early stages of colorectal cancer, tracking two of the main biomarkers for tumor development: CD44 and HIF-1α. We optimized the fabrication process to obtain alginate micro-beads with controlled size and stiffness, mimicking the early stages of colorectal cancer. Human colorectal HCT-116 cancer cells were encapsulated with controlled initial number, and cell viability and protein expression of said 3Din vitromodels was compared to that of current gold standards (cell monolayers and spheroids). Our results evidenced that encapsulated HCT-116 demonstrated a high viability, increase in stem-like cell populations (increased expression of CD44) and reduced hypoxic regions (lower HIF-1a expression) compared to spheroid cultures. In conclusion we show that our biofabricated system is a highly reproducible and easily accessible alternative to study cell behavior, allowing to better mimic the early stages of colorectal cancer in comparison to otherin vitromodels. The use of biofabricatedin vitromodels will improve the translatability of results, in particular when testing strategies for therapeutic intervention.Creative Commons Attribution license.