Abstract：
Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.
摘要：
细胞外基质（ECM）在细胞行为和发育中起着至关重要的作用。由人诱导多能干细胞（hiPSC）产生的类器官是许多研究领域的焦点。然而,在经典细胞培养材料中缺乏生理线索阻碍了有效的iPSC分化。将市售ECM结合到干细胞培养物中提供了有益于细胞维持的物理和化学线索。动物来源的市售基底膜产品由支持细胞维持的ECM蛋白和生长因子组成。由于ECM具有可以调节细胞命运的组织特异性特性，无异种基质用于将其转化为临床研究。虽然市售基质广泛用于hiPSC和类器官工作，这些矩阵的等价性尚未评估。这里,在四种不同的基质中，hiPSC维持和人类肠道类器官（hIO）生成的比较研究：Matrigel（Matrix1-AB），Geltrex（矩阵2-AB），Cultrex(矩阵3-AB)，和体外凝胶(基质4-XF)进行。虽然殖民地缺乏完美的圆形，有最小的自发分化，其中超过85%的细胞表达干细胞标志物SSEA-4。矩阵4-XF导致3D圆形团块的形成。此外，增加用于制备基质4-XF水凝胶溶液的培养基中的补充物和生长因子的浓度将SSEA-4的hiPSC表达提高了1.3倍。与其他动物来源的基底膜相比，基质2-AB维持的hiPSC的分化导致在中肠/后肠阶段球状体释放较少。与其他人相比,无异种类器官基质（Matrix4-O3）导致更大和更成熟的hIO，表明可以利用无异种水凝胶的物理性质来优化类器官的生成。总之,结果表明，不同基质组成的变化会影响IO分化的阶段。这项研究提高了人们对商用矩阵差异的认识，并为iPSC和IO工作期间的矩阵优化提供了指导。
