Bioengineering Graduate Program, University of Puerto Rico Mayaguez, Call Box 9000, Mayaguez, Puerto Rico 00681-9000, United States.
Bioengineering Department, Moffitt Cancer Center, Tampa, Florida 32611, United States.
ACS Biomater Sci Eng. 2024 Sep 9;10(9):5739-5751. doi: 10.1021/acsbiomaterials.4c01008. Epub 2024 Aug 26.
The increasing cost of high-volume cultures and dependence on serum and growth factor supplementation limit the affordability of mesenchymal stromal cell (MSC) therapies. This has spurred interest in developing strategies that support adherent cell expansion while reducing raw material costs. Culture surfaces coated with sulfated glycosaminoglycans (GAGs), specifically heparan sulfate (HS), are an alternative to prolong growth factor retention in cell cultures. Unlike heparin, recombinant HS (rHS) offers strong binding affinity for multiple growth factors and extracellular matrix components, such as collagen I, without undesirable anticoagulant effects or xenobiotic health risks. The potential of rHS as a factor reservoir in MSC cultures remains underexplored. This study investigated the impact of rHS on the growth and anti-inflammatory properties of undifferentiated bone marrow MSCs in both planar and microcarrier-based cultures. It was hypothesized that rHS would enable MSC growth with minimal growth factor supplementation in a sulfation level-dependent manner. Cell culture surfaces were assembled via the layer-by-layer (LbL) method, combining alternating collagen I (COL) and rHS. These bilayers support cell adhesion and enable the incorporation of distinct sulfation levels on the culture surface. Examination of pro-mitogenic FGF and immunostimulatory IFN-γ release dynamics confirmed prolonged availability and sulfate level dependencies. Sulfated surfaces supported cell growth in low serum (2% FBS) and serum-free (SF) media at levels equivalent to standard culture conditions. Cell growth on rHS-coated surfaces in SF was comparable to that on heparin-coated surfaces and commercial surface-coated microcarriers in low serum. These growth benefits were observed in both planar and microcarrier (μCs) cultures. Additionally, rHS surfaces reduced β-galactosidase expression relative to uncoated surfaces, delaying cell senescence. Multivariate analysis of cytokines in conditioned media indicated that rHS-containing surfaces enhanced cytokine levels relative to uncoated surfaces during IFN-γ stimulation and correlated with decreased pro-inflammatory macrophage activity. Overall, utilizing highly sulfated rHS with COL reduces the need for exogenous growth factors and effectively supports MSC growth and anti-inflammatory potency on planar and microcarrier surfaces under minimal factor supplementation.
高容量培养的成本不断增加，以及对血清和生长因子补充的依赖，限制了间充质基质细胞 (MSC) 治疗的可负担性。这激发了人们开发支持贴壁细胞扩增同时降低原材料成本的策略的兴趣。用硫酸化糖胺聚糖 (GAGs)，特别是硫酸乙酰肝素 (HS) 涂层的培养表面是延长细胞培养中生长因子保留时间的替代方法。与肝素不同，重组 HS (rHS) 对多种生长因子和细胞外基质成分（如 I 型胶原）具有很强的结合亲和力，而没有不良的抗凝作用或异源生物健康风险。rHS 作为 MSC 培养物中的因子库的潜力尚未得到充分探索。本研究调查了 rHS 对未分化骨髓 MSC 在平面和微载体培养中的生长和抗炎特性的影响。假设 rHS 将以最小的生长因子补充以依赖于硫酸化水平的方式实现 MSC 生长。细胞培养表面通过层层 (LbL) 方法组装，交替结合 I 型胶原 (COL) 和 rHS。这些双层支持细胞粘附，并能够在培养表面上掺入不同的硫酸化水平。对促有丝分裂的 FGF 和免疫刺激性 IFN-γ 释放动力学的研究证实了延长的可用性和硫酸化水平依赖性。硫酸化表面在低血清（2% FBS）和无血清（SF）培养基中支持细胞生长，水平相当于标准培养条件。在 SF 中，rHS 涂层表面上的细胞生长与肝素涂层表面和商业表面涂层微载体在低血清中的生长相当。这些生长优势在平面和微载体 (μCs) 培养中均观察到。此外，rHS 表面相对于未涂层表面降低了β-半乳糖苷酶的表达，从而延缓了细胞衰老。条件培养基中细胞因子的多元分析表明，与未涂层表面相比，rHS 表面在 IFN-γ 刺激下增强了细胞因子水平，并且与促炎巨噬细胞活性降低相关。总的来说，在 COL 中使用高度硫酸化的 rHS 减少了对外源性生长因子的需求，并在最小因子补充下有效支持 MSC 在平面和微载体表面的生长和抗炎效力。
