Injectable Hydrogels to Protect Transplanted Cells from Hypoxia

项目摘要 PAR-18-206：可注射水凝胶保护移植细胞耐缺氧 直接局部注射细胞移植是许多再生医学治疗的一种很有前途的策略； 然而，无论临床适应症如何，这种策略的治疗潜力都受到了极大的限制。 由于细胞输送效率低下和移植细胞长期存活不佳。我们最近设计了一款 可注射水凝胶，通过在注射过程中提供(1)机械屏蔽来改善细胞输送 防止细胞膜破裂的过程，(2)体内快速凝胶化以在预定的交付时定位细胞 以及(3)促进移植细胞扩散和迁移到宿主体内的细胞黏附配体 组织。在脊髓损伤(SCI)的临床前模型中，使用这种水凝胶移植雪旺细胞(SCs) 导致成功的细胞输送显著增加，这与改进的治疗方法有关 结果。然而，移植细胞的长期存活率较差仍然是一个未得到满足的挑战，原因是 低氧的宿主环境。因此，我们提出了两种正交生物材料设计的发展方向 策略(目标1中的生物力学策略和目标2中的生化策略)以创建可注射 水凝胶可以改善移植细胞的输送，并促进在低氧条件下的长期存活。这些材料， 名为Shield(用于注射包囊和长期给药的剪切稀释型水凝胶)完全 化学定义，以便于FDA未来的研究。作为概念证明，盾牌将在 脊髓损伤的临床前模型，在该模型中，移植的SC疗法已知患有严重的低氧细胞 死亡。在目标1中，我们评估了矩阵力学可以改变支持生存的分泌体的假设。 被包裹的细胞，从而产生可溶的自分泌信号，从而改善低氧生存。单元格将是 包裹在具有一定硬度的屏蔽材料中，在常氧和低氧条件下培养 (分别为5%和1%O2)，并评估活性、增殖、神经营养因子的分泌和生长 细胞坏死(亲环素A和Fodrin分解产物)和凋亡(caspase-3)的因子和标记物 和Tunel)。作为一种平行的方法，在目标2中，我们评估了持续的、局部的交付 有利于生存的因素可以通过设计稳定的脂囊库来实现， 交联剂进入我们的可注射水凝胶。双层间共价稳定多层脂胶囊 交联度用于调节释放速率。因此，这种模块化设计 该策略可用于独立控制多种促生存因子的给药动力学。 被包裹的细胞将按照目标1进行评估。在目标3中，我们在临床前大鼠身上验证了我们的体外研究结果 脊髓干细胞移植建立颈髓挫伤模型。干细胞的存活和分布，天然组织反应， 将评估神经再生和前肢功能恢复。总而言之，因为 基于细胞的再生医学疗法取决于移植细胞的存活，这项技术 直接解决缺氧引起的细胞死亡可以显著改善临床结果。

Project Summary PAR-18-206: Injectable Hydrogels to Protect Transplanted Cells from Hypoxia Cell transplantation by direct local injection is a promising strategy for many regenerative medicine therapies; however, regardless of clinical indication, the therapeutic potential of this strategy has been drastically limited by inefficient cell delivery and poor long-term survival of transplanted cells. We have recently designed an injectable hydrogel that improves cell delivery by providing (1) mechanical shielding during the injection process to prevent cell membrane rupture, (2) rapid gelation in vivo to localize cells at the intended delivery site, and (3) cell-adhesive ligands that promote the spreading and migration of transplanted cells into the host tissue. In a preclinical model of spinal cord injury (SCI), use of this hydrogel to transplant Schwann cells (SCs) resulted in a significant increase in successful cell delivery, which correlated with improved therapeutic outcomes. However, poor long-term survival of transplanted cells continues to be an unmet challenge due to the hypoxic host environment. Therefore, we propose the development of two orthogonal biomaterial design strategies (a biomechanical strategy in Aim 1 and a biochemical strategy in Aim 2) to create injectable hydrogels that improve transplanted cell delivery and promote long-term survival in hypoxia. These materials, named SHIELD (Shear-thinning Hydrogels for Injectable Encapsulation and Long-term Delivery) are fully chemically defined to facilitate future FDA studies. As a proof of concept, SHIELD will be evaluated in a preclinical model of SCI, where transplanted SC therapies are known to suffer from significant hypoxic cell death. In Aim 1, we evaluate the hypothesis that matrix mechanics can alter the pro-survival secretome of encapsulated cells, thereby creating soluble, autocrine signals that improve hypoxic survival. Cells will be encapsulated in SHIELD materials with a range of stiffness, cultured under normoxic and hypoxic conditions (5% and 1% O2, respectively), and assessed for viability, proliferation, secretion of neurotrophins and growth factors, and markers of cell necrosis (cyclophilin A and fodrin breakdown product) and apoptosis (caspase-3 and TUNEL). As a parallel approach, in Aim 2, we evaluate the hypothesis that sustained, localized delivery of pro-survival factors can be achieved through the design of stabilized, lipid-vesicle depots that physically crosslink into our injectable hydrogel. The multi-lamellar lipid capsules are stabilized by inter-bilayer covalent crosslinking, and the degree of crosslinking is used to tune the release rate. Thus, this modular design strategy can be used to independently control the delivery kinetics of multiple pro-survival factors. Encapsulated cells will be evaluated as in Aim 1. In Aim 3, we validate our in vitro findings in a preclinical rat model of cervical, contusive SCI with SC transplantation. SC survival and distribution, native tissue response, neuro-regeneration, and functional forelimb recovery will be assessed. In summary, because the success of cell-based regenerative medicine therapies hinges on the survival of transplanted cells, technologies that directly address cell death by hypoxia can significantly improve clinical outcomes.