Novel Bioprinted Neural Stem Cell-Embedded Hydrogel Matrices for Enhanced Treatment of Glioblastoma

胶质母细胞瘤(GBM)是影响成人的最常见的原发恶性脑肿瘤，中位生存期 时间为12-15个月。治疗基底节瘤的标准是最大限度地切除肿瘤，其次是 配合放射治疗和替莫唑胺治疗。然而，肿瘤细胞在切除后仍留在大脑中， 构成疾病复发的威胁。系统实施的治疗，如放射和化疗 不是针对手术后大脑中存在的微小肿瘤病变，因此对 预防90%的GBM患者复发。我们的团队和其他人已经证明了 治疗性神经干细胞(TNSCs)作为治疗术后GBM的药物输送平台 与生俱来的特性称为肿瘤嗜性。TNSCs与GBM细胞分泌的细胞因子相互作用，启动 导致tNSC向肿瘤方向迁移的信号级联反应。这种定向迁移可以是 作为一种靶向机制，用于输送由基因工程tNSC分泌的药物。 然而，平台的耐用性受到直接植入基底膜切除的tNSCs的快速清除的限制 空洞。TNSCs被包裹在生物材料中，当被输送到腔内时，可以阻止这种快速的清除 延长治疗效果的持续时间。我们的团队已经证明了生物兼容材料，如 由于商业止血剂能够支持tNSC在体内的长期存活。然而，这些 基质可以对tNSC的迁移构成障碍，与注射tNSC相比，导致的肿瘤杀伤作用微乎其微。 仅在公共广播公司。因此，我们发现在增强的tNSC活性和未损伤的细胞之间取得了平衡 必须达到迁移，以优化tNSCs进行长期的GBM治疗。为了做到这一点，我们将开发一部小说 适应3D打印技术，连续液体界面生产(CLIP)，其中tNSC为3D 在被称为生物打印的过程中打印成水凝胶。这导致了细胞嵌入的3D水凝胶，它可以 无需任何中间细胞种植步骤即可植入基底膜切除腔内。我们已经证明了 生物印染的细胞水凝胶比体外种植的细胞具有更高的种子密度 表面。然而，在生物打印中，细胞的行为和功能还没有被表征或优化 水凝胶。此外，支持最长细胞存活率的生物相容性最好的水凝胶表现出最低的 打印分辨率。因此，我们建议通过开发一种生物相容性来优化这一新的生物打印策略 以及可印刷的树脂，可支持细胞存活至少一个月。此外，我们将描述细胞的特征 生物打印前后的健康和功能，以确保有毒树脂单体和紫外线没有 损害了植入细胞的有效性或安全性。最后，我们将描述细胞负载的功效。 生物印迹水凝胶在无包膜细胞作为对照的GBM小鼠切除后模型中的应用。 我们假设优化的生物打印策略将在制造过程中产生更高的一致性， 临床处理更容易，肿瘤抑制持续时间更长，从而改善了患者的预后。

Glioblastoma (GBM) is the most common primary malignant brain tumor affecting adults, with a median survival time of 12-15 months. The standard of care for treating GBMs is maximum tumor resection followed by concomitant radiation and temozolomide therapy. However, tumor cells remain in the brain after resection, posing the threat of disease recurrence. Systemically administered treatments like radiation and chemotherapies are not targeted to the microscopic tumor lesions present in the brain post-surgery and are thus ineffective at preventing recurrence for 90% of GBM patients. Our group and others have demonstrated the promise of therapeutic neural stem cells (tNSCs) as a drug delivery platform for treating post-operative GBM due to an innate property known as tumor tropism. tNSCs interact with cytokines secreted by GBM cells, initiating a signaling cascade which results in tNSC migration in the direction of the tumor. This directional migration can be leveraged as a targeting mechanism for the delivery of drugs secreted by genetically engineered tNSCs. However, the platform's durability is limited by rapid clearance of tNSCs implanted directly into the GBM resection cavity. Encapsulation of tNSCs in biomaterials when delivered into the cavity could prevent this rapid clearance and lengthen the duration of therapeutic efficacy. Our group has demonstrated that biocompatible materials such as commercially available hemostats are able to support long-term in vivo tNSC viability. However, these matrices can pose a barrier to tNSC migration, resulting in insignificant tumor killing compared to tNSCs injected in PBS alone. Thus, we discovered that a balance between enhanced tNSC viability and unimpaired cell migration must be reached to optimize tNSCs for long-term GBM therapy. To do so, we will develop a novel adaptation of the 3D printing technology, continuous liquid interface production (CLIP), in which tNSCs are 3D printed into hydrogels in a process known as bioprinting. This results in cell-embedded 3D hydrogels which could be implanted into the GBM resection cavity without any intermediate cell seeding steps. We have shown that bioprinted cell-laden hydrogels exhibit higher seeding consistency than cells seeded externally onto hydrogel surfaces. However, cell behavior and function has not been characterized or optimized inside bioprinted hydrogels. Moreover, the most biocompatible hydrogels which support the longest cell viability exhibit the lowest printing resolution. Thus, we propose to optimize this novel bioprinting strategy by developing a biocompatible and printable resin that can support cell viability for at least one month. Furthermore, we will characterize cell health and functionality pre- and post-bioprinting to ensure that toxic resin monomers and UV light have not compromised the efficacy or safety of the embedded cells. Finally, we will characterize the efficacy of cell-laden bioprinted hydrogels in a post-resection GBM mouse model with unencapsulated cells serving as a comparator. We hypothesize that the optimized bioprinting strategy will result in higher consistency during manufacturing, easier clinical handling, and a longer duration of tumor suppression, leading to improved patient outcomes.