Biomanufacturing of Axon-Based Tissue Engineered Nerve Grafts Using Porcine GalSafe Neurons
Biomanufacturing of Axon-Based Tissue Engineered Nerve Grafts Using Porcine GalSafe Neurons
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DOI：
10.1089/ten.tea.2020.0303
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发表时间：
2021-04-09
影响因子：
4.1
通讯作者：
Cullen, D. Kacy
中科院分区：
文献类型：
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作者：
Katiyar, Kritika S.;Burrell, Justin C.;Cullen, D. Kacy
用于修复主要周围神经损伤（PNI）的现有策略在促进轴突再生和功能恢复方面是低效的，并且对于> 5cm的神经损伤通常是无效的。为了满足这一需求，我们先前已经通过轴突拉伸生长的过程开发了组织工程神经移植物（TENGs）。TENG由活的、厘米级的、对齐的轴突束组成，其通过提供被称为轴突促进轴突再生（AFAR）的新发现的作用机制，以与PNI的小型和大型动物模型中的金标准自体移植物相当的速率加速轴突再生。为了能够实现TENG的临床级生物制造，必须利用低免疫原性、表现出低批次间变异性并且能够耐受轴突拉伸生长的合适的细胞来源。为了满足这些要求，由Revivicor，Inc.生产的基因工程的、FDA批准的异种细胞源GalSafe（R）神经元，已被选定为推进TENG生物制品的最终临床使用。为此，从遗传工程化的GalSafe第40天猪胚胎收获感觉和运动神经元，在定制的机械生物反应器中培养，并且轴突束在25天内成功拉伸生长至5cm。重要的是，观察到感觉和运动GalSafe神经元耐受>= 1 mm/天的已建立的轴突拉伸生长方案，以产生跨越1、3或5cm的连续、健康的轴突束。一旦拉伸生长，将1 cm GalSafe TENG移植到无胸腺大鼠坐骨神经中的1 cm病变中。在2周和8周的终末时间点通过组织学测量评估再生。来自GalSafe TENG的神经元存活并引起AFAR，如使用野生型TENG时所观察到的。在修复后8周，在损伤部位远端的神经切片中观察到有髓鞘再生轴突，证实了穿过损伤的轴突再生。这些实验首次证明了GalSafe神经元的成功收获和轴突拉伸生长，可用作生物工程神经移植物的起始生物质，以及在已建立的临床前模型中的初始安全性和有效性-这是推进临床级TENG用于未来监管测试和最终临床试验的重要步骤。
Existing strategies for repair of major peripheral nerve injury (PNI) are inefficient at promoting axon regeneration and functional recovery and are generally ineffective for nerve lesions >5 cm. To address this need, we have previously developed tissue engineered nerve grafts (TENGs) through the process of axon stretch growth. TENGs consist of living, centimeter-scale, aligned axon tracts that accelerate axon regeneration at rates equivalent to the gold standard autograft in small and large animal models of PNI, by providing a newfound mechanism-of-action referred to as axon-facilitated axon regeneration (AFAR). To enable clinical-grade biomanufacturing of TENGs, a suitable cell source that is hypoimmunogenic, exhibits low batch-to-batch variability, and able to tolerate axon stretch growth must be utilized. To fulfill these requirements, a genetically engineered, FDA-approved, xenogeneic cell source, GalSafe(R) neurons, produced by Revivicor, Inc., have been selected to advance TENG biofabrication for eventual clinical use. To this end, sensory and motor neurons were harvested from genetically engineered GalSafe day 40 swine embryos, cultured in custom mechanobioreactors, and axon tracts were successfully stretch-grown to 5 cm within 25 days. Importantly, both sensory and motor GalSafe neurons were observed to tolerate established axon stretch growth regimes of >= 1 mm/day to produce continuous, healthy axon tracts spanning 1, 3, or 5 cm. Once stretch-grown, 1 cm GalSafe TENGs were transplanted into a 1 cm lesion in the sciatic nerve of athymic rats. Regeneration was assessed through histological measures at the terminal time point of 2 and 8 weeks. Neurons from GalSafe TENGs survived and elicited AFAR as observed when using wild-type TENGs. At 8 weeks postrepair, myelinated regenerated axons were observed in the nerve section distal to the injury site, confirming axon regeneration across the lesion. These experiments are the first to demonstrate successful harvest and axon stretch growth of GalSafe neurons for use as starting biomass for bioengineered nerve grafts as well as initial safety and efficacy in an established preclinical model-important steps for the advancement of clinical-grade TENGs for future regulatory testing and eventual clinical trials.