人骨骼肌源细胞体外效力测定法的建立。
Development of an in vitro potency assay for human skeletal muscle derived cells.
机构信息
Innovacell Biotechnologie AG, Science Park, Innsbruck, Austria.
Daniel Swarovski Research Laboratory, Department of Visceral, Transplant, and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria.
出版信息
PLoS One. 2018 Mar 22;13(3):e0194561. doi: 10.1371/journal.pone.0194561. eCollection 2018.
BACKGROUND
Potency is a quantitative measure of the desired biological function of an advanced therapy medicinal product (ATMP) and is a prerequisite for market approval application (MAA). To assess the potency of human skeletal muscle-derived cells (SMDCs), which are currently investigated in clinical trials for the regeneration of skeletal muscle defects, we evaluated acetylcholinesterase (AChE), which is expressed in skeletal muscle and nervous tissue of all mammals.
METHODS
CD56+ SMDCs were separated from CD56- SMDCs by magnetic activated cell sorting (MACS) and both differentiated in skeletal muscle differentiation medium. AChE activity of in vitro differentiated SMDCs was correlated with CD56 expression, fusion index, cell number, cell doubling numbers, differentiation markers and compared to the clinical efficacy in patients treated with SMDCs against fecal incontinence.
RESULTS
CD56- SMDCs did not form multinucleated myotubes and remained low in AChE activity during differentiation. CD56+ SMDCs generated myotubes and increased in AChE activity during differentiation. AChE activity was found to accurately reflect the number of CD56+ SMDCs in culture, their fusion competence, and cell doubling number. In patients with fecal incontinence responding to SMDCs treatment, the improvement of clinical symptoms was positively linked with the AChE activity of the SMDCs injected.
DISCUSSION
AChE activity was found to truly reflect the in vitro differentiation status of SMDCs and to be superior to the mere use of surface markers as it reflects not only the number of myogenic SMDCs in culture but also their fusion competence and population doubling number, thus combining cell quality and quantification of the expected mode of action (MoA) of SMDCs. Moreover, the successful in vitro validation of the assay proves its suitability for routine use. Most convincingly, our results demonstrate a link between clinical efficacy and the AChE activity of the SMDCs preparations used for the treatment of fecal incontinence. Thus, we recommend using AChE activity of in vitro differentiated SMDCs as a potency measure in end stage (phase III) clinical trials using SMDCs for skeletal muscle regeneration and subsequent market approval application (MAA).
背景
效价是评估先进治疗药物产品（ATMP）所需生物功能的定量指标，是市场准入申请（MAA）的前提条件。为了评估目前正在临床试验中用于骨骼肌缺陷再生的人骨骼肌源性细胞（SMDC）的效价，我们评估了乙酰胆碱酯酶（AChE），它在所有哺乳动物的骨骼肌和神经组织中表达。
方法
通过磁激活细胞分选（MACS）将 CD56+ SMDC 从 CD56- SMDC 中分离出来，并在骨骼肌分化培养基中进行分化。体外分化的 SMDC 的 AChE 活性与 CD56 表达、融合指数、细胞数量、细胞倍增数、分化标志物相关，并与接受 SMDC 治疗的粪便失禁患者的临床疗效进行比较。
结果
CD56- SMDC 未形成多核肌管，在分化过程中 AChE 活性保持较低水平。CD56+ SMDC 生成肌管，并在分化过程中 AChE 活性增加。AChE 活性准确反映了培养物中 CD56+ SMDC 的数量、融合能力和细胞倍增数。在对 SMDC 治疗有反应的粪便失禁患者中，临床症状的改善与注射的 SMDC 的 AChE 活性呈正相关。
讨论
AChE 活性真实反映了 SMDC 的体外分化状态，优于单纯使用表面标志物，因为它不仅反映了培养物中肌源性 SMDC 的数量，还反映了其融合能力和群体倍增数，从而结合了细胞质量和量化 SMDC 的预期作用机制（MoA）。此外，该测定法的成功体外验证证明了其适合常规使用。最令人信服的是，我们的结果表明，粪便失禁治疗中使用的 SMDC 制剂的临床疗效与 AChE 活性之间存在关联。因此，我们建议将体外分化的 SMDC 的 AChE 活性作为使用 SMDC 进行骨骼肌再生的终末期（III 期）临床试验中的效价测量指标，以及随后的市场准入申请（MAA）。
Zotero 插件