MICA: Moving From Autologous to Allogeneic Cell Therapy: Developing Up-Scale GMP Protocols for Orthopaedics.

受损的软骨可以在罗伯特·琼斯和艾格尼丝·亨特（RJAH）骨科医院使用NHS批准的软骨细胞（软骨细胞）疗法进行治疗，称为自体软骨细胞植入（ACI）或作为临床试验的一部分使用“干细胞”。在这两种技术中，患者自己的细胞在第一次手术时从健康的软骨或骨髓中取出，在实验室中生长2-3周，并在第二次手术中植入关节以修复软骨。这个过程既耗时又昂贵;更重要的是，一些患者的细胞生长不好，或者具有意味着它们在植入时不再能治愈患者自己的软骨的特性。为了降低成本和改善患者的结果，理想的是使用“现成的”治疗，其中来自不同供体的细胞已经被测试和选择其治疗潜力（效力），并储存在生物库中用于一个阶段的程序。目前在我们的良好生产规范（GMP）实验室中培养细胞的方法涉及使用几种对空气“开放”的程序，可能会增加感染的风险。此外，传统的培养技术在生长阶段使用多个塑料瓶，这是劳动密集型的，并且监测和维护耗时。该项目提出使用一种称为生物反应器（Quantum）的最先进的半自动机器来培养细胞。这台机器的内表面积比我们目前使用的大得多（大50-100倍），因此可以更快地生长更多的细胞。生物反应器单元是“封闭的”，这意味着污染的风险大大降低。Quantum还大大降低了每剂量的劳动力和制造成本。我们在实验室中对Quantum生物反应器系统进行了评估，用于扩增从健康骨髓或脐带中分离的人类干细胞和从全膝关节置换术中分离的人类软骨细胞。使用专业技术评估蛋白质和基因的细胞扩展表征表明，在Quantum中生长的细胞和使用传统技术的细胞显示出相当的治疗特性。此外，量子产生了非常大的干细胞数量，平均在13天内产生了1.32亿个骨髓干细胞，在8天内产生了1.69亿个脐带干细胞，软骨细胞的细胞数在8天内也为7500万（即使用生物反应器产生的干细胞数量是现有方法的40倍，软骨细胞数量是现有方法的近20倍）量子已被用于生产干细胞在大型国际临床试验治疗条件，如糖尿病和肾脏疾病，无论是在英国和其他地方。在当前的提案中，我们将建立在Quantum系统中生长的软骨细胞的GMP生产方案。该研究的目的是从捐赠给研究的健康青少年和成人组织中培养大量细胞，以建立治疗软骨缺损和骨关节炎的最佳来源。此外，我们将通过全面评估Quantum中生长的软骨和干细胞在实验室中生长和产生软骨的能力，并将其特性与目前临床使用的细胞进行比较，确保它们至少与我们目前使用的细胞一样好。

Damaged cartilage can be treated at the Robert Jones and Agnes Hunt (RJAH) Orthopaedic hospital using an approved cartilage cell (chondrocyte) therapy on the NHS called autologous chondrocyte implantation (ACI) or with 'stem cells' as part of a clinical trial. In both techniques a patient's own cells are taken from healthy cartilage or bone marrow at first surgery, grown up in the lab over 2-3 weeks and implanted back into the joint to repair the cartilage at the second surgery. This procedure is time consuming and costly; what is more, some patients' cells do not grow well or have properties which mean they can no longer heal the patient's own cartilage when implanted back. To reduce cost and improve patient outcome it would be ideal to use an 'off the shelf' treatment, whereby cells from different donors have been tested and chosen for their therapeutic potential (potency) and stored in a biobank for use in a one stage procedure. The current method of growing cells in our Good Manufacturing Practice (GMP) laboratory involves the use of several procedures which are 'open' to the air and could increase the risk of infection. In addition, traditional culture techniques utilise multiple plastic flasks for growth phases, which are labour intensive and time consuming to monitor and maintain. This project proposes the use of a state of the art semi-automated machine called a bioreactor (Quantum) in which to grow the cells. This machine has a much larger internal surface area compared to what we currently use (50-100 times larger), so allowing more cells to be grown quicker. The bioreactor units are 'closed' which means that the risk of contamination is significantly reduced. Labour and manufacturing costs per dose are also reduced considerably with the Quantum. We have carried out assessments of the Quantum bioreactor system in our laboratory for the expansion of human stem cells isolated from healthy bone marrow or umbilical cords and human chondrocytes isolated from total knee replacements. Extended characterisation of cells using specialist techniques to assess proteins and genes showed that cells grown in the Quantum and using traditional techniques displayed comparable therapeutic properties. Moreover, the Quantum generated very large stem cell numbers resulting, on average, in 132 million bone marrow stem cells being produced in 13 days and 169 million umbilical cord stem cells in 8 days, cell numbers for chondrocytes were 75 million also in 8 days (i.e. using the bioreactor produced over forty times as many stem cells and nearly twenty times as many chondrocytes as we would get with current methods).The Quantum has been used to produce stem cells in large international clinical trials treating conditions such as diabetes and kidney disease, both in the UK and elsewhere. In the current proposal we will establish GMP manufacturing protocols for cartilage cells grown in the Quantum system. The aim of the study is to grow large numbers of cells from healthy juvenile and adult tissues donated to research in order to establish the best source for treating cartilage defects and osteoarthritis. In addition, we will ensure they are at least as good as the cells we currently use by comprehensively assessing the characteristics of the cartilage and stem cells grown in the Quantum in terms of their ability to grow and produce cartilage in the laboratory and by comparing their properties to cells currently used in the clinic.