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

受损的软骨可以在Robert Jones and Agnes Hunt(RJAH)整形外科医院使用NHS批准的软骨细胞(软骨细胞)疗法进行治疗，这种疗法被称为自体软骨细胞植入(ACI)，或者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提取，在实验室中生长2-3周，然后在第二次手术中重新移植到关节中修复软骨。这一过程既耗时又昂贵；更重要的是，一些患者的细胞不能很好地生长，或者具有一些特性，这意味着当它们被重新植入患者自己的软骨时，它们不再能够愈合。为了降低成本和改善患者预后，理想的做法是使用现成的治疗方法，即对来自不同捐赠者的细胞进行测试和选择，以确定其治疗潜力(效力)，并将其存储在生物库中，以便在一步程序中使用。目前在我们的良好制造规范(GMP)实验室中培养细胞的方法涉及到几种程序的使用，这些程序对空气是开放的，可能会增加感染的风险。此外，传统的栽培技术在生长阶段使用多个塑料烧瓶，这是劳动密集型的，监测和维护也很耗时。该项目建议使用一种最先进的半自动机器，称为生物反应器(Quantum)，在其中培养细胞。这台机器的内表面积比我们目前使用的要大得多(50-100倍)，因此可以更快地生长更多的细胞。生物反应器单元是关闭的，这意味着污染的风险大大降低。随着Quantum的推出，每剂的劳动力和制造成本也大大降低。我们在实验室对Quantum生物反应器系统进行了评估，以扩增从健康骨髓或脐带分离的人类干细胞和从全膝关节置换手术中分离的人类软骨细胞。使用专家技术评估蛋白质和基因的扩展细胞特征表明，在Quantum中生长的细胞和使用传统技术的细胞显示出类似的治疗特性。此外，Quantum产生了非常多的干细胞数量，平均在13天内生产了1.32亿个骨髓干细胞，在8天内生产了1.69亿个脐带干细胞，软骨细胞的细胞数量在8天内也达到了7500万个(即使用生物反应器生产的干细胞是我们目前方法生产的干细胞数量的40多倍和近20倍)。在英国和其他地方，Quantum已被用于治疗糖尿病和肾脏疾病等大型国际临床试验中的干细胞生产。在目前的提案中，我们将为在量子系统中生长的软骨细胞建立GMP制造协议。这项研究的目的是从健康的青少年和成人组织中培养大量捐赠给研究的细胞，以建立治疗软骨缺陷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，并通过将它们的特性与目前临床使用的细胞进行比较，来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.