Cell labelling for in vivo use and in process quality control

该项目的临床部分是一项基于新型细胞标记系统的喷雾培养异体角质形成细胞在加速危及生命的烧伤（和其他非烧伤大面积皮肤脱落）的再上皮化方面的功效的单一临床试验。虽然培养的角质细胞异体细胞不能在移植中长期存活，但它们似乎在急性和慢性伤口愈合中都具有有用的临床作用。在前者中，它们加速了部分厚度皮肤伤口的上皮化，而在后者中，它们似乎将伤口表型转化为更急性的表型。这些作用是由多种细胞因子和生长因子的释放介导的。同种异体移植物细胞丢失的过程不是经典的，被描述为蠕变替代，并且了解很少。在没有活体标记的情况下，喷涂细胞的伤口分布、它们的附着和存活以及它们随后的迁移几乎是不可能跟踪的（我们过去曾使用性别不匹配和y探针）。这种效果可以在临床上看到，但以一种非常主观的方式缩短了“愈合时间”。随着时间的推移，细胞标签将允许评估异体和自体角质形成细胞对表皮屏障功能加速恢复的相对贡献。本临床研究将在一个新的客观水平上确立喷雾培养异体角化细胞辅助治疗的疗效，同时为临床环境引入一种新的细胞标记系统。选择危及生命的烧伤人群[bbbb30 % TBSA]有多种原因。这是培养角化细胞干细胞技术历来应用的人群，因为及时缝合伤口限制了败血症相关的死亡率。大的供体部位被创造出来，病人经常回到手术室，通常是每天，进行进一步的手术或大的换药。这将允许纵向检索穿孔活检材料后，喷雾应用。将包括三个臂，每个臂以1% TBSA供体部位伤口为代表：-对照供体部位伤口，用Jelonet敷料以标准方式处理，-喷雾培养异体角化细胞处理供体部位伤口，-喷雾培养异体角化细胞包括细胞标记处理伤口。对伤口进行一系列临床评估，并在换药时进行标准化摄影。在供体部位创建后立即从每个部位进行6mm穿孔活检，并在接下来的两周内再进行两个时间点-理想情况下为48小时和5天，但出于道德原因，有时需要进行全身麻醉手术。切片将被切开并快速冷冻或固定以供评估。所有患者均为成年人，且仅在知情同意的情况下招募。将在统计设备上进行功率计算，目前预计患者人数在6至10人之间。这项工作将由一名外科研究员在PI的指导下攻读更高的学位，硕士学位或博士学位。我们的目标是建立一种检测系统，从宏观上展示标记异体细胞的分布、存活和迁移。组织将被处理以检测细胞标记、异体反应或其他形态学证据、同种异体与自体移植物对体积的相对贡献，以及角质细胞迁移、增殖、分化和凋亡的标记。

The clinical component of the project is a single clinical trial of the efficacy of spray cultured allogeneic keratinocytes in acceleration of re-epithelialisation of life-threatening burn injury [and other non-burn large area skin loss] split-thicknessskin graft donor sites based on a novel cell labelling system.Whilst cultured keratinocyte allogeneic cells do not survive transplantation longterm, they appear to have a useful clinical role in both acute and chronic wound healing. In the former, they accelerate epithelialisation of partial-thickness cutaneouswounds, and in the latter, they appear to convert the wound phenotype to a more acute one. These effects are mediated by the release of a wide range of cytokines and growth factors in an orchestrated way. The process of allograft cell loss is notclassical, has been described as creeping substitution and is poorly understood. The wound distribution of sprayed cells, their attachment and survival, and their subsequent migration have been almost impossible to follow in the absence of an invivo label [we have used sex-mismatching and a Y-probe in the past]. The effects can be seen clinically, but in a very subjective way as shortened "time to healing". A cell label will allow an assessment of the relative contribution of allogeneicand autologous keratinocytes to accelerated restoration of epidermal barrier function over time.This clinical study will establish the efficacy of spray cultured allogeneic keratinoyte adjuvant therapy at a new level of objectivity, and at the same time introduce a novel cell labelling system to the clinical environment. The population of lifethreateningburn injury [>30% TBSA] has been chosen for a variety of reasons. This is the population for whom cultured keratinocyte stem cell technology has historically applied, since timely wound closure limits sepsis related mortality. Largedonor sites are created, and the patients return to theatre regularly, often daily, for further surgery or large dressing changes. This will allow longitudinal retrieval of punch biopsy material following spray application.Three arms will be included, each represented by a 1% TBSA donor site wound:- control donor site wound, managed in a standard way with Jelonet dressings,- spray cultured allogeneic keratinocyte treated donor site wound,- spray cultured allogeneic keratinocyte including cell label treated wound.The wounds will be followed by serial clinical evaluation and standardised photography at dressing changes. 6mm punch biopsies will be retrieved from each site at T0, immediately following creation of the donor site, and two further time pointsover the following two weeks - T48hrs and T5days ideally, but for ethical reasons at times otherwise when general anaesthetic procedures are required. Biopsies will be bisected and snap frozen or fixed for evaluation.All patients will be adult, and recruited only with informed consent. A power calculation will be performed on statistical dvice, and a patient number of between 6 and 10 is currently anticipated. The work will be conducted by a Surgical research fellow toward a higher degree, MDRes or PhD under PI supervision.The aspiration is for a detection system that will demonstrate the distribution, survival and migration of labelled allogeneic cells macroscopically. Tissue will be processed for detection of the cell label, for morphological evidence of foreign bodyreaction or other, for the relative contribution of allo- vs autograft to volume, and for markers of keratinocyte migration, proliferation, differentiation and apoptosis.