Development and validation of a procedure to isolate viable bone marrow cells from the vertebrae of cadaveric organ donors for composite organ grafting.
Development and validation of a procedure to isolate viable bone marrow cells from the vertebrae of cadaveric organ donors for composite organ grafting.
复制标题
DOI：
10.3109/14653249.2011.605350
复制
发表时间：
2012-01
期刊：
影响因子：
4.5
通讯作者：
Donnenberg AD
中科院分区：
文献类型：
--
作者：
Gorantla VS;Schneeberger S;Moore LR;Donnenberg VS;Zimmerlin L;Lee WP;Donnenberg AD
供体来源的椎骨骨髓（BM）已被提议促进实体器官移植与尸体器官的嵌合。接受定向供体移植并结合供体骨髓或血细胞以及新型移植周围免疫抑制的患者成功摆脱免疫抑制的报告重新引起了人们对尸体器官实施类似方案的兴趣。我们进行了六次临床前全面分离，以使椎骨骨髓制剂适应良好生产规范 (GMP) 环境。 L4-T8 椎体被运送到 10 000 级洁净室，清除软组织，在原型骨磨机中分割和粉碎。用含有盐水、白蛋白、DNAse和庆大霉素的培养基冲洗骨碎片，并通过不锈钢筛过滤。使用原型 BM 滚筒搅拌两轮后，洗脱额外的细胞。大多数回收的细胞（70.9 ± 14.1%，平均值±标准差）直接从碎骨中洗脱，而分别有 22.3% 和 5.9% 在第一轮和第二轮翻滚后洗脱。使用 BM 收集试剂盒汇集并过滤细胞（500、200 μm）。较大的腰椎产生的细胞约为胸椎的 1.6 倍。平均产物产生 5.2 ± 1.2×1010 个总细胞，其中 6.2 ± 2.2×108 个为 CD45+ CD34+。活力分别为 96.6 ± 1.9% 和 99.1 ± 0.8%。多色流式细胞术显示不同的 CD34+ CD90+ CD117 暗淡造血干细胞群（占 CD34 + 细胞的 15.5 ± 7.5%）和 CD45− CD73+ CD105+ 间充质基质细胞（占总细胞的 0.04 ± 0.04%）。该程序可用于制备适合在 GMP 环境下进行人类同种异体移植的临床级细胞。
Donor-derived vertebral bone marrow (BM) has been proposed to promote chimerism in solid organ transplantation with cadaveric organs. Reports of successful weaning from immunosuppression in patients receiving directed donor transplants in combination with donor BM or blood cells and novel peri-transplant immunosuppression has renewed interest in implementing similar protocols with cadaveric organs. We performed six pre-clinical full-scale separations to adapt vertebral BM preparations to a good manufacturing practice (GMP) environment. Vertebral bodies L4–T8 were transported to a class 10 000 clean room, cleaned of soft tissue, divided and crushed in a prototype bone grinder. Bone fragments were irrigated with medium containing saline, albumin, DNAse and gentamicin, and strained through stainless steel sieves. Additional cells were eluted after two rounds of agitation using a prototype BM tumbler. The majority of recovered cells (70.9 ± 14.1%, mean ± SD) were eluted directly from the crushed bone, whereas 22.3% and 5.9% were eluted after the first and second rounds of tumbling, respectively. Cells were pooled and filtered (500, 200 μm) using a BM collection kit. Larger lumbar vertebrae yielded about 1.6 times the cells of thoracic vertebrae. The average product yielded 5.2 ± 1.2×1010 total cells, 6.2 ± 2.2×108 of which were CD45+ CD34+. Viability was 96.6 ± 1.9% and 99.1 ± 0.8%, respectively. Multicolor flow cytometry revealed distinct populations of CD34+ CD90+ CD117 dim hematopoietic stem cells (15.5 ± 7.5% of the CD34 + cells) and CD45− CD73+ CD105+ mesenchymal stromal cells (0.04 ± 0.04% of the total cells). This procedure can be used to prepare clinical-grade cells suitable for use in human allotransplantation in a GMP environment.