Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Cytotherapy. 2012 Jan;14(1):104-13. doi: 10.3109/14653249.2011.605350. Epub 2011 Sep 12.
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 × 10(10) total cells, 6.2 ± 2.2 × 10(8) 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.
已有研究表明，供体来源的椎骨骨髓（BM）可促进尸体器官来源的实体器官移植中的嵌合体形成。在接受定向供体移植并联合供体 BM 或血细胞以及新型移植前免疫抑制治疗的患者中，成功实现免疫抑制脱除的报道，重新激发了人们对在尸体器官中实施类似方案的兴趣。
我们进行了六次临床前全规模分离，以适应 BM 制剂在良好生产规范（GMP）环境中的应用。L4-T8 椎体被运送到 10000 级洁净室，清除软组织，在原型骨研磨机中分割和粉碎。骨碎片用含有生理盐水、白蛋白、DNA 酶和庆大霉素的培养基冲洗，并通过不锈钢筛过滤。用原型 BM 搅拌器进行两轮搅拌后，洗脱更多的细胞。
大多数回收的细胞（70.9 ± 14.1%，平均值 ± 标准差）直接从粉碎的骨中洗脱，而 22.3%和 5.9%分别在第一轮和第二轮搅拌后洗脱。细胞经 BM 收集试剂盒（500、200 μm）过滤和合并。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 ± 1.2×10(10)个总细胞，其中 6.2 ± 2.2×10(8)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出明显的 CD34(+) CD90(+) CD117(dim)造血干细胞群体（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%）。
该程序可用于在 GMP 环境中制备适合人类同种异体移植的临床级细胞。
