Wan Chao, He Qiling, McCaigue Mervyn, Marsh David, Li Gang
Department of Trauma and Orthopaedic Surgery, Institute of Medical Research, Queen's University Belfast, Musgrave Park Hospital, Belfast BT9 7JB, United Kingdom.
J Orthop Res. 2006 Jan;24(1):21-8. doi: 10.1002/jor.20023.
To obtain enough quantity of osteogenic cells is a challenge for successful cell therapy in bone defect treatment, and cell numbers were usually achieved by culturing bone marrow cells in a relatively long duration. This study reports a simple and cost-effective method to enhance the number of mesenchymal stem cells (MSCs) by collecting and replating the nonadherent cell population of marrow MSCs culture. Bone marrow MSCs were isolated from 11 patients, cultured at a density of 1 x 10(5)/cm(2) to 1 x 10(6)/cm(2) in flasks. For the first three times of media change, the floating cells were centrifuged and replated in separate flasks. The total number of cells in both the primary and replating flasks were counted at day 21. Cell proliferation rate, potentials for osteogenic, chondrognenic, and adipogenic differentiation were examined in both cell types in vitro. In vivo osteogenic potentials of the cells were also tested in mice implantation model. The results showed that MSCs derived from nonadherent cell population of marrow cell cultures have similar cell proliferation and differentiation potentials as the originally attached MSCs in vitro. When implanted with hydroxyapatite/tricalcium phosphate (HA-TCP) materials subcutaneously in serve combined immune deficiency (SCID) mice, newly formed bony tissues were found in both cell type groups with osteocalcin expression. We have obtained 36.6% (20.70%-44.97%) more MSCs in the same culture period when the nonadherent cell populations were collected. The findings confirmed that the nonadherent cell population in the bone marrow culture is a complementary source of MSCs, collecting these cells is a simple and cost-effective way to increase MSCs numbers and reduce the time required for culturing MSCs for clinical applications.
获取足够数量的成骨细胞是骨缺损治疗中细胞治疗成功的一项挑战，细胞数量通常通过在相对较长时间内培养骨髓细胞来实现。本研究报告了一种简单且经济高效的方法，即通过收集并重新接种骨髓间充质干细胞（MSC）培养物中的非贴壁细胞群体来增加间充质干细胞的数量。从11名患者中分离出骨髓间充质干细胞，以1×10(5)/cm(2)至1×10(6)/cm(2)的密度在培养瓶中培养。在前三次更换培养基时，将漂浮的细胞离心并重新接种到单独的培养瓶中。在第21天对原代培养瓶和重新接种培养瓶中的细胞总数进行计数。在体外对两种细胞类型的细胞增殖率、成骨、成软骨和成脂分化潜能进行了检测。还在小鼠植入模型中测试了细胞的体内成骨潜能。结果表明，源自骨髓细胞培养物非贴壁细胞群体的间充质干细胞在体外具有与最初贴壁的间充质干细胞相似的细胞增殖和分化潜能。当将其与羟基磷灰石/磷酸三钙（HA-TCP）材料皮下植入重症联合免疫缺陷（SCID）小鼠时，在两个细胞类型组中均发现了新形成的骨组织且有骨钙素表达。当收集非贴壁细胞群体时，在相同培养期内我们获得的间充质干细胞数量增加了36.6%（20.70%-44.97%）。这些发现证实，骨髓培养中的非贴壁细胞群体是间充质干细胞的一个补充来源，收集这些细胞是增加间充质干细胞数量并减少临床应用中间充质干细胞培养所需时间的一种简单且经济高效的方法。
