应用不同方法进行人牙髓细胞原代培养的比较研究

目的比较并建立人牙髓细胞原代培养的理想方法.方法分别采用组织块法、酶消化法和组织块消化法进行人牙髓细胞的体外原代培养,倒置显微镜及台盼蓝染色观察并比较三者的培养成活率、细胞活力、细胞形态和数量、培养耗时等,免疫组化法鉴定细胞来源.结果三种方法均可从人牙髓组织分离培养获得外胚间充质来源的牙髓细胞.组织块法耗时较长,4周能进行首次传代,传代成功率50%,细胞形态较单一;酶消化法能在较短时间内获得形态多样的人牙髓细胞,但培养成活率较低,且细胞数量较少,8天时传代成功率为20%;组织块消化法不仅培养成活率(75%)和8天传代成功率(62.5%)较高,而且在较短时间内可多量获得多种形态类型的人牙髓细胞.结论组织块消化法是一种更加科学可行的人牙髓细胞原代培养的优化方法,可为进行人牙髓干细胞的分离培养及特性研究提供实验方法学基础.

Comparing study on the effects of different methods in primary cultures of human dental pulp cells

Objective To compare the effects of different methods in primary cultures of human dental pulp cells in vitro.Methods The pulp explants were dissected out from healthy human dental pulp, cultured by the tissue explants method, tissue-explants collagenase/dispase digestion method and the combined use of explants technique and enzymatic separation method, respectively. The cell culture efficiency, cell vitality, cell morphology, and culture duration were observed and evaluated by phase-contract microscopy and trypan-blue dying methods.Results The primary dental pulp cells cultured by the tissue explants method could reach confluence after 4 weeks. The culture successfulness of dental pulp cells obtained by tissue-explants collagenase/dispase digestion method was only 20%. Compared with the two methods separately, the effective combined use of both methods could obviously promote culture efficiency, a larger number of human dental pulp cells with more vitality and more different morphologies could be obtained in a shorter time (5-7 days). Conclusion The established combined use of explants technique and enzymatic separation method is the optimal new method for the primary culture of human dental pulp cells in vitro, it may provide a methodological foundation for isolation and culture of stem cells from human dental pulp.