Tsagias N, Koliakos I, Karagiannis V, Eleftheriadou M, Koliakos G G
3rd University Obstetrics and Gynaecology Clinic, Ippokration General Hospital, Medical School, Aristotle University, Thessaloniki, Greece.
Transfus Med. 2011 Aug;21(4):253-61. doi: 10.1111/j.1365-3148.2011.01076.x. Epub 2011 May 30.
Umbilical cord (UC) mesenchymal cells have the ability to differentiate into various cell types, which make them an easily obtainable source for therapeutic uses. Different approaches have been used to isolate mesenchymal stem cells (MSC).
Here, we report a detailed enzymatic method where large number of cells can be efficiently isolated from the cord matrix and cryopreserved on the same day of arrival at the laboratory.
METHODS/MATERIALS: Cells were successfully isolated from 12 samples, with a new procedure that uses the total length of the UC. MSC have been isolated using a detailed enzymatic method with collagenase and hyaluronidase followed by trypsin, without removing the vessels and without mincing the cord. Stem cells were measured with flow cytometry before cryopreservation and post-thaw. Cultured cells were assessed for MSC marker expression and adherence plasticity for three passages. Multilineage differentiation was performed.
Nucleated cell yield was calculated at 0·95 × 10(6) /cm. MSC yield was calculated at 0·65 × 10(6) /cm of cord with flow cytometry while the mean length was 31 cm. Cultured cells expressed the mesenchymal markers CD29, CD90, CD105 and CD44. Mesenchymal marker expression remained intact over the three passages and post-thaw. Osteogenic and adipogenic differentiation was evaluated.
Our findings provide a fast and efficient method for mesenchymal cell isolation from Wharton's jelly using the total length of the UC. This method resulted in a large number of cells while the cells retained their mesenchymal character after thawing. This method can be easily applied, along with UC blood, for UC banking.
脐带间充质细胞具有分化为多种细胞类型的能力，这使其成为易于获取的治疗用途细胞来源。已采用不同方法分离间充质干细胞（MSC）。
在此，我们报告一种详细的酶法，可从脐带基质中高效分离出大量细胞，并在抵达实验室当天进行冷冻保存。
方法/材料：采用一种使用脐带全长的新程序，成功从12个样本中分离出细胞。使用胶原酶和透明质酸酶，随后用胰蛋白酶的详细酶法分离MSC，无需去除血管且无需切碎脐带。在冷冻保存前和解冻后用流式细胞术检测干细胞。对培养的细胞进行三代传代培养，评估其MSC标志物表达和黏附可塑性。进行多向分化。
有核细胞产量计算为0.95×10⁶/cm。用流式细胞术计算MSC产量为0.65×10⁶/cm脐带，平均长度为31 cm。培养的细胞表达间充质标志物CD29、CD90、CD105和CD44。间充质标志物表达在三代传代培养及解冻后保持完整。评估了成骨和成脂分化。
我们的研究结果提供了一种从华通氏胶中使用脐带全长快速高效分离间充质细胞的方法。该方法可获得大量细胞，且细胞解冻后仍保留其间充质特性。该方法可与脐带血一起轻松应用于脐带储存。
