BACKGROUND: 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).AIM: 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.RESULTS: 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.CONCLUSIONS: 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.