Mixed enzymatic-explant protocol for isolation of mesenchymal stem cells from Wharton’s jelly and encapsulation in 3D culture system

Abstract

We report combination of explants and enzymatic protocol as mixed enzymatic-explant procedure to faster extraction of MSCs from WJ. Umbilical cords (UC) were collected from Imam Khomini Hospital. For explant outgrowth, 6 - 9 pieces of WJ were transferred onto tissue culture flask and waited for attachment. For mixed enzymatic-explant, 1 cm 3 pieces WJ were placed in enzymatic cocktail comprising 4 mg/ml Collagenase Type I and 1 mg/ml Hyaluronidase and 0.1% trypsin-EDTA. Then isolated cells were analyzed for surface cell markers such as CD73, CD31. Isolated 1.0 × 10 6 MSCs/ml were encapsulated in alginate hydrogel. Cells with MSCs phenotype were isolated from mixed enzymatic-explant and explant procedures within 24 - 48 hrs and 7 - 10 days, respectively. Both of procedures were shown to form clumps and colonies with dense centers. Phenotypic changes gradually appeared as round cell in UC pieces into homogeneous spindle-shaped and typical fibroblast-like shape cells. By using flow cytometery MSCs showed positive for CD73, and negative for CD31. the morphology of viable MSCs in the beads did not significantly show a different morphology pattern before and after the bead formation process. These findings are indicated that when mixed enzymatic-explant procedure is performed MSCs can be isolated faster and much higher from WJ. These finding is important in comparing with time consuming explants culture for isolation of MSCs.