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.

Comparison of Preterm and Term Wharton’s Jelly-Derived Mesenchymal Stem Cell Properties in Different Oxygen Tensions

“…Cells were isolated from the umbilical cord Wharton’s jelly using an enzyme digestion method [Bruyn et al, 2011; Azandeh et al, 2012; Rostamzadeh et al, 2015]. The enzymatic method for extraction of cells was chosen over explant technique for several reasons, including: high popularity of this method among other researchers, concern of umbilical tissue detaching and thereby reducing cell migration onto culture dish, potentially higher risk for plate contamination from intact tissue pieces, cell yield is comparable, and excellent success with this method in our laboratory [Hua et al, 2014; Arutyunyan et al, 2016a; Salehinejad et al, 2012; Han et al, 2013].…”

Resveratrol promotes secretion of wound healing related growth factors of mesenchymal stem cells originated from adult and fetal tissues

“…Umbilical cord in 3 cm length and amnion membrane were collected after the mothers have consented. Wharton's jelly MSCs and AM-MSCs were isolated using mixed enzymatic-explant method based on protocol developed by Azendeh et al [15] with our modification. Briefly, blood vessels were removed to allow only the WJ and AM to come into direct contact with the enzymatic solution.…”

Therapeutic potential of mesenchymal stem cells for diabetes

“…The supernatant is removed and the cells are plated on a tissue culture dish with stem cell media. Collagenase, hyaluronidase, trypsin, and dispase are examples of enzymes used to dissociate WJ-MSCs from the matrix (Bruyn et al 2011; Azandeh et al 2012; Rostamzadeh et al 2015). …”