Isolation of mesenchymal stromal/stem cells from cryopreserved umbilical cord blood cells

Abstract

JC EH lin xp ematopathol Original Article INTRODUCTIONCell therapy using human mesenchymal stromal/stem cells (MSCs) has been extensively explored for the treatment of a variety of diseases based on their multiple biological and therapeutic effects.1 An off-the-shelf allogeneic bone marrow (BM)-derived MSC product is currently available in the clinic for the treatment of the intractable acute graft-versushost disease after hematopoietic stem cell transplantation (HSCT). 2,3 BM is the most commonly used source of MSCs. 4 However, invasive procedures are required to obtain BM cells from donors. Although other sources of MSCs that can be obtained without invasive treatments have been sought, including expulsed placenta and exfoliated teeth, complex procedures with enzymatic digestion are necessary to prepare cells from these tissues. 5,6 The demand for allogeneic MSCs is expected to increase in the future; therefore, appropriate sources of MSCs that can be alternatives to BM need to be explored for the further development of MSCbased therapy.In a previous study, we demonstrated that MSCs can be isolated from freshly donated umbilical cord blood (UCB) units that do not qualify for the Japanese banking system as a hematopoietic stem cell (HSC) source because of their small volume.7 UCB has advantages over other tissues because it can be obtained from donors without invasive procedures, and a simple adhesion method without complex procedures can be used to isolate MSCs. In addition, infrastructure for processing and cryopreserving UCB cells is established in the banking system for HSCT in Japan. More importantly, about 90% of donated UCB units are discarded because they do not meet the requirements for an HSC source in terms of volume and/or cell number. 8 We have further extended our investigation of the availability of such UCB units as a Umbilical cord blood (UCB) has advantages over other tissues because it can be obtained without an invasive procedure and complex processing. We explored the availability of cryopreserved UCB cells as a source of mesenchymal stromal/stem cells (MSCs). MSCs were successfully isolated from six of 30 UCB units (median volume, 34.0 mL; median nucleated cell number, 4.4×10 8 ) that were processed and cryopreserved using CP-1/human serum albumin. This isolation rate was lower than that (57%) from non-cryopreserved UCB cells. The number of nucleated cells before and after hydroxyethyl starch separation, UCB unit volume, and cell viability after thawing did not significantly differ between UCB units from which MSCs were successfully isolated and those from which they were not. When CryoSure-DEX40 was used as a cryoprotectant, MSCs were isolated from two of ten UCB units. Logistic regression analysis demonstrated that the cryopreservation method was not significantly associated with the success of MSC isolation. The isolated MSCs had a similar morphology and surface marker expression profile as bone marrow-derived MSCs and were able to differentiate into osteogenic, adipogenic, and chondro...

Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stem Cells from Sheep: Culture Characteristics

“…The transportation and maintenance of the samples at lower temperature for around 30–45 min might have affected the tissue samples. Some of the studies have been able to demonstrate isolation of MSCs from cryopreserved foetal membranes and umbilical cord blood, although with limited success [ 24 , 25 , 26 ]. The success rate of MSCs’ isolation from umbilical cord blood had been <60% and a delay in achieving the culture expansion has been demonstrated [ 24 , 25 ].…”

Success rates in isolating mesenchymal stem cells from permanent and deciduous teeth

“…The success rate of BMMSCs and ADSCs was reported to be 100% 22 , whereas the success rate of UCB-MSCs is only 29%. However, this could be increased by 63% by precoating the culture dish with foetal calf serum taking into account only units of optical quality 23 . The success rate of MSC isolation from cryopreserved UCB is below 57% 24 .…”

A simple, efficient and economical method for isolating and culturing human umbilical cord blood‑derived mesenchymal stromal cells

“…The medium is an important factor in cultivating hucB-MScs. Most researchers have adopted dMeM (low or high glucose) supplemented with 5-20% FBS, though the success rate is low (17,18,26). Bieback et al (19) used Mesencult™ medium to improve culture results.…”

“…Previously, hucB-MScs were cultured in media including dMeM/F12, dMeM, and α-MeM (17,18). Some methods added supplements including hydrocortisone, granulocyte-macrophage colony stimulating factor and insulin, while others used only Mesencult™ medium (19,20).…”