Optimization of human umbilical cord blood-derived mesenchymal stem cell isolation and culture methods in serum- and xeno-free conditions

Abstract

Background Although umbilical cord blood (UCB) is identified as a source of mesenchymal stem cells (MSCs) with various advantages, the success in cell isolation is volatile. Therefore, it is necessary to optimize methods of cord blood-derived MSC (UCB-MSC) isolation and culture. In this study, we evaluated the efficiency of UCB-MSC isolation and expansion using different commercially available serum- and xeno-free media and investigated the capacity of autologous serum and plasma as a supplement to support cell proliferation. Additionally, we defined the presence of multilineage-differentiating stress-enduring (Muse) cells in the UCB-MSC population. Functions of UCB-MSC in in vitro angiogenesis processes and anti-cancer were also verified. Methods Mononuclear cells were isolated using density gradient separation and cultured in four commercial media kits, as well as four surface coating solutions. UCB-MSCs were characterized and tested on tube formation assay, and co-cultured with SK-MEL cells in a transwell system. Results The results showed that only StemMACS™ MSC Expansion Media is more appropriate to isolate and culture UCB-MSCs. The cells exhibited a high cell proliferation rate, CFU forming capability, MSC surface marker expression, trilineage differentiate potential, and chromosome stability. In addition, the culture conditions with autologous serum coating and autologous plasma supplement enhanced cell growth and colony forming. This cell population contained Muse cells at rate of 0.3%. Moreover, UCB-MSCs could induce the tube formation of human umbilical vein endothelial cells and inhibit more than 50% of SK-MEL cell growth. Conclusions UCB-MSCs could be high-yield isolated and expanded under serum- and xeno-free conditions by using the StemMACS™ MSC Expansion Media kit. Autologous serum coating and plasma supplement enhanced cell proliferation. These UCB-MSCs had effected the tube formation process and an anti-cancer impact.

Manufacture and Quality Control of Human Umbilical Cord-Derived Mesenchymal Stem Cell Sheets for Clinical Use

“…Ideally, the culture medium in the whole process of cell sheet manufacture, including cell culture, should be chemically defined and not contain animal extracts, research-grade human extracts, or research-grade exogenous growth factors. Although several serum-free media have been investigated for human mesenchymal stem cell (MSC) expansion [ 21 , 22 , 23 , 24 ], ingredients of these mediums could not be risk-evaluated and controlled in final products due to their confidentiality, which does not meet the GMP guidelines and drug standards. In addition, these media contain a variety of research-grade exogenous growth factors or human/animal extracts, which would also cause safety risks for clinical applications.…”

Advance of Mesenchymal Stem Cells in Chronic End-Stage Liver Disease Control

“…The immunophenotypes of MSCs make it possible to be transplanted in an autologous or an allogeneic way, which broadens clinical applications. However, there are differences in the surface markers of MSCs due to the MSC sources [12], donor age [13], isolation methods [14], and culture conditions [15]. In vitro, MSCs of different origins differ in their ability to expand.…”

Human Mesenchymal Stromal Cells Derived from Perinatal Tissues: Sources, Characteristics and Isolation Methods

“…Due to the low recovery of MSCs circulate in UCB, it is difficult to isolate and culture them compared to haematopoietic stem cells. Besides that, the yield is also inversely affected by gestation age ( 38 ). UCB-MSCs also underwent senescence earlier than other PL-MSCs.…”

“…One of the major setbacks contributing to the wide-ranging success rate between 10% and 90% in culturing UCB-MSCs is inconsistency in the isolation process ( 38 , 42 ). Many researchers have collectively shown that sample volume, cellular content and processing duration after CB collection play an important role in determining the success rate ( 42 , 43 ).…”