Good manufacturing practice-compliant isolation and culture of human umbilical cord blood-derived mesenchymal stem cells

Abstract

BackgroundMesenchymal stem cells (MSCs) are an attractive source of stem cells for clinical applications. These cells exhibit a multilineage differentiation potential and strong capacity for immune modulation. Thus, MSCs are widely used in cell therapy, tissue engineering, and immunotherapy. Because of important advantages, umbilical cord blood-derived MSCs (UCB-MSCs) have attracted interest for some time. However, the applications of UCB-MSCs are limited by the small number of recoverable UCB-MSCs and fetal bovine serum (FBS)-dependent expansion methods. Hence, this study aimed to establish a xenogenic and allogeneic supplement-free expansion protocol.MethodsUCB was collected to prepare activated platelet-rich plasma (aPRP) and mononuclear cells (MNCs). aPRP was applied as a supplement in Iscove modified Dulbecco medium (IMDM) together with antibiotics. MNCs were cultured in complete IMDM with four concentrations of aPRP (2, 5, 7, or 10%) or 10% FBS as the control. The efficiency of the protocols was evaluated in terms of the number of adherent cells and their expansion, the percentage of successfully isolated cells in the primary culture, surface marker expression, and in vitro differentiation potential following expansion.ResultsThe results showed that primary cultures with complete medium containing 10% aPRP exhibited the highest success, whereas expansion in complete medium containing 5% aPRP was suitable. UCB-MSCs isolated using this protocol maintained their immunophenotypes, multilineage differentiation potential, and did not form tumors when injected at a high dose into athymic nude mice.ConclusionThis technique provides a method to obtain UCB-MSCs compliant with good manufacturing practices for clinical application.

Good manufacturing practice-compliant isolation and culture of human adipose derived stem cells

“…Moreover, PRP reduced the ADSCs doubling time, and increased the slope value for cell proliferation. Similar effects were also observed in previous studies on ADSC culture (Kocaoemer et al, 2007;Koellensperger et al, 2014;Van Pham et al, 2013a) and in other MSC culture (Iudicone et al, 2014;Pham et al, 2014). The increased proliferation rate is thought to originate from possible combinatorial effects of the growth factor pool within PRP.…”

Venous Blood Derivatives as FBS-Substitutes for Mesenchymal Stem Cells: A Systematic Scoping Review

“…While the natural-formed blood clot contains 95% of red blood cells, 5% platelets, less than 1% white blood cells and fibrin strands, PRP holds 4% red blood cells, 95% platelets and 1% white blood cells (28). PRP should be chemically activated by the addition of human/bovine thrombin or Calcium Chloride -CaCl 2 , affording activated PRP -aPRP (24,25,29). To obtain PRP, whole anticoagulated blood, must be submitted to a double-centrifugation; the first one (soft spin) results in a three-layer suspension where the red blood cells are found at the bottommost layer.…”

“…Then, superficial layer is discarded and the remaining material (PRP) is shaken. To release its platelet content, PRP must be activated (aPRP) by the addition of human/bovine thrombin or Calcium Chloride (CaCl 2 ) (24,25). Human Platelet Lysate (HPL) results from a lysis of high platelet concentrate (traditionally the PRP), being the platelet mechanical lysis induced by susceptive freeze-thaw cycles at -80 or -20 °C (typically 2 or 3 cycles).…”

“…In contrast, umbilical cord blood is more difficult to obtain donors and provide few volume of blood available (12,(21)(22)(23). Therefore, VBD presetting an important source easily accessed to clinicians providing a potential xeno-free supplementation for MSC expansion (24,25). In addition, a wide range of different serum/plasma blood derivatives has been reported as FBS substitutes showing promising results.…”

Induced Pluripotent and Mesenchymal Stem Cells as a Promising Tool for Articular Cartilage Regeneration

“…More precisely, products in compliance with GMP should include the highest standards of sterility, quality control, and documentation, following a standard operating procedure (SOP), at each phase of production from cell isolation to freezing and storage [78,79]. The human pluripotent stem cell-derived products must be developed under well-defined conditions, ensuring the maintenance of pluripotency and/or differentiation of the original stem cells [80].…”