Human umbilical cord blood plasma as an alternative to animal sera for mesenchymal stromal cells in vitro expansion – A multicomponent metabolomic analysis

Abstract

Mesenchymal Stromal cells (MSCs) have a potential role in cell-based therapies. Foetal bovine serum (FBS) is used to supplement the basal cell culture medium but presents several disadvantages and risks. Other alternatives have been studied, including human umbilical cord blood plasma (hUCBP), aiming at the development of xeno-free culturing protocols. A comparative characterization of multicomponent metabolic composition of hUCBP and commercial FBS based on Nuclear Magnetic Resonance (NMR) spectroscopy and multivariate statistical analysis was performed. The analysis of 1H-NMR spectra revealed both similarities and differences between the two proposed supplements. Similar metabolites (amino acids, glucose, lipids and nucleotides) were found in the hUCBP and FBS NMR spectra. The results show that the major difference between the metabolic profiles of the two proposed supplements are due to the significantly higher levels of glucose and lower levels of lactate, glutamate, alanine and branched chain amino acids in hUCBP. Similar or slightly different levels of important proteinogenic amino acids, as well as of nucleotides, lipids were found in the hUCBP and FBS. In order to validate it’s suitability for cell culture, umbilical cord-MSCs (UC-MSCs) and dental pulp stem cells (DPSCs) were expanded using hUCBP. In both hMSCs, in vitro culture with hUCBP supplementation presented similar to improved metabolic performances when compared to FBS. The two cell types tested expressed different optimum hUCBP percentage content. For DPSCs, the optimum hUCBP content was 6% and for UC-MSCs, 4%. Cultured hMSCs displayed no changes in senescence indicators, as well as maintained characteristic surface marker’s expression. FBS substitution was associated with an increase in early apoptosis events, in a dose dependent manner, as well as to slight up- and down-regulation of targeted gene’s expression. Tri-lineage differentiation capacity was also influenced by the substitution of FBS by hUCBP.

Mesenchymal Stem/ Stromal Cells metabolomic and bioactive factors profiles: A comparative analysis on the umbilical cord and dental pulp derived Stem/ Stromal Cells secretome

“…The surface marker profiles of UC-MSCs and DPSCs were confirmed though Flow Cytometry, with anti-positive (CD90, CD105, CD44) and negative marker (CD34, CD11b, CD19, CD45, MHC class II) antibodies (Human MSC Analysis Kit, 562245, BD Biosciences), as described in detail in [43]. Data was acquired using BD FACSCalibur ™ 3 CA Becton Dickinson, BD Biosciences, and data was processed using FlowJo Engine X10.4 (v3.05478, LLC).…”

Derivation of clinical-grade mesenchymal stromal cells from umbilical cord under chemically defined culture condition – platform for future clinical application

“…To reduce these risks, efforts are devoted toward the development of human supplements including human serum [10], platelet lysate [11], and cord blood plasma [12]. However, the usage of humansourced supplements is quite controversial, mainly because of their limited availability.…”

Dental pulp stem cells and Bonelike® for bone regeneration in ovine model

“…Furthermore, hDPSCs have been demonstrated to secrete an extensive array of bioactive factors (growth factors and cytokines) [30] that modulate the activity of native cells and inflammatory populations at lesion sites and therefore unleash the regenerative response.…”

“…Surface marker profiles of hDPSCs were assessed by flow cytometry. hDPSCs populations were cultured for 5 days, as described previously [30], harvested using Accutase TM Cell detachment solution (561527, BD Biosciences © ), being counted and resuspended in Stain Buffer (554676, BD Biosciences © ). hDPSCs in Passage 5 and 7 (P5 and P7) were incubated with anti-positive (CD90, CD105 and CD44) and anti-negative marker (CD34, CD11b, CD19, CD45 and MHC Class II) antibodies and assayed as per manufacturer’s instructions (hMSC Analysis Kit, 562245, BD Biosciences © ), using a BD FACSCalibur™ 3 CA Becton Dickinson (BD Biosciences © ).…”