Adipose tissue is an attractive source of stem cells due to its wide availability. They contribute to the stromal vascular fraction (SVF), which is composed of pre-adipocytes, tissue-progenitors, and ...pericytes, among others. Because its direct use in medical applications is increasing worldwide, new quality control systems are required. We investigated the ability of the Non-Equilibrium Earth Gravity Assisted Dynamic Fractionation (NEEGA-DF) method to analyze and separate cells based solely on their physical characteristics, resulting in a fingerprint of the biological sample. Adipose tissue was enzymatically digested, and the SVF was analyzed by NEEGA-DF. Based on the fractogram (the UV signal of eluting cells versus time of analysis) the collection time was set to sort alive cells. The collected cells (F-SVF) were analyzed for their phenotype, immunomodulation ability, and differentiation potential. The SVF profile showed reproducibility, and the alive cells were collected. The F-SVF showed intact adhesion phenotype, proliferation, and differentiation potential. The methodology allowed enrichment of the mesenchymal component with a higher expression of mesenchymal markers and depletion of debris, RBCs, and an extracellular matrix still present in the digestive product. Moreover, cells eluting in the last minutes showed higher circularity and lower area, proving the principles of enrichment of a more homogenous cell population with better characteristics. We proved the NEEGA-DF method is a "gentle" cell sorter that purifies primary cells obtained by enzymatic digestion and does not alter any stem cell function.
Advanced therapy medicinal products (ATMP) are complex medicines based on gene therapy, somatic cell therapy, and tissue engineering. These products are rapidly arising as novel and promising ...therapies for a wide range of different clinical applications. The process for the development of well-established ATMPs is challenging. Many issues must be considered from raw material, manufacturing, safety, and pricing to assure the quality of ATMPs and their implementation as innovative therapeutic tools. Among ATMPs, cell-based ATMPs are drugs altogether. As for standard drugs, technologies for quality control, and non-invasive isolation and production of cell-based ATMPs are then needed to ensure their rapidly expanding applications and ameliorate safety and standardization of cell production. In this review, emerging approaches and technologies for quality control of innovative cell-based ATMPs are described. Among new techniques, microfluid-based systems show advantages related to their miniaturization, easy implementation in analytical process and automation which allow for the standardization of the final product.
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•Advanced therapy medicinal products (ATMPSs) as new promising therapies.•Quality control (QC) in ATMPs product development.•Sterility, identity, purity, viability, potency, and reproducibility of ATMPs products.•Characterization of raw material, stability, and release of ATMPs products.•Microfluidic tools for the QC of ATMPs.•Label-free approaches for QC of ATMPs.•Integration of machine learning and AI tools to improve data management and development process of ATMPs.
Glucose transport activity and its possible regulation by reactive oxygen species in two Glut1-expressing megakaryocytic cell lines, MO7e and B1647, differing in cytokine sensitivity were compared. ...Results show that: (1) In MO7e cells, glucose transport rate increased in response to thrombopoietin, granulocyte–macrophage colony-stimulating factor, or stem cell factor, due to a decreased
K
m. (2) A higher
V
max value was determined in B1647 cells, owing to the relative higher abundance of Glut1 on the plasmalemma; in these cells no change in glucose transport rate was observed on cytokine treatment. (3) The basal level of intracellular ROS was higher in B1647 than in M07e cells, where ROS production was enhanced upon cytokine exposure. (4) Basal or stimulated ROS production and Glut1 activity were significantly reduced by pretreating both cell lines with EUK-134, a superoxide dismutase and catalase mimetic. (5) In MO7e cells, EUK-134 brought back to control levels the
K
m values obtained on cytokine treatment, whereas in B1647 cells the antioxidant drastically reduced
V
max by decreasing the Glut1 content of the plasma membrane. Our data suggest that differences in acute regulation of glucose transport activity in the two cell lines may be related to differences in amplitude and spatial organization of ROS production.
Bone marrow mesenchymal stem cells (MSCs) are currently being investigated in preclinical and clinical settings because of their multipotent differentiative capacity or, alternatively, their ...immunosuppressive function. The aim of this study was to evaluate dental pulp (DP) as a potential source of MSCs instead of bone marrow (BM).
Flow cytometric analysis showed that DP-MSCs and BM-MSCs were equally SH2, SH3, SH4, CD29 and CD 166 positive. The in vitro proliferative kinetics of MSCs were measured by 3H-thymidine incorporation uptake. The immunosuppressive function of MSCs was then tested by coculturing PHA-stimulated allogeneic T cells with or without MSCs for 3 days.
BM-MSCs could be differentiated in vitro into osteogenic, chondrogenic and adipogenic lineages. DP-MSCs showed osteogenic and adipocytic differentiation, but did not differentiate into chondrocytes. Although DP-MSCs grow rapidly in vitro between day 3 and day 8 of culture and then decrease their proliferation by day 15, BM-MSCs have a stable and continuous proliferation over the same period of time. The addition of DP-MSCs or BM-MSCs resulted in 91 +/- 4% and 75 +/- 3% inhibition of T cell response, respectively, assessed by a 3H-thymidine assay.
Dental pulp is an easily accessible and efficient source of MSCs, with different kinetics and differentiation potentialities from MSCs as isolated from the bone marrow. The rapid proliferative capacity together with the immunoregulatory characteristics of DP-MSCs may prompt future studies aimed at using these cells in the treatment or prevention of T-cell alloreactivity in hematopoietic or solid organ allogeneic transplantation.
Abstract
Background
New microfat preparations provide material suitable for use as a regenerative filler for different facial areas. To support the development of new robust techniques for ...regenerative purposes, the cellular content of the sample should be considered.
Objectives
To evaluate the stromal vascular fraction (SVF) cell components of micro-superficial enhanced fluid fat injection (SEFFI) samples via a technique to harvest re-injectable tissue with minimum manipulation. The results were compared to those obtained from SEFFI samples.
Methods
Microscopy analysis was performed to visualize the tissue structure. Micro-SEFFI samples were also fractionated using Celector,® an innovative non-invasive separation technique, to provide an initial evaluation of sample fluidity and composition. SVFs obtained from SEFFI and micro-SEFFI were studied. Adipose stromal cells (ASCs) were isolated and characterized by proliferation and differentiation capacity assays.
Results
Microscopic and quality analyses of micro-SEFFI samples by Celector® confirmed the high fluidity and sample cellular composition in terms of red blood cell contamination, the presence of cell aggregates, and extracellular matrix fragments. ASCs were isolated from adipose tissue harvested using SEFFI and micro-SEFFI systems. These cells were demonstrated to have a good proliferation rate and differentiation potential towards mesenchymal lineages.
Conclusions
Despite the small sizes and low cellularity observed in micro-SEFFI-derived tissue, we were able to isolate stem cells. This result partially explains the regenerative potential of autologous micro-SEFFI tissue grafts. In addition, using this novel Celector® technology, tissues used for aging treatment were characterized analytically, and the adipose tissue composition was evaluated with no need for extra sample processing.
Level of Evidence: 5
Term Amniotic membrane (AM) is a very attractive source of Mesenchymal Stem Cells (MSCs) due to the fact that this fetal tissue is usually discarded without ethical conflicts, leading to high ...efficiency in MSC recovery with no intrusive procedures. Here we confirmed that term AM, as previously reported in the literature, is an abundant source of hMSCs; in particular we further investigated the AM differentiation potential by assessing whether these cells may also be committed to the angiogenic fate. In agreement with the recommendation of the International Society for Cellular Therapy, the mesenchymal cells herein investigated were named Amniotic Membrane-human Mesenchymal Stromal Cells (AM-hMSC).
The recovery of hMSCs and their in vitro expansion potential were greater in amniotic membrane than in bone marrow stroma. At flow cytometry analysis AM-hMSCs showed an immunophenotypical profile, i.e., positive for CD105, CD73, CD29, CD44, CD166 and negative for CD14, CD34, CD45, consistent with that reported for bone marrow-derived MSCs. In addition, amniotic membrane-isolated cells underwent in vitro osteogenic (von Kossa stain), adipogenic (Oil Red-O stain), chondrogenic (collagen type II immunohistochemichal detection) and myogenic (RT-PCR MyoD and Myogenin expression as well as desmin immunohistochemical detection) differentiation. In angiogenic experiments, a spontaneous differentiation into endothelial cells was detected by in vitro matrigel assay and this behaviour has been enhanced through Vascular Endothelial Growth Factor (VEGF) induction. According to these findings, VEGF receptor 1 and 2 (FLT-1 and KDR) were basally expressed in AM-hMSCs and the expression of endothelial-specific markers like FLT-1 KDR, ICAM-1 increased after exposure to VEGF together with the occurrence of CD34 and von Willebrand Factor positive cells.
The current study suggests that AM-hMSCs may emerge as a remarkable tool for the cell therapy of multiple diseased tissues. AM-hMSCs may potentially assist both bone and cartilage repair, nevertheless, due to their angiogenic potential, they may also pave the way for novel approaches in the development of tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required.
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