Advanced Therapy Medicinal Products as Potential Therapeutic Strategy against COVID-19 and Immune-Related Disorders

Advanced Therapy Medicinal Products (ATMPs) comprise a heterogenous class of innovative medicinal products, which further require extensive preclinical and clinical assessments before their broader use in the general population [...

Optimization of Decellularization Procedure in Rat Esophagus for Possible Development of a Tissue Engineered Construct

Background: Current esophageal treatment is associated with significant morbidity. The gold standard therapeutic strategies are stomach interposition or autografts derived from the jejunum and colon. However, severe adverse reactions, such as esophageal leakage, stenosis and infection, accompany the above treatments, which, most times, are life threating. The aim of this study was the optimization of a decellularization protocol in order to develop a proper esophageal tissue engineered construct. Methods: Rat esophagi were obtained from animals and were decellularized. The decellularization process involved the use of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) buffers for 6 h each, followed by incubation in a serum medium. The whole process involved two decellularization cycles. Then, a histological analysis was performed. In addition, the amounts of collagen, sulphated glycosaminoglycans and DNA content were quantified. Results: The histological analysis revealed that only the first decellularization cycle was enough to produce a cellular and nuclei free esophageal scaffold with a proper extracellular matrix orientation. These results were further confirmed by biochemical quantification. Conclusions: Based on the above results, the current decellularization protocol can be applied successfully in order to produce an esophageal tissue engineered construct

Matrix-Induced Adipose-Derived Mesenchymal Stem Cells Implantation for Knee Articular Cartilage Repair. Two Years Follow-up.

Objective. The aim of this study was to evaluate the efficacy of cartilage repair using matrix-induced adipose-derived mesenchymal stem cells (MSCs) for focal chondral knee lesions.Materials and Methods. Twenty consecutive patients treated for symptomatic full-thickness chondral defects were prospectively followed for two years. All patients underwent a single-stage procedure consisting in filling each defect with autologous culture-expanded mesenchymal stem cells embedded in a trimmed-to-fit commercially available biodegradable matrix. Knee-related function was evaluated based on subjective scores given by two self-reported questionnaires (KOOS and IKDC).  Results. The data analysis recorded significant improvements (p<0.001) in all the values. The mean preoperative scores in the subscales of KOOS, as well as in the IKDC subjective score were constantly increased during the follow-up period with statistical significant differences at 6, 12 and 24 months review.Conclusions. Matrix-induced adipose-derived mesenchymal stem cells implantation is an efficient and safe single-staged cell-based procedure to manage full-thickness focal chondral lesions of the knee. 

Improved Repopulation Efficacy of Decellularized Small Diameter Vascular Grafts Utilizing the Cord Blood Platelet Lysate

Background: The development of functional bioengineered small-diameter vascular grafts (SDVGs), represents a major challenge of tissue engineering. This study aimed to evaluate the repopulation efficacy of biological vessels, utilizing the cord blood platelet lysate (CBPL). Methods: Human umbilical arteries (hUAs, n = 10) were submitted to decellularization. Then, an evaluation of decellularized hUAs, involving histological, biochemical and biomechanical analysis, was performed. Wharton’s Jelly (WJ) Mesenchymal Stromal Cells (MSCs) were isolated and characterized for their properties. Then, WJ-MSCs (1.5 × 106 cells) were seeded on decellularized hUAs (n = 5) and cultivated with (Group A) or without the presence of the CBPL, (Group B) for 30 days. Histological analysis involving immunohistochemistry (against Ki67, for determination of cell proliferation) and indirect immunofluorescence (against activated MAP kinase, additional marker for cell growth and proliferation) was performed. Results: The decellularized hUAs retained their initial vessel’s properties, in terms of key-specific proteins, the biochemical and biomechanical characteristics were preserved. The evaluation of the repopulation process indicated a more uniform distribution of WJ-MSCs in group A compared to group B. The repopulated vascular grafts of group B were characterized by greater Ki67 and MAP kinase expression compared to group A. Conclusion: The results of this study indicated that the CBPL may improve the repopulation efficacy, thus bringing the biological SDVGs one step closer to clinical application

Evaluation of HLA-G Expression in Multipotent Mesenchymal Stromal Cells Derived from Vitrified Wharton’s Jelly Tissue

Background: Mesenchymal Stromal Cells (MSCs) from Wharton’s Jelly (WJ) tissue express HLA-G, a molecule which exerts several immunological properties. This study aimed at the evaluation of HLA-G expression in MSCs derived from vitrified WJ tissue. Methods: WJ tissue samples were isolated from human umbilical cords, vitrified with the use of VS55 solution and stored for 1 year at −196 °C. After 1 year of storage, the WJ tissue was thawed and MSCs were isolated. Then, MSCs were expanded until reaching passage 8, followed by estimation of cell number, cell doubling time (CDT), population doubling (PD) and cell viability. In addition, multilineage differentiation, Colony-Forming Units (CFUs) assay and immunophenotypic analyses were performed. HLA-G expression in MSCs derived from vitrified samples was evaluated by immunohistochemistry, RT-PCR/PCR, mixed lymphocyte reaction (MLR) and immunofluorescence. MSCs derived from non-vitrified WJ tissue were used in order to validate the results obtained from the above methods. Results: MSCs were successfully obtained from vitrified WJ tissues retaining their morphological and multilineage differentiation properties. Furthermore, MSCs from vitrified WJ tissues successfully expressed HLA-G. Conclusion: The above results indicated the successful expression of HLA-G by MSCs from vitrified WJ tissues, thus making them ideal candidates for immunomodulation

Evaluation of the Regenerative Potential of Platelet-Lysate and Platelet-Poor Plasma Derived from the Cord Blood Units in Corneal Wound Healing Applications: An In Vitro Comparative Study on Corneal Epithelial Cells

Background: Cord blood platelet lysate (CB-PL) and cord blood platelet poor plasma (CB-PPP) have been applied with success in wound healing applications. Pathologies such as Sjogrens’s Syndrome (SS) and chronic graft versus host disease (cGVHD) can lead to severe ophthalmology issues. The application of CB-PL and CB-PPP may be strongly considered for damaged cornea healing. This study aimed to the evaluation of the beneficial properties of CB-PL and CB-PPP in corneal wound healing applications. Methods: Initially, the CB-PL and CB-PPP were produced from donated cord blood units (CBUs), followed by biochemical analysis. Corneal epithelial cells (CECs) were isolated from wistar rats and then cultured with medium containing 20% v/v either of CB-PL or CB-PPP. To define the impact of CB-PL and CB-PPP, biochemical, morphological analysis, scratch-wound assays, and immunoassays in CECs were performed. Results: CB-PL and CB-PPP were characterized by good biochemical parameters, regarding their quality characteristics and biomolecule content. CECs’ morphological features did not change after their cultivation with CB-PL or CB-PPP. A scratch wound assay and molecular analysis of CECs expanded with CB-PL indicated higher migratory capacity compared to those cultured with CB-PPP. Conclusion: CB-PL and CB-PPP exhibited good properties with respect to cell migration and proliferation, and could be considered an alternative source for eye drop production, to possibly be used in cornea wound healing applications

Evaluation of Peripheral Blood and Cord Blood Platelet Lysates in Isolation and Expansion of Multipotent Mesenchymal Stromal Cells

Background: Multipotent Mesenchymal Stromal Cells (MSCs) are used in tissue engineering and regenerative medicine. The in vitro isolation and expansion of MSCs involve the use of foetal bovine serum (FBS). However, many concerns have been raised regarding the safety of this product. In this study, alternative additives derived either from peripheral or cord blood were tested as an FBS replacement. Methods: Platelet lysates (PL) from peripheral and cord blood were used for the expansion of MSCs. The levels of growth factors in peripheral blood (PB) and cord blood (CB) PLs were determined using the Multiple Reaction Monitoring (MRM). Finally, the cell doubling time (CDT), tri-lineage differentiation and phenotypic characterization of the MSCs expanded with FBS and PLs were determined. Results: MSCs treated with culture media containing FBS and PB-PL, were successfully isolated and expanded, whereas MSCs treated with CB-PL could not be maintained in culture. Furthermore, the MRM analysis yielded differences in growth factor levels between PB-PL and CB-PL. In addition, the MSCs were successfully expanded with FBS and PB-PL and exhibited tri-lineage differentiation and stable phenotypic characteristics. Conclusion: PB-PL could be used as an alternative additive for the production of MSCs culture medium applied to xenogeneic-free expansion and maintenance of MSCs in large scale clinical studies

Successful short-term cryopreservation of volume-reduced cord blood units in a cryogenic mechanical freezer: effects on cell recovery, viability, and clonogenic potential

BackgroundCord blood (CB) units are stored from weeks to years in liquid- or vapor-phase nitrogen until they are used for transplantation. We examined the effects of cryostorage in a mechanical freezer at -150 degrees C on critical quality control variables of CB collections to investigate the possible use of mechanical freezers at -150 degrees C as an alternative to storage in liquid- (or vapor-) phase nitrogen. Study Design and MethodsA total of 105 CB units were thawed and washed at different time intervals (6, 12, 24, and 36 months). For every thawed CB unit, samples were removed and cell enumeration (total nucleated cells [TNCs], mononuclear cells [MNCs], CD34+, CD133+) was performed. In addition, viability was obtained with the use of flow cytometry, and recoveries were calculated. Also, total absolute colony-forming unit counts were performed and progenitor cell recoveries were studied by clonogenic assays. ResultsSignificant differences (p<0.05) were observed in certain variables (TNCs, MNC numbers, viability) when they were examined in relation with time intervals, while others (CD34+, CD133+) were relatively insensitive (p=NS) to the duration of time interval the CB units were kept in cryostorage condition. ConclusionsThe data presented suggest that cryopreservation of CB units in a mechanical freezer at -150 degrees C may represent an alternative cryostorage condition for CB cryopreservation

Selection Criteria of Cord Blood Units for Platelet Gel Production: Proposed Directions from Hellenic Cord Blood Bank. Comment on Mallis et al. Short Term Results of Fibrin Gel Obtained from Cord Blood Units: A Preliminary in Vitro Study. Bioengineering 2019, 6, 66

This article provides additional knowledge for cord blood platelet gel (CBPG) production. Recently, it has been shown that CBPG exerts beneficial properties in wound healing applications. CBPG is produced after a two-step centrifugation process, following the addition of calcium gluconate. Clinical-grade CBPG can be produced in public cord blood banks, worldwide. However, standardization of the CBPG production process must be established in order to reduce discrepancies that occurred due to different platelet gel preparations. This article aims to provide an update regarding the selection criteria of cord blood units (CBUs), and to provide evidence for the improvement of the CBPG production process. (Comment on “Short Term Results of Fibrin Gel Obtained from Cord Blood Units: A Preliminary in Vitro Study” Bioengineering 2019, 6, 66)