Standardized xeno- and serum-free culture platform enables large-scale expansion of high-quality mesenchymal stem/stromal cells from perinatal and adult tissue sources

Background aimsMesenchymal stem/stromal cells (MSCs) are of interest for the treatment of graft-versus-host disease, autoimmune diseases, osteoarthritis and neurological and cardiovascular diseases. Increasing numbers of clinical trials emphasize the need for standardized manufacturing of these cells. However, many challenges related to diverse isolation and expansion protocols and differences in cell tissue sources exist. As a result, the cell products used in numerous trials vary greatly in characteristics and potency.MethodsThe authors have established a standardized culture platform using xeno- and serum-free commercial media for expansion of MSCs derived from umbilical cord (UC), bone marrow and adipose-derived (AD) and examined their functional characteristics.ResultsMSCs from the tested sources stably expanded in vitro and retained their biomarker expression and normal karyotype at early and later passages and after cryopreservation. MSCs were capable of colony formation and successfully differentiated into osteogenic, adipogenic and chondrogenic lineages. Pilot expansion of UC-MSCs and AD-MSCs to clinical scale revealed that the cells met the required quality standard for therapeutic applications.ConclusionsThe authors’ data suggest that xeno- and serum-free culture conditions are suitable for large-scale expansion and enable comparative study of MSCs of different origins. This is of importance for therapeutic purposes, especially because of the numerous variations in pre-clinical and clinical protocols for MSC-based products.     

IMMOBILIZATION OF BOVINE CATALASE ONTO MAGNETIC NANOPARTICLES

The scope of this study is to achieve carrier-bound immobilization of catalase onto magnetic particles (Fe₃O₄ and Fe₂O₃NiO₂ · H₂O) to specify the optimum conditions of immobilization. Removal of H₂O₂ and the properties of immobilized sets were also investigated. To that end, adsorption and then cross-linking methods onto magnetic particles were performed. The optimum immobilization conditions were found for catalase: immobilization time (15 min for Fe₃O₄; 10 min for Fe₂O₃NiO₂ · H₂O), the initial enzyme concentration (1 mg/mL), amount of magnetic particles (25 mg), and glutaraldehyde concentration (3%). The activity reaction conditions (optimum temperature, optimum pH, pH stability, thermal stability, operational stability, and reusability) were characterized. Also kinetic parameters were calculated by Lineweaver–Burk plots. The optimum pH values were found to be 7.0, 7.0, and 8.0 for free enzyme, Fe₃O₄-immobilized catalases, and Fe₂O₃NiO₂ · H₂O-immobilized catalases, respectively. All immobilized catalase systems displayed the optimum temperature between 25 and 35°C. Reusability studies showed that Fe₃O₄-immobilized catalase can be used 11 times with 50% loss in original activity, while Fe₂O₃NiO₂ · H₂O-immobilized catalase lost 67% of activity after the same number of uses. Furthermore, immobilized catalase systems exhibited improved thermal and pH stability. The results transparently indicate that it is possible to have binding between enzyme and magnetic nanoparticles.     

Synthesis and evaluation of N-substituted indole-3-carboxamide derivatives as inhibitors of lipid peroxidation and superoxide anion formation

The in vitro antioxidant effects of novel N-substituted indole-3-carboxamides (I3CDs) 1-10 on rat liver microsomal NADPH-dependent lipid peroxidation (LP) levels and their free radicals scavenging properties were determined by the inhibition of superoxide anion formation (SOD). Among the synthesized compounds, 4, 5, 8 and 9 significantly inhibited SOD with an inhibition range at 84–100% at 10^{? 3} M concentration. The presence of halo substituents both ortho- and para- positions of these compounds resulted 100% inhibition of SOD. Comparison the activity results of halogenated and non-halogenated derivatives suggested that the halogenated compounds are more active than the non-halogenated compounds. On the other hand, the introduction of a para fluoro benzyl in the 1-position of indole (compounds 7, 8) has more impact on the SOD inhibition when the benzamide ring was mono halogenated. However, none of other compounds had a significant inhibitory effects on the level of lipid peroxidation.     

Investigation of the in vitro antioxidant behaviour of some 2-phenylindole derivatives: discussion on possible antioxidant mechanisms and comparison with melatonin

Oxidative stress has been implicated in the development of many neurodegenerative diseases and also responsible from aging and some cancer types. Indolic compounds are a broad family of substances present in microorganisms, plants and animals. They are mainly related to tryptophan metabolism, and present particular properties that depend on their respective chemical structures. Due to free radical scavenger and antioxidant properties of indolic derivatives such as indolinic nitroxides and melatonin, a series of 2-phenyl indole derivatives were prepared and their in vitro effects on rat liver lipid peroxidation levels, superoxide formation and DPPH stable radical scavenging activities were determined against melatonin, BHT and α-tocopherol. The compounds significantly inhibited (72–98%) lipid peroxidation at 10^{? 3} M. These values were similar to that observed with BHT (88%). Possible structure–activity relationships of the compounds were discussed.     

Wee1 kinase as a target for cancer therapy

Wee1, a protein kinase, regulates the G₂ checkpoint in response to DNA damage. Preclinical studies have elucidated the role of wee1 in DNA damage repair and the stabilization of replication forks, supporting the validity of wee1 inhibition as a viable therapeutic target in cancer. MK-1775, a selective and potent small-molecule inhibitor of wee1, is under clinical development as a potentiator of DNA damage caused by cytotoxic chemotherapies. We present a review of the role of wee1 in the cell cycle and DNA replication and summarize the clinical development to date of this novel class of anticancer agents.     

Comparative Characteristics of Porous Bioceramics for an Osteogenic Response In Vitro and In Vivo

Porous calcium phosphate ceramics are used in orthopedic and craniofacial applications to treat bone loss, or in dental applications to replace missing teeth. The implantation of these materials, however, does not induce stem cell differentiation, so suitable additional materials such as porous calcium phosphate discs are needed to influence physicochemical responses or structural changes. Rabbit adipose-derived stem cells (ADSC) and mouse osteoblastic cells (MC3T3-E1) were evaluated in vitro by the MTT assay, semi-quantitative RT-PCR, and immunoblotting using cells cultured in medium supplemented with extracts from bioceramics, including calcium metaphosphate (CMP), hydroxyapatite (HA) and collagen-grafted HA (HA-col). In vivo evaluation of the bone forming capacity of these bioceramics in rat models using femur defects and intramuscular implants for 12 weeks was performed. Histological analysis showed that newly formed stromal-rich tissues were observed in all the implanted regions and that the implants showed positive immunoreaction against type I collagen and alkaline phosphatase (ALP). The intramuscular implant region, in particular, showed strong positive immunoreactivity for both type I collagen and ALP, which was further confirmed by mRNA expression and immunoblotting results, indicating that each bioceramic material enhanced osteogenesis stimulation. These results support our hypothesis that smart bioceramics can induce osteoconduction and osteoinduction in vivo, although mature bone formation, including lacunae, osteocytes, and mineralization, was not prominent until 12 weeks after implantation.