Mesenchymal stromal cells (MSCs) are promising candidates for novel cell therapeutic applications. For clinical scale manufacturing, human factors from serum or platelets have been suggested as alternatives to fetal bovine serum (FBS). We have previously shown that pooled human serum (HS) and thrombin-activated platelet releasate in plasma (tPRP) support the expansion of adipose tissuederived MSCs. Contradictory results with bone marrow (BM)-derived MSCs have initiated a comprehensive comparison of HS, tPRP, and pooled human platelet lysate (pHPL) and FBS in terms of their impact on MSC isolation, expansion, differentiation, and immunomodulatory activity. In addition to conventional Ficoll density gradient centrifugation, depletion of lineage marker expressing cells (RosetteSep) and CD271 1 sorting were used for BM-MSC enrichment. Cells were cultured in medium containing either 10% FBS, HS, tPRP, or pHPL. Colony-forming units and cumulative population doublings were determined, and MSCs were maximally expanded. Although both HS and tPRP comparable to FBS supported isolation and expansion, pHPL significantly accelerated BM-MSC proliferation to yield clinically relevant numbers within the first two passages. MSC quality and functionality including cell surface marker expression, adipogenic and osteogenic differentiation, and immunosuppressive action were similar in MSCs from all culture conditions. Importantly, spontaneous cell transformation was not observed in any of the culture conditions. Telomerase activity was not detected in any of the cultures at any passage. In contrast to previous data from adipose tissue-derived MSCs, pHPL was found to be the most suitable FBS substitute in clinical scale
Mesenchymal stromal cells (MSCs) are promising candidates for innovative cell therapeutic applications. For clinical scale manufacturing regulatory agencies recommend to replace fetal bovine serum (FBS) commonly used in MSC expansion media as soon as equivalent alternative supplements are available. We already demonstrated that pooled blood group AB human serum (HS) and thrombin-activated platelet releasate plasma (tPRP) support the expansion of multipotent adipose tissue-derived MSCs (ASCs). Slight differences in size, growth pattern and adhesion prompted us to investigate the level of equivalence by compiling the transcriptional profiles of ASCs cultivated in these supplements. A whole genome gene expression analysis was performed and data verified by polymerase chain reaction and protein analyses. Microarray-based screening of 34,039 genes revealed 102 genes differentially expressed in ASCs cultured with FBS compared to HS or tPRP supplements. A significantly higher expression in FBS cultures was found for 90 genes (fold change ≥2). Only 12 of the 102 genes showed a lower expression in FBS compared to HS or tPRP cultures (fold change ≤0.5). Differences between cells cultivated in HS and tPRP were hardly evident. Supporting previous observations of reduced adhesion of cells cultivated in the human alternatives we detected a number of adhesion and extracellular matrix-associated molecules expressed at lower levels in ASCs cultivated with human supplements. Confirmative assays analyzing transcript or protein expression with selected genes supported these results. Likewise a number of mesodermal differentiation-associated genes were higher expressed in cells grown in FBS. Quantifying adipogenic and osteogenic differentiation lacked to demonstrate a clear correlation to the supplement due to donor-specific variances. Our results emphasize the necessity of comparability studies as they indicate that FBS induces a culture adaptation exceeding that of ex vivo culture in human supplements and thus may contribute to the therapeutic potential.
Background: Contamination of cell culture and biological material by mollicute species is an important safety issue and requires testing. We have developed a singletube real-time polymerase chain reaction (PCR) assay for rapid detection of Mollicutes species stipulated by the European Pharmacopeia. Methods: Primers and TaqMan probes (FAM-labeled) were deduced from 16S rDNA sequence alignment of 18 mollicutes species. A synthetic internal control (IC) DNA and an IC-specific TaqMan probe (VIC-labeled) were included. The analytical sensitivity of the assay was determined on DNA dilutions from 12 mollicute strains. Specificity was proven by the use of DNA from other bacteria. Results: Analytical sensitivities of the PCR assay were in the range of 405-2,431 genomes/ml for 11 of the 12 tested mollicute DNA samples. The lowest sensitivity was found for Ureaplasma urealyticum (19,239 genomes/ml). Negative results for DNA samples from 3 different ubiquitous bacteria demonstrated the specificity of the PCR assay for Mollicutes. Direct testing of cell culture supernatants spiked with Mycoplasma orale revealed similar sensitivity compared to isolated DNA. Conclusions: Our single-tube real-time PCR assay with internal reaction control enables rapid and specific detection of mollicute contaminants. The test protocol is suitable for routine quality control of cell therapeutics.
Ziel: Die vorliegende Arbeit hat zum Ziel, 20 Jahre nach Einrichtung der Nationalen Stillkommission anhand der in Deutschland publizierten Stilldaten einen Einblick in die Entwicklung des Stillverhaltens zu geben. Methodik: Zur Identifizierung relevanter Publikationen wurde eine umfassende Literaturrecherche in den Datenbanken PubMed und Web of Science mit den Suchbegriffen ?breast feeding" oder ?breastfeeding? in Kombination mit ?Germany? durchgef?hrt. Der Publikationszeitraum wurde daf?r auf die Jahre 1995?2014 begrenzt. Ergebnisse: Insgesamt wurden 35 Studien mit Daten zum Stillen f?r die Geburtsjahrg?nge 1990?2012 erfasst. Die Daten stammen mehrheitlich aus regionalen oder lokalen Erhebungen und sind ?berwiegend retrospektiv erhoben worden. Etwa 60% der Studien waren mit dem prim?ren Ziel durchgef?hrt worden, Daten zum Stillen bzw. zur Ern?hrung im S?uglingsalter zu erheben. Trotz hoher Stillraten zu Beginn (72?97%) ist nach wie vor in den ersten 2 Monaten der st?rkste Abfall der (Voll-)Stillraten zu verzeichnen, so dass bis zum Alter von 6 Monaten nur noch etwa 50% der S?uglinge ?berhaupt gestillt werden. Schlussfolgerung: Verst?rkte Stillf?rderma?nahmen und unter anderem der Ausbau Fr?her Hilfen zum Stillen sind notwendig, um den Stillerfolg in Deutschland nachhaltig zu sichern. Mit Blick auf die Defizite bei der Stilldatenerhebung erscheint die Errichtung eines effizienten standardisierten Stillmonitorings in Deutschland unverzichtbar.
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