The regulatory requirements in cell processing, in the choice of a biomaterial scaffold and in quality control analysis, have to be followed in the clinical application of tissue-engineered grafts. Confirmation of sterility during quality control studies requires prolonged storage of the cell-based construct. After storage, preservation of the functional properties of the cells is an important prerequisite if the cells are to be used for cell-based tissue therapies. The study presented here shows the generation of 3D constructs based on Wharton's jelly multipotent mesenchymal stromal cells (WJ-MSCs) and the clinically-acceptable HyaloFast® scaffold, and the effect of two- and six-day hypothermic storage of 3D cell-based constructs on the functional properties of populated cells. To study the viability, growth, gene expression, and paracrine secretion of WJ-MSCs within the scaffolds before and after storage, xeno-free culture conditions, metabolic, qPCR, and multiplex assays were applied. The WJ-MSCs adhered and proliferated within the 3D HyaloFast®. Our results show different viability of the cells after the 3D constructs have been stored under mild (25 °C) or strong (4 °C) hypothermia. At 4 °C, the significant decrease of metabolic activity of WJ-MSCs was detected after 2 days of storage, with almost complete cell loss after 6 days. In mild hypothermia (25 °C) the decrease in metabolic activity was less remarkable, confirming the suitability of these conditions for cell preservation in 3D environment. The significant changes were detected in gene expression and in the paracrine secretion profile after 2 and 6 days of storage at 25 °C. The results presented in this study are important for the rapid transfer of tissue engineering approaches into clinical applications.
- Publikační typ
- časopisecké články MeSH
Kmenové buňky se stávají účinným nástrojem v léčbě řady defektů a onemocnění. V poslední době se ukazuje, že terapeutický efekt kmenových buněk spočívá nejenom v jejich integraci a diferenciaci do buněk příslušné tkáně, ale především v jejich parakrinní aktivitě, tj. schopnosti sekretovat trofické a růstové faktory, cytokiny a chemokiny, které mají regenerativní a protizánětlivé účinky. Kondiciované médium (KM) obsahující sekreční produkty kmenových buněk lze proto využít v tzv. bezbuněčné terapii, která představuje alternativu k terapii buněčné s výhodou nižších rizik, možnosti alogenního podání a velkoobjemové výroby. Výsledky preklinických studií potvrzují, že terapeutický efekt KM je srovnatelný s přímou aplikací kmenových buněk. Cílem této práce je shrnout výsledky studií využívající KM z různých typů kmenových buněk v regenerativní medicíně a současně vypracovat přehled faktorů, kterými lze modifikovat buněčnou sekreci a složení KM.
Stem cells become an effective tool for treatment of a variety of defects and diseases. Recently, it appears that the therapeutic effect of stem cells lies not only in their integration and differentiation into cells of the tissue, but especially in their paracrine activity, i.e. the ability to secrete trophic and growth factors, cytokines and chemokines that have regenerative and anti-inflammatory effects. Conditioned medium (CM) containing secretory products of stem cells can thus be used in cell-free therapy which represents an alternative to the cell-based therapy, with advantage of lower risks, the possibility of allogenic administration and mass production. Preclinical studies confirm that the therapeutic effect of CM is comparable to the effect of the application of stem cells. The aim of this paper is to summarize the results of studies using CM from different types of stem cells in regenerative medicine and simultaneously develop an overview of the factors that can modify cellular secretion and the composition of CM.
- Klíčová slova
- bezbuněčná terapie,
- MeSH
- buněčná a tkáňová terapie metody MeSH
- buněčné kultury MeSH
- embryonální kmenové buňky MeSH
- hojení ran MeSH
- kmenové buňky MeSH
- kultivační média speciální * MeSH
- kultivované buňky MeSH
- lidé MeSH
- mezenchymální kmenové buňky MeSH
- parakrinní signalizace MeSH
- preklinické hodnocení léčiv MeSH
- sekretom MeSH
- transplantace kmenových buněk * MeSH
- transplantace mezenchymálních kmenových buněk MeSH
- výzkum kmenových buněk MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
The present study was designed to extensively characterize cell lines derived from porcine blastocysts by several methodical approaches, including morphological observation, cytogenetic analysis, estimation of alkaline phosphatase activity and detection of specific marker expression at the mRNA/protein level. A comparison was made between the properties of cell lines isolated from in vivo- and in vitro-obtained blastocysts. Our results showed that 57.1% of the in vivo-obtained blastocysts attached to the feeder layer and that 33.3% of them started to grow in a monolayer. The percentage of attached in vitro-produced blastocysts was lower (24.6%), and only 6.9% of them started to grow. Outgrowths from the in vitro-produced blastocysts formed mainly trophectoderm or epithelial-like monolayer, whereas the in vivo-obtained blastocysts formed heterogeneous outgrowths that also contained cells with embryonic stem (ES)-like morphology. Detailed analyses showed that the primary outgrowths with ES-like morphology expressed the pluripotency markers OCT-4 and NANOG and revealed intensive alkaline phosphatase staining, while they did not express markers of differentiation. The majority of passaged cells, including those with ES-like morphology, lacked OCT-4 protein and revealed expression of specific differentiation markers (cytokeratin 18, lamins A/C, transferrin, α-fetoprotein and GATA-4), although they still expressed NANOG and exhibited weak alkaline phosphatase activity. Moreover, these cells spontaneously differentiated into neural, fibroblast or epithelial-like cells, even in the presence of leukaemia inhibitory factor. Our results show that complex analysis of markers of pluripotency as well as differentiation markers is necessary for proper interpretation of data in porcine embryonic stem cell studies.
- MeSH
- biologické markery analýza metabolismus MeSH
- blastocysta cytologie metabolismus MeSH
- buněčné linie MeSH
- embryo savčí MeSH
- fertilizace in vitro MeSH
- fertilizace genetika fyziologie MeSH
- homeodoménové proteiny genetika metabolismus MeSH
- hypoxanthinfosforibosyltransferasa genetika metabolismus MeSH
- kultivace embrya MeSH
- oktamerní transkripční faktor 3 genetika metabolismus MeSH
- prasata embryologie genetika metabolismus MeSH
- stanovení celkové genové exprese MeSH
- těhotenství MeSH
- validační studie jako téma MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
