Adipose - Derived Stem Cell Exosomes & Their Relevance In Regenerative Medicine
Author
Papaioannou, Stavros
Term
4. term
Publication year
2014
Pages
59
Abstract
Adipose-derived stem cells (ASCs) exhibit strong immunomodulatory and regenerative effects that are thought to be mediated in part by the small extracellular vesicles (exosomes) they release. This thesis addresses the need for standardized workflows for culture, serum preparation, isolation, and characterization of ASC exosomes. Using ASCs from three donors, a particle-depleted fetal calf serum protocol was established to reduce background. Two commonly used isolation methods were then compared: sequential ultracentrifugation and polymer-based precipitation (Invitrogen kit). Isolated fractions were quantified and characterized by nanoparticle tracking analysis (NTA), transmission and immuno-electron microscopy (TEM/IEM), atomic force microscopy (AFM), and an extracellular vesicle array (EV array). Hypoxic preconditioning (1% O2) was also applied to examine whether low oxygen alters exosome production and features. The serum depletion protocol produced virtually particle-free FCS, enabling unbiased vesicle analysis. While all isolates contained 30–100 nm particles by NTA, only kit-isolated samples displayed canonical exosome markers (CD9, CD63, CD81) by EM and EV array, indicating that polymer-based precipitation was the most suitable approach in this setting. Hypoxia at 1% O2 did not yield morphological or quantitative benefits for ASC-derived exosomes compared with normoxia. Overall, the work supports standardized serum preparation combined with polymer-based isolation to obtain robust, marker-positive ASC exosomes, informing the development of exosome-based, cell-free therapies in regenerative medicine.
Adipøse stamceller (ASCs) har lovende immunmodulerende og regenerative egenskaber, som i høj grad formidles af de små ekstracellulære vesikler (eksosomer), de udskiller. Denne afhandling adresserer behovet for standardiserede arbejdsgange til dyrkning, serumforberedelse, isolering og karakterisering af ASC-eksosomer. Med ASCs fra tre donorer blev der først udviklet en protokol til partikel-depleteret føtalt kalveserum for at minimere baggrundsstøj. Herefter blev to udbredte isoleringsmetoder sammenlignet: sekventiel ultracentrifugering og polymer-baseret udfældning (Invitrogen-kit). De isolerede fraktioner blev kvantificeret og karakteriseret ved nanopartikel-sporingsanalyse (NTA), transmissions- og immuno-elektronmikroskopi (TEM/IEM), atomkraftmikroskopi (AFM) og en extracellular vesicle-array (EV-array). Desuden blev hypoksisk præ-konditionering (1% O2) anvendt for at undersøge, om lavt iltniveau påvirker eksosomproduktion og -egenskaber. Serumdepletionsprotokollen gav praktisk talt partikelfrit FCS og muliggjorde upartisk analyse af vesikler. Alle isolater indeholdt 30–100 nm partikler ifølge NTA, men kun kit-isolerede prøver udviste klassiske eksosommarkører (CD9, CD63, CD81) ved EM og EV-array, hvilket indikerer, at polymer-baseret udfældning var den mest velegnede metode i denne sammenhæng. Hypoksi ved 1% O2 gav ingen tydelige morfologiske eller kvantitative forbedringer af ASC-eksosomer sammenlignet med normoksi. Samlet peger arbejdet på, at standardiseret serumforberedelse kombineret med polymer-baseret isolering giver robuste, markør-positive ASC-eksosomer, med relevans for videre udvikling af eksosom-baserede, cellefrie terapier i regenerativ medicin.
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