Isolation and ex-vivo activation of adipose-derived cells

Abstract

In recent years stem cell research has become increasingly important for regenerative medicine and tissue engineering. The isolation of stem cells from adipose tissue evades ethical concerns with which embryonic stem cells and induces pluripotent stem cells (iPS) are afflicted,because of its declaration as clinical waste material. Tumescent liposuction is a minimally invasive procedure providing high amounts of adipose tissue rich in therapeutically relevantcells within a short time. The isolated stromal vascular fraction (SVF) and the adipose derived stromal/stem cells (ASC) contained there in show a high regenerative potential and have been successfully used in many clinical studies. Maintaining SVF cells in their natural environment and therefore providing the maximum possible regenerative potential of adipose tissue-derived cells is a prerequisite for successful autologous clinical application. With an improved gentle and fast isolation process by minor manipulation it is possible to obtain a therapeutically relevant cell population. A physical stimulus already used in clinics is the extracorporeal shockwave therapy (ESWT), shockwaves are characterized by their high rise in pressure within a very short time followed by cavitation wave with a negative amplitude. By applyinglow-energy ESWT on freshly obtained human liposuction material and isolated SVF cells (invitro) we aimed to equalize and enhance stem cell properties and their functionality. We were able to show an increased adenosine tri-phosphate (ATP) concentration after applying ESWT on adipose tissue as well as a significantly increased expression of single mesenchymal and vascular surface markers in comparison with the untreated group. Additionally, the protein secretion of insulin-like growth factor 1 (IGF-1) and placental growth factor (PLGF) was significantly enhanced. Further it was investigated if there is the same beneficial effect when applying ESWT on the adipose tissue harvest site before liposuction to improve cell propertiesin situ. We showed a significantly enhanced viability, ATP concentration and population doublings after 3 weeks in culture for cells isolated from ESW treated adipose tissue harvest site. Further the expression of mesenchymal and endothelial/pericytic markers was elevated collaborating with the increased angiogenic differentiation potential as well as the increased secretion of certain angiogenic proteins after ESWT in situ. Besides ESWT the effect of another physical stimulus on SVF/ASC cells was tested - Low level laser therapy (LLLT) has already shown beneficial effects. Therefore, we investigated effects of pulsed blue (475nm), green(516nm) and red (635nm) light from light-emitting diodes (LEDs) applied on freshly isolated SVF cells. Cells had a stronger capacity to vascular tube formation after exposure to greenand red light concomitant with an increased concentration of vascular endothelial growth factor(VEGF) in the secretome. In a side project during the PhD program the hormone-relatedwomens disease lipedema was investigated. The SVF cell properties of healthy and lipedemapatients were investigated and a significant enhancement in cell yield as well as a reduction in adipogenic differentiation capacity of lipedema SVF cells was revealed. Within this work different physical forces applied on adipose tissue and adipose tissue-derived cells were presented as well as an improved isolation method and characteristics of degenerated adiposetissue. This are promising applications for the clinical use in the field of regenerative medicine and tissue regeneration.