Abstract
Vascularized adipose tissue models are highly demanded as alternative to existing animal models to elucidate the mechanisms of widespread diseases, screen for new drugs or asses corresponding safety levels. Standardly used animal-derived sera therein, are associated to ethical concerns, the risk of contaminations and many uncertainties in their composition and impact on cells. Therefore their use should be completely omitted. In this study we developed a serum-free, defined co-culture medium and implemented it to set up an adipocyte-endothelial cell (EC) co-culture model. Human adipose-derived stem cells were differentiated under defined conditions (diffASCs) and, like human microvascular ECs (mvECs), cultured in a developed defined co-culture medium in mono-, indirect or direct co-culture for 14 days. The developed defined co-culture medium was superior to compared mono-culture media and facilitated the functional maintenance and maturation of diffASCs including perilipin A expression, lipid accumulation and glycerol and leptin release. The medium equally allowed mvEC maintenance, confirmed by the expression of CD31 and vWF and acLDL uptake. Thereby mvECs showed a strong dependency on EC-specific factors. Additionally the development of vascular structures by mvECs was facilitated when directly co-cultured with diffASCs. The completely defined co-culture system allows for the serum-free setup of adipocyte/EC co-cultures and thereby represents a valuable and ethically acceptable tool for the setup of vascularized adipose tissue models.
Highlights
Cell and tissue based in vitro models continue to gain momentum in the replacement of animal trials
Abbreviations 3D, three-dimensional; AcLDL, acetylated low-density lipoprotein; ACM, defined adipocyte maintenance medium; ASC, adipose-derived stem cell; AT, adipose tissue; CD31, cluster of differentiation 31; CoM, defined adipocyte/endothelial cell co-culture medium; DAPI, 4‘,6-diamidino-2-phenylindole; diffASC, adipogenic differentiated adipose-derived stem cell; EC, endothelial cell; EC basal medium (ECBM), endothelial cell basal medium; ECM, defined endothelial cell growth medium; EGM-2mv, endothelial cell growth medium 2, microvascular; FBS, fetal bovine serum; FDA, fluorescein diacetate; MAIN, defined adipocyte maintenance mix; MSCGM, mesenchymal stem cell growth medium; MvEC, microvascular endothelial cell; PBS, phosphate buffered saline; PI, propidium iodide; RT, room temperature; vascular endothelial growth factor (VEGF), vascular endothelial cell growth factor; von Willebrand factor (vWF), von Willebrand Factor of plastic surgeries received from Dr Ziegler (Volz et al, 2017; Huber et al, 2015a)
We evaluated the suitability of a defined co-culture medium for an adipocyte/EC co-culture model with regard to cell maintenance in diffASC and microvascular ECs (mvECs) mono-cultures, as well as in an indirect and direct co-culture model of both cell types
Summary
Cell and tissue based in vitro models continue to gain momentum in the replacement of animal trials. Animal sera include the risk of potential contaminations, and their constitution may vary from batch to batch (van der Valk et al, 2004; Gstraunthaler, 2003; Gstraunthaler et al, 2013) Both potential contaminations and an unstable composition causing fluctuation in product quality are considered major challenges to overcome before engineered models may be produced under Good Manufacturing Practice or Good Cell Culture Practice conform conditions and, e.g., be applied as human tissue implants (Hartung et al, 2002). The composition of blood serum, whether of animal or human origin, is not completely identified – neither qualitatively nor quantitatively To avoid these drawbacks, the culture of in vitro engineered cell and tissue models has to be performed under completely defined conditions by excluding serum and all other animal-derived components, e.g., bovine brain extract. A defined medium by definition only contains factors of known structure, such as recombinant growth factors and hormones, and must exclude complex proteins or hydrolysates (van der Valk et al, 2010)
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