Xeno-free pre-vascularized spheroids for therapeutic applications

Abstract

Spheroid culture has gained increasing popularity, arising as a promising tool for regenerative medicine applications. Importantly, spheroids may present advantages over single-cell suspensions in cell-based therapies (CT). Unfortunately, most growth media used for spheroid culture contain animal origin-components, such as fetal bovine serum (FBS). The presence of FBS compromises the safety of CT and presents economic and ethical constraints. SCC (supplement for cell culture) is a novel xeno-free (XF) industrial cell culture supplement, derived from well-controlled pooled human plasma and processed under good manufacturing practice rules. Here, we developed a XF SCC-based formulation for 2D-culture of outgrowth endothelial cells (OEC), and then used it for generating co-culture spheroids of OEC and mesenchymal stem cells (MSC). XF MSC-OEC spheroids were characterized in detail and compared to spheroids cultured in FBS-supplemented medium. XF spheroids presented comparable integrity, size and morphology as the reference culture. The use of both media resulted in spheroids with similar structure, abundant extracellular matrix deposition and specific patterns of OEC distribution and organization. Notably, XF spheroids presented significantly enhanced angiogenic potential, both in vitro (fibrin sprouting assay) and in vivo (CAM assay). These findings are particularly promising in the context of potential therapeutic applications.

Fetal bovine serum-free culture of endothelial progenitor cells-progress and challenges

“…More recently, an industrial, pharmaceutical‐grade human plasma‐derived supplement for cell culture (SCC), currently under development at Grifols (Barcelona, Spain), has been described as a suitable candidate for XF culture of several cell types, including EC (Bauman, Feijão, Carvalho, Granja, & Barrias, ; Blazquez‐Prunera, Diez, Gajardo, & Grancha, ; Castells‐Sala, Martorell, & Balcells, ; Cimino et al, ; Diez, Bauman, Gajardo, & Jorquera, ; Rodríguez‐Pizà et al, ). SCC is a ready‐to‐use lyophilized product, obtained through cold‐ethanol fractionation of industrial human plasma pools from healthy donors.…”

“…The supplement is characterized by high safety, resulting from rigorous testing of donations, and specific viral inactivation steps during production, in addition to purification steps with pathogen removal capacity. Bauman et al () developed a SCC‐based XF media for OEC culture, which was subsequently used for generating 3D spheroids of cocultured OEC and MSC. XF MSC‐OEC 3D spheroids presented comparable integrity, size, and morphology as EGM‐2MV‐cultured spheroids.…”

Advanced Bottom‐Up Engineering of Living Architectures

“…The established 3D spheroids demonstrated a significant pro‐angiogenic potential in in vivo chick chorioallantoic membrane (CAM). assays, accompanied by close tissue integration, and their culture under xeno‐free conditions potentiates their applicability in bottom‐up tissue engineering (Figure B) . It is worth to reference that in vitro culture of cell‐rich assemblies under xeno‐free conditions is an important parameter for cell‐based therapies application in a more realistic clinical setting.…”

“…B) Co‐culture OEC–MSC 3D spheroid improved angiogenic sprouting and integration into CAM under xeno‐free conditions. Reproduced under the terms of the CC‐BY Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/) . Copyright 2017, The Authors, published by Springer Nature.…”

Analysis of the Effect of Increased α2,3-Sialylation on RTK Activation in MKN45 Gastric Cancer Spheroids Treated with Crizotinib

“…Gastric multicellular tumor spheroids (MCTS) formation was conducted by two different approaches, either in ULA plates (Corning, New York, NY, USA) or in 3D Petri Dish ® (MICROTISSUES ® technology, MicroTissues Inc., Sigma-Aldrich, St. Louis, MO, USA) following the protocol described in [30,64]. Briefly, 12-series agarose micro-molds were prepared using 2% agarose in 0.9% of NaCl.…”