Systematic review on spheroids from adipose-derived stem cells: Spontaneous or artefact state?

Abstract

Three-dimensional (3D) cell cultures represent the spontaneous state of stem cells with specific gene and protein molecular expression that are more alike the in vivo condition. In vitro two-dimensional (2D) cell adhesion cultures are still commonly employed for various cellular studies such as movement, proliferation and differentiation phenomena; this procedure is standardized and amply used in laboratories, however their representing the original tissue has recently been subject to questioning. Cell cultures in 2D require a support/substrate (flasks, multiwells, etc.) and use of fetal bovine serum as an adjuvant that stimulates adhesion that most likely leads to cellular aging. A 3D environment stimulates cells to grow in suspended aggregates that are defined as "spheroids." In particular, adipose stem cells (ASCs) are traditionally observed in adhesion conditions, but a recent and vast literature offers many strategies that obtain 3D cell spheroids. These cells seem to possess a greater ability in maintaining their stemness and differentiate towards all mesenchymal lineages, as demonstrated in in vitro and in vivo studies compared to adhesion cultures. To date, standardized procedures that form ASC spheroids have not yet been established. This systematic review carries out an in-depth analysis of the 76 articles produced over the past 10 years and discusses the similarities and differences in materials, techniques, and purposes to standardize the methods aimed at obtaining ASC spheroids as already described for 2D cultures.

Keywords: 3D cultures; adipose stem cells; biomaterials; hanging drop; spheroids; spinner flask.

Figure 1

Summary of characteristics to form spheroids of adipose stem cells. Spheroids of adipose stem cells can derive from liposuction fat and can be differentiated in several mesenchymal lineages such as angiogenic, chondrogenic and osteogenic ones. Size and hypoxia effects are the major features concerning the formation and abilities of spheroids. Size mainly plays internal effects such as: central necrosis, low‐nutrient supply and waste accumulation whilst hypoxia has external ones such as the autocrine and/or paracrine factors production of HIF1, IL‐6, IL‐8, VEGF, MCP‐1, aSMA, and angiopoietin1 factors.

Figure 2

Summary of methods to form spheroids of adipose stem cells. Representative culture methods listed in the text to form spheroids of adipose stem cells. Common culture methods include the use of ultralow adhesion flask, hanging drop and spinner flask. Novel culture methods, for example, include lockyballs (microscaffolds with a porous wall with interlockable hooks) and GMP (microwells plate employed with gelatin microparticles) techniques. Moreover, spheroid formation can take place through scaffold‐free methods (e.g., CMS [centrifugal microfluidic‐based spheroid] or plates treated with Pluronic F‐127 aqueous solution) or by using several materials such as PLGA (Poly [l‐glutamic acid]) with OEGs (Oligo(ethylene glycol), Collagen, OPF (oligo(poly(ethylene glycol) fumarate) hydrogels and HA (hyaluronic acid) gel.