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.

This questioning has lead to a progressive replacement of 2D cultures in favor of three-dimensional (3D) cell cultures with protein expression patterns and intercellular junctions that are more alike in vivo conditions. Today, these 3D cell cultures have become a very attractive system for the scientific community (Abbott, 2003), however experts do not all agree on these in vitro procedures. ASCs represent promising cell therapies for regenerative medicine and immunomodulation (Agrawal et al., 2014;Pappalardo et al., 2019). Laboratories throughout the world that are currently studying 3D cultures adopt a personal, nonstandardised technique albeit often achieving the same final evaluations, namely that 3D spheroids feature superior characteristics compared to 2D classical ASCs (A. Di Stefano, Montesano et al., 2020;A. B. Di Stefano et al., 2018;A. B. Di Stefano, Grisafi et al. 2020;A. B. Di Stefano et al., 2015;S. Kim, Han, et al., 2018).
Does the spheroid already exist in the tissue or is it the result of a later union or rearrangement of individual cells? This question continues to stand as specialist literature currently available is not conclusive.
This systematic review aims to summarize evidence from studies carried out in the past decade on ASC spheroids. This study reviews all the techniques used for their isolation and/or formation with specific plate, scaffold systems, or nonadhesion methods and discusses the benefits and drawbacks of the different techniques. (i) Iinclusion criteria: preclinical or clinical research papers concerning the potential regenerative role of S-ASCs culture.

| MATERIALS AND METHODS
(ii) Exclusion criteria: reviews and papers that did not directly refer to human ASC spheroids studies.
Two reviewers independently screened all search results, abstracts, and full texts. Further searches included relevant references from selected articles. Data concerning the type of paper, cell isolation, culture methods, and results were extrapolated from the selected articles.
Data were analyzed to summarize current evidence on the following queries: (i) What technical methods that have been developed in the past decade to create 3D cellular ASC aggregates/spheroids? (ii) What are the benefits in terms of regenerative capacities of 3D-ASCs compared to traditional 2D-ASC cultures?
(iii) Why does not a standardized method of 3D-ASC isolation and in vitro culture exist today? 3 | RESULTS 3.1 | Common culture methods using specific technical conditions (ultralow/low adhesion or concave multiwell) Many authors isolate ASCs from liposuction, culture them in traditional adhesion conditions, trypsinize them and then take the cells to 3D conditions. Table 1 lists the selected studies using common culture methods including ultralow/low adhesion or concave multiwell to form ASC spheroids featuring different sizes and oxygen concentrations.
Using agarose micromolds with basic medium, Fennema et al. (2018) generated ASC spheroids adding LG-DMEM (Low-Glucose Dulbecco's Modified Eagle's Medium) and a 400 µm diameter well rubber. Guo et al. instead formed 3D spheroids whose aggregates were then removed and transferred to the nonadhesive plastic Petri dishes called agarose 3D Petri dishes. This innovative method was used to overcome poor post thaw cells and improve the viability and neural differentiation potential of 3D-ASCs (Guo et al., 2015).  (Cho et al., 2017). No and collaborators instead coated the plates with 3% BSA obtaining spheroids with a different diameter (500 μm), fabricating them by employing the soft lithography technique. They also discovered that hepatocyte spheroids acquired an improved liver-specific function when cocultured with hASC spheroids . Finally, Lee et al. used (Hong et al., 2020).
Small spheroids with average spherical shapes (100 μm diameter) were obtained when a seeding density of 100.000 cells/cm was reached in 96-well plates containing circular adhesive domains. In this study, the 3D condition of the ASCs was associated with an increase of the VEGF-A and IL-8 expressions as regards to wound healing (Furuhata et al., 2016). The capacity to form spheroids in response to different oxygen concentrations is a consolidated fact. Culture geometry, depending on spheroid size, is essential to hypoxia effects; each spheroid counted a different number of cells and was placed at different oxygen concentrations to observe the VEGF expression. An increased VEGF secretion was observed in the spheroids in 2% oxygen conditions compared to a 20% oxygen culture condition (Skiles et al., 2013), (Skiles et al., 2015). Oberringer  3.2 | Common culture methods of ASC spheroids (hanging drop or spinner flask)  Commercially III−VI passages of 2D 5000 (5 k), 10,000 (10 k), 20,000 (20 k), or 60,000 (60 k) cells were pipetted into 0.5 ml, siliconized, screw-cap microcentrifuge tubes and centrifuged at 500 rcf for 2 min. encapsulated within the PEG hydrogels Spheroid size in normoxic (20%) and hypoxic (1% and 2%) condition in monoculture and HUVEC cocultures.    Several studies tested different hydrogels to form ASC spheroids by employing state of the art materials in the field of tissue engineering such as hyaluronic acid (Feng et al., 2017;Hu et al., 2020) and chitosan (Chen et al., 2018;Cheng, Chang, et al., 2012;Cheng, Wang, et al., 2012;Hsu et al., 2014;Huang et al., 2011;Lin et al., 2020), demonstrating an improved stemness gene  Loading spheroids onto acellular, lyophilized and sterile human dermis thin sectioned acellular dermal matrix (tsADM). 8 mm diameter round tsADM was fixed at the center of the coverslip.

| DISCUSSION
Spheroid is an element with its own defined and unique characteristics regardless of the method used for its formation. The size of the spheroid is critical because, increasing in size, the levels of expression of hypoxic factors stimulating angiogenesis and of antiapoptotic genes increase as well. Therefore, spheroids contrast the anoikis phenomena better than monolayer cultures. Another important factor to consider in in vitro cultures is that during the detachment process, the trypsin action on the single-layer of cells causes a great In the last 10 years, scientific literature has established that 3D spheroids properly represent the original cells condition in in vivo tissues (Abbott, 2003). This would lead us to assume that spheroids already exist in the tissue as a 3D structure and can therefore be directly isolated, most likely through tissue disintegration. We believe that the ideal technique would be to F I G U R E 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.
culture cells in suspension without preliminary adhesion isolation or additional processes. In fact, this would lead to an upstream selection of cells that could form different spheroids. Among the classic techniques, only three studies (A. B. Di Stefano et al., 2015;Gimeno et al., 2017;Winter et al., 2003) and, amongst the canonical techniques, the hanging drop and spinner flasks, all obtain spheroids without requiring the first in vitro selection step under adherence conditions.
In the section concerning these innovative methods, we included multiple strategies for spheroid formation; from those featuring a physical nature such as LLLI, a bio-chemical-physical nature (PAAm with CMMPs), to scaffolds such as GNF hydrogel, PLLA-G/mff, PLGA-ADH hydrogel, PLGA-OEGs, HA and HA-chitosan, ELP-PEI, and Pd/Si NWs systems. Furthermore, there is the engineeringstructural nature such as Bioreactors, Lockyballs, CellSaic, StemFit 3D, and pTS. Among these studies, only two involve the immediate use of 3D without passing through the adherence conditions such as the centrifugal microfluidic disk formulated to generate finelycontrolled multicellular spheroids (Park et al. 2017) and the Pd/Si NWs system performed with 3D-ASCs obtained from the hanging drop method (Seo et al., 2014).
We believe that standardizing a single 3D-ASC isolation technique, such as the spontaneous formation in ultra-low culturing conditions without the need of special instruments, would be more reliable and reproducible solution.
Although we propose an ideal spheroid isolation technique, it is crucial to evaluate two others important aspects in this discussion: one highlights the importance of these techniques for the in vitro characterization of spheroids, however, on the other hand, it is important to find innovative techniques that involve the least manipulation and the highest yield of cells for prompt use in the clinical field.
In the near future, clinical applications in regenerative medicine will feature the use of bio-printers and bioreactors capable of creating ad personam organ or tissue replacement. The results of this systematic review suggest that 3D-ASCs could represent the ideal candidates for clinical studies of regenerative medicine and, given their variety, ad hoc scaffolds could be chosen according to the specific requirements. Figure 1  F I G U R E 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.