Stem cell spheroids – A vital tool in regenerative medicine

Stem cells serve as the primary resource in regenerative medicine including tissue engineering and transplantation therapy. To this effect human mesenchymal stem cells contain secretory factors with anti-inflammatory, angiogenic and immune regulation factors that makes them an efficient resource for biological and clinical applications.

Stem cells are characterised by their ability to self renew (or stemness), yielding new cells from the original stem cells, thus being a useful tool in cell based therapy, high throughput screening, and tissue engineering. In order to preserve the “stemness” of stem cells, they need to be cultivated in a medium that replicates the in-vivo microenvironment, which contains growth factors, and extracellular matrix (ECM) interactions (1,2). Additionally they are also multi-potent (able to differentiate into other types of cells) in response different transcription factors.

Three dimensional cell culture systems have enabled methods where the in-vivo microenvironment can be replicated in culture conditions. The cell-cell and cell-ECM interactions observed in 3D cell culture helps to maintain cellular mechanisms and properties thus maintaining the key characteristics of stem cells including stemness (3).

Spheroids as a 3D culturing technique

Spheroids are an aggregation of cells into a round cell cluster that is a three-dimensional in structure. An anti-adhesive surface such as BIOFLOATTM Flex plates is helpful for the formation of round and uniform spheroids. Spheroids can form with a variety of cell types such as primary cells or immortalized cell lines. Other spheroid culture techniques include, pellet cultures, liquid overlay method, hanging drop method and rotating wall vessels and spinner flasks for large scale cultures.

Spheroid cultures of stem cells

To date, spheroid cultures have become an important tool in stem cell 3D cultures. Mesenchymal stem cell spheroids indicated preserved intrinsic phenotype, completed with cell- cell and cell -ECM interactions. These cultures indicated enhanced expression of transcription factors necessary to maintain stemness (4). Furthermore spheroid stem cells indicated high levels of cytokines and chemokine that plays a role in proliferation, cell viability and cell migration together with enhanced angiogenic factors compared with traditional monolayer cultures (4,5).

3D cell cultures of induced pluripotent stem cells (iPSCs) mimic both cell-cell and cell-matrix interactions in studying the differentiation of cells. iPSCs can be cultured with or without a supporting scaffold. Spheroid cultures of iPSCs are cultured using cell repellant-coated plates such as BIOFLOAT 96 well plates, to generate uniform spheroids in a relatively short period of time. These cell cultures remain viable for longer periods with the inclusion of scaffolds such as hydrogels, thus making it possible to create disease models where progressive changes can be observed, such as in neurodegenerative diseases (6).


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2. Ramalho-Santos M., Yoon S., Matsuzaki Y., Mulligan R.C., Melton D.A. “Stemness”: Transcriptional profiling of embryonic and adult stem cells. Science. 2002;298:597–600.

3. Rosso F., Giordano A., Barbarisi M., Barbarisi A. From cell-ECM interactions to tissue engineering. J. Cell Physiol. 2004;199:174–180.

4. Murphy K.C., Hoch A.I., Harvestine J.N., Zhou D., Leach J.K. Mesenchymal stem cell spheroids retain osteogenic phenotype through alpha2beta1 signaling. Stem Cells Transl. Med. 2016;5:1229–1237.

5. Zhang Q., Nguyen A.L., Shi S., Hill C., Wilder-Smith P., Krasieva T.B., Le A.D. Three-dimensional spheroid culture of human gingiva-derived mesenchymal stem cells enhances mitigation of chemotherapy-induced oral mucositis. Stem Cells Dev. 2012;21:937–947.

6. Haishuang Lin, Qiang Li, and Yuguo Lei (2017. “Three-dimensional tissues using human pluripotent stem cell spheroids as biofabrication building blocks”. Biofabrication 9 025007.

7. Kouroupis D and Correa D (2021) Increased Mesenchymal Stem Cell Functionalization in Three-Dimensional Manufacturing Settings for Enhanced Therapeutic Applications. Front. Bioeng. Biotechnol. 9:621748.