Summary

Explore the benefits of MSC spheroids over conventional 2D MSC culturing. Compared to conventional 2D MSCs, there are fewer preclinical studies on MSC spheroids. While 2D methods often compromise MSC functionality, 3D spheroid cultures maintain their qualities, leading to improved therapeutic outcomes. 3D spheroid formation mimics the in vivo MSC niche, ensuring viability and facilitating homing to affected tissues. Thus, MSC spheroids offer insights into MSC biology in their native niche and improve therapeutic outcomes following ex vivo expansion.

Cell therapy represents a pioneering approach that utilises living cells to treat medical conditions and restore tissue function. This innovative method leverages the regenerative potential of cells to address a diverse array of diseases, spanning from cancer and autoimmune disorders to degenerative conditions. Cells for therapy can be sourced from various origins, including the patient’s own body, donors, or stem cell banks. These cells are often subjected to manipulation, expansion, and sometimes genetic modification in laboratory settings before administration to patients. Cell therapy heralds the era of personalised medicine, offering novel treatment avenues where conventional therapies may falter.

Conventionally, monodispersed cells are administered directly into the body via intravenous (IV) injection, intramuscular (IM) injection, intra-articular injection, or other routes, depending on the intended target. These cells traverse the bloodstream to reach specific tissues or organs, where they exert their therapeutic effects. However, the efficacy of monodispersed cells may sometimes be eclipsed by spheroids, three-dimensional (3D) aggregates of cells that closely mimic tissue architecture and organisation. 

Improved therapeutic potential of MSC spheroids

MSC spheroids, in particular, have garnered significant interest in cell therapy due to their multifaceted advantages. They excel in enhancing paracrine signalling, improving cell survival and functionality, producing a complex extracellular matrix (ECM), exerting a broader spectrum of therapeutic effects, and facilitating retention and engraftment. These attributes position MSC spheroids as promising candidates for applications in regenerative medicine and tissue engineering, offering a transformative approach to address clinical challenges and advance patient care.

• Improved stemness and differentiation potential

MSCs spheroids induced significant upregulation of pluripotency marker genes (Sox-2, Oct-4 and Nanog), and increased their clonogenicity. It also enhanced differentiation potential into multi-lineage cells.

• Improved survival

MSCs spheroids have a better tolerance to hypoxia, as they express higher levels of hypoxia-inducible factor 1 and manganese superoxide dismutase. MSC speriods also upregulate the antiapoptotic molecule Bcl-2 and downregulate the proapoptotic molecule, BAX, confirming the prosurvival molecular profile in spheroidal cells.

• Improved secretion of anti-inflammatory and angiogenic factors

Spheroid culture of MSCs leads to increased secretion of angiogenic growth factors and cytokines, such as angiogenin (ANG), fibroblast growth factor 2 (FGF-2), angiopoietin 2 (ANGPT-2), VEGF and hepatocyte growth factor (HGF), and anti-inflammatory cytokines such as transforming growth factor- β1 (TGF-β1) and interleukin-6 (IL-6) compared to the monolayer cultures.

What are the mechanisms of the functional improvement of MSC spheroids?

MSC spheroids enhanced therapeutic potential compared to traditional monolayer cultures prompts exploration into the underlying mechanisms driving their functional improvement. Here are some factors that could play pivotal roles in reshaping the properties of MSCs within spheroids: 

• Altered Cellular Organisation:

The aggregation of MSCs into spheroids fundamentally alters cellular organisation, including cell-matrix and cell-cell interactions. This restructuring influences cellular morphology, cytoskeletal organisation, and polarisation, potentially impacting cell behaviour and function.

• Substrate Stiffness and Elasticity:

The mechanical properties of the substrate significantly influence the paracrine properties of MSCs. Changes in rigidity and elasticity occur during spheroid formation, which may modulate cell behaviour and the secretion of bioactive factors regulating the immunomodulatory effects.

• Enhanced Cell-Cell Contact:

Increased cell-cell contact within spheroids upregulates the expression of cell adhesion molecules such as cadherins (E-cadherin, N-cadherin, cadherin-11) and gap junction proteins (Connexin-43). These interactions not only define the lineage specificity of MSCs but also facilitate intercellular communication and signaling within the spheroid microenvironment.

• Hypoxic Microenvironment: 

The core of MSC spheroids experiences mild hypoxia, which serves as a potent stimulus for the upregulation of pro-survival and angiogenic factors. This hypoxic niche within spheroids initiates adaptive responses, promoting cell survival, proliferation, and the expression of angiogenic factors crucial for neovascularization and tissue regeneration. 

• Enhanced Extracellular Matrix (ECM) Secretion: 

MSC spheroids exhibit enhanced secretion of extracellular matrix (ECM) components, fostering a favourable microenvironment enriched with growth factors and cytokines. This localised deposition of ECM proteins supports autocrine signalling and provides structural support for cellular processes crucial for tissue repair and regeneration. 

• Autophagy Regulation: 

Increased autophagic activity within MSC spheroids protects cells from environmental stresses and may contribute to improved survival and the prevention of premature senescence. Autophagy serves as a cellular quality control mechanism, ensuring the maintenance of cellular homeostasis and functionality. 

Techniques for producing ex vivo MSC spheroids

The translation of MSC spheroids from laboratory-scale production to large-scale manufacturing represents a pivotal step in harnessing their therapeutic potential for clinical applications. However, the transition from bench to bedside necessitates the development of robust and scalable production methods capable of generating consistent and clinically relevant spheroid populations. In this context, optimising large-scale production processes for MSC spheroids presents a multifaceted challenge, encompassing considerations such as cell sourcing, culture conditions, scalability, and regulatory compliance.

Various spheroid generation techniques have already been established. Among these, the most widespread are the hanging drop approach, application of low-adhesive substrates, membrane-based aggregation, and the forced aggregation method. 

Hanging drop technique:

The hanging drop technique stands as one of the most prevalent methods for generating spheroids, offering a straightforward means to create controlled cell populations and spheroid sizes. However, its viability for large-scale spheroid production is hindered by the confined space of standard culture dishes and the labour-intensive processes involved in establishment and harvesting. Addressing these limitations, CellHD-256 emerges as an innovative chip-based technology facilitating the uniform production of 3D cell spheroids. With simplicity and cost-effectiveness at its core, CellHD-256 boasts the capability to generate up to 256 spheroids efficiently.

Low-attachment surface: 

While low-attachment surfaces provide another accessible approach for spheroid formation, they often result in significant variations in size and morphology among the produced spheroids. To counter this challenge, SphericalPlate 5D enters the scene with a revolutionary design aimed at cultivating perfectly round spheroids characterized by high uniformity and reproducibility. Leveraging patented technology, SphericalPlate 5D enables the generation of up to 9000 standardized spheroids per plate, paving the way for seamless translation to clinical and diagnostic applications.

In vivo therapeutic effect of MSC spheroids

Various preclinical study models are employed to investigate the in vivo therapeutic effects of MSC spheroids, encompassing wound healing, pro-inflammatory diseases, bone and osteochondral defects, knee synovitis, cardiovascular diseases, hepatic regeneration, kidney injury, neurogenic pain, and ischemia models.

Studies consistently demonstrate that the application of MSC spheroids in vivo elicits enhanced therapeutic effects. For instance, Deng et al. showcased in a study that 3D spheroids of human placenta-derived MSCs attenuated spinal cord injury (SCI). Transplanted MSCs survived throughout the experiment, maintaining their advantageous secretory properties and exerting significant neuroprotective effects by reducing lesion cavities, suppressing inflammation and astrogliosis, and promoting angiogenesis. These findings suggest that MSC spheroids hold immense potential for SCI treatment.

In a recent investigation by Lee et al., they established a neuropathic pain murine model via chronic constriction injury of the right sciatic nerve. Their study revealed that transplantation of spheroids alleviated chronic pain more effectively and demonstrated prolonged in vivo survival compared to monolayer-cultured cells. Additionally, MSC spheroids modulated proinflammatory cytokines and genes associated with chronic inflammatory responses, indicating their potential to enhance pain alleviation and motor function improvement. These findings underscore the promising role of spheroids in facilitating MSC-based cell therapies for induced neuropathic pain injuries.

References

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Hazrati A, Malekpour K, Soudi S, Hashemi SM. Mesenchymal stromal/stem cells spheroid culture effect on the therapeutic efficacy of these cells and their exosomes: A new strategy to overcome cell therapy limitations. Biomed Pharmacother. 2022;152:113211. doi:10.1016/j.biopha.2022.113211

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Lee N, Park GT, Lim JK, et al. Mesenchymal stem cell spheroids alleviate neuropathic pain by modulating chronic inflammatory response genes. Front Immunol. 2022;13:940258. Published 2022 Aug 8. doi:10.3389/fimmu.2022.940258

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