The primary novel finding of this study was the development of a novel process to generate a substantial quantity of muscle-derived Progenitor Cells (mdP-Cells), keeping the interesting regenerative properties of muscle-derived mesenchymal stem cells, while using an innovative autologous 3D cell culture method from a single muscle micro-biopsy. We firmly believe that 3D cell culture holds large potential for future applications in tissue engineering, regenerative medicine, drug development, toxicity testing, and organoid formation.
While the conventional 2D monolayer in vitro cell culture system has been invaluable, there is growing evidence suggesting that 3D non-adherent conditions better mimic the in vivo environment. Although mesenchymal stem cells (MSCs) have traditionally been characterized by their ability to adhere to plastic in vitro, such 2D culture conditions do not fully replicate the complex cell-cell and cell-extracellular matrix (ECM) interactions observed in vivo. This is particularly relevant for MSCs, which are progenitor/stem cells involved in skeletal and connective tissue formation (Yen et al., 2023).
In comparison to conventional 2D mdMSCs culture described by Ceusters et al. (2017), the culture duration was reduced, the technique was simpler and the growth rate higher. During this 3D culture process, we respect the propensity of the cells to grow in a complex autologous 3D environment, allowed by the growth factors presented in the horse plasma, which is more physiological than 2D culture in fetal bovine serum. Regarding the growth of the cells, we properly do not cell passages, as we do not detach the cells from their support, which is shown by the absence of expression of CD44, an adhesion protein, when cells are cultured in plasma. The cells are nevertheless able to quickly express this protein if they need to attach to their growing support, as shown by a 24h classical culture of our cells in 2D serum based medium. This point of not passaging the cells during their amplification is particularly noteworthy for mitigating the risk of cell senescence observed in some conditions following multiple passages (Yang et al., 2018; Krasnova et al., 2023). Indeed, senescence is a cellular response to endogenous and exogenous stressors that can be induced by a number of stimuli, including oxidative stress, irradiation, chemicals, or replicative exhaustion. Senescence is a progressive, dynamic, and multistep process that includes chromatin remodeling, epigenetic modifications, mitochondrial alterations, and the production of a proinflammatory secretome and ends in an irreversible state of growth arrest (Neri & Borzì, 2020).
Progression of senescence is a continuous process associated to a progressive decline in telomere length of MSCs throughout culturing and the secretome's gradual phenotypic transition to release increasingly greater pro-inflammatory factors (Baxter et al., 2004; Coppé et al., 2010).
Another innovative aspect was the utilization of an enzymatic composition designed for dissolving the gel, rather than employing trypsin. It is widely acknowledged that trypsin induced a degradation of the surface proteins. The detachment of cells by trypsin altered the morphology and metabolic pathways (Kapiszewska et al., 1991; Sharma et al., 2019)
The trilineage differentiation properties and the immunomodulatory properties exhibited similarity between the two methods.
Another key advantage of this protocol is the elimination of FBS. Despite being accepted by regulatory agencies, the use of FBS in eukaryotic cell culture is associated with various concerns (Duarte Rojas et al., 2024). In the context of mammalian cell culture, particularly with stem cells, the presence of animal-derived proteins in the serum introduces xenogenic elements into the cell culture medium. Such "contaminations" may give rise to health issues and elicit immune or inflammatory responses. For instance, in equine studies, Rowland et al. (2021a) demonstrated a heightened inflammatory reaction following the administration of MSCs cultured with FBS compared to those cultured without FBS.
Research endeavors have focused on devising alternative culture media devoid of serum, with the aim of enriching the media with synthetic compounds instead (Ragny et al. 2024). In our study, we successfully utilized a gel derived from autologous plasma as a source of growth factors. This significant discovery enabled us to establish a fully autologous process for therapeutic applications in horses, thereby minimizing the risk of cell rejection upon transplantation. In addition, the use of autologous plasma collected concurrently with the microbiopsy, thereby bringing us closer to the conditions required for the development of a minimally manipulated product (Serteyn et al., 2021)
MSCs are generally recognized as having low immunogenicity, allogeneic cell products are utilized in equine conditions. Nonetheless, some studies have reported inflammatory and immunological reactions in cases of mismatch between the recipient and donor cells (Rowland et al., 2021 b).
In summary, this study introduced a simplified 3D culture technique aimed at generating minimally manipulated autologous cell products designed for musculoskeletal applications. Our method provided a straightforward procedure for isolating progenitor cells capable of differentiation and exhibiting immunomodulatory properties. Moving forward, our focus will be on further enhancing the technology to transform the 3D culture system into an efficient and automated platform.