Background

Musculoskeletal disorders are the leading cause of loss of use in athletic horses. In a study of 126 elite show jumpers, 55% of training days lost for medical reasons were due to non-acute orthopedic injuries and 22% to acute injuries [1]. Prevalence increases with age, affecting 51% of horses over 15 years and up to 77% of geriatric horses over 30 [2]. In aged horses, osteoarthritis and chronic laminitis are most common [3–4]. In Thoroughbreds, fracture risk is markedly elevated, prompting research on early warning markers [5]. Standardbreds show lower fracture incidence but higher rates of tendon and suspensory ligament injuries [6].

The limited healing capacity of bone, joint, tendon, and ligament injuries, coupled with high recurrence rates and modest efficacy of conventional therapies, highlights the need for regenerative strategies. Regenerative medicine aims to restore or replace damaged tissues through approaches such as tissue engineering, stimulation of endogenous repair, or mesenchymal stem cell (MSC) therapy [7–8]. In equine practice, several regenerative modalities—platelet-rich plasma (PRP), autologous conditioned serum (ACS), autologous protein solution (APS), and MSCs—are already in clinical use [9–12]. Adult MSCs have been isolated from multiple tissues, with bone marrow and adipose tissue as the main sources. Muscle-derived MSCs (mdMSCs) have also been described across species [13–16]. Ceusters et al. (2017) introduced a simple sampling method using skeletal muscle microbiopsies. Traditionally, mdMSCs are expanded from explants via density-gradient selection and 2D culture in fetal bovine serum (FBS)-supplemented medium, with FBS being an accepted source of growth factors [17].

However, 2D cultures fail to replicate the architecture and physiological conditions of native tissues [18]. In contrast, 3D cultures provide a more biomimetic environment that better reflects tissue architecture, cell–cell interactions, and functional behavior. Advantages include improved modeling of proliferation, differentiation, disease mechanisms, and drug responses, as well as the ability to study complex processes such as angiogenesis and immune interactions. Nevertheless, 3D systems face challenges: technical complexity, variability across protocols, higher costs, and difficulties in imaging and analysis [19–20]. Recently, our group demonstrated the feasibility of expanding mdMSCs in 2D and 3D cultures using 10% equine allogenic platelet lysate as an alternative to FBS [21].

The present study had two main objectives: (i) to establish a novel method for isolating muscle-derived progenitor cells (mdPg cells) in a 3D plasma-based gel (3D plasma) combined with a safe retrieval solution, thereby addressing the need for simplified culture methods to generate minimally manipulated autologous cell products for equine regenerative medicine; and (ii) to evaluate whether cells cultured under these minimally manipulated 3D-plasma conditions retain key mdMSC properties—namely immunophenotype, immunomodulatory activity, and trilineage differentiation capacity—comparable to conventional 2D serum culture.