Recently, research is undergoing a drastic change in the application of the animal model as a unique investigation strategy, considering an alternative approach for the development of science for the future. Although conventional monolayer cell cultures represent an established and widely used in vitro method, the lack of tissue architecture and the complexity of such a model fails to inform true biological processes in vivo. Recent advances in cell culture techniques have revolutionized in vitro culture tools for biomedical research by creating powerful three-dimensional (3D) models to recapitulate cell heterogeneity, structure and functions of primary tissues. These models also bridge the gap between traditional two-dimensional (2D) single-layer cultures and animal models. 3D culture systems allow researchers to recreate human organs and diseases in one dish and thus holds great promise for many applications such as regenerative medicine, drug discovery, precision medicine, and cancer research, and gene expression studies. Bioengineering has made an important contribution in the context of 3D systems using scaffolds that help mimic the microenvironments in which cells naturally reside, supporting the mechanical, physical and biochemical requirements for cellular growth and function. We therefore speak of models based on organoids, bioreactors, organ-on-a-chip up to bioprinting and each of these systems provides its own advantages and applications. All of these techniques prove to be excellent candidates for the development of alternative methods for animal testing, as well as revolutionizing cell culture technology. 3D systems will therefore be able to provide new ideas for the study of cellular interactions both in basic and more specialized research, in compliance with the 3R principle. In this review, we provide a comparison of 2D cell culture with 3D cell culture, provide details of some of the different 3D culture techniques currently available by discussing their strengths as well as their potential applications.

Regenerative medicine aims to restore the normal function of diseased or damaged cells, tissues, and organs using a set of different approaches, including cell-based therapies. In the veterinary field, regenerative medicine is strongly related to the use of mesenchymal stromal cells (MSCs), which belong to the body repair system and are defined as multipotent progenitor cells, able to self-replicate and to differentiate into different cell types. This review aims to take stock of what is known about the MSCs and their use in the veterinary medicine focusing on clinical reports on dogs and horses in musculoskeletal diseases, a research field extensively reported in the literature data. Finally, a perspective regarding the use of the secretome and/or extracellular vesicles (EVs) in the veterinary field to replace parental MSCs is provided. The pharmaceuticalization of EVs is wished due to the realization of a Good Manufacturing Practice (GMP product suitable for clinical trials.

Laminitis, a highly debilitating disease of the foot in ungulates, is characterized by pathological changes of the complex lamellar structures that maintain the appendicular skeleton within the hoof. Laminitis is a multifactorial disease that involves perturbation of the vascular, hematological, and inflammatory homeostasis of the foot. Interestingly, the pathogenesis of the disease resembles what is observed in metabolic syndromes and sepsis-induced organ failure in humans and animals. We hypothesized that local administration of mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) might contribute to establishing an anti-inflammatory and pro-angiogenic environment, and could stimulate the injured tissue in order to restore its functional integrity. According to this assumption, an experimental protocol based on the local intravenous administration of adipose tissue-derived MSCs (aMSCs) in combination with PRP was developed for the treatment of horses affected by chronic laminitis. Nine horses with severely compromised venograms (showing grade III and IV laminitis) that had been unsuccessfully treated with conventional therapies were enrolled. aMSCs and PRP (15 × 106 cells resuspended in 15 mL of PRP) were injected into the lateral or medial digital vein three times, at one-month intervals. The first administration was performed with allogeneic aMSCs, while for the following administrations, autologous aMSCs were used. There was no adverse short-term reaction to the intravenous injection of aMSCs. In the long term, venograms outlined, in all subjects, a progressive amelioration of the vascularization of the foot. An improvement in the structure and function of the hoof was also observed. No adverse events were reported during the follow-up, and the horses returned to a comfortable quality of life. Although the number of animals enrolled in the study is limited, both clinical observations and venography demonstrated an enhancement in the condition of all horses, suggesting that the regenerative therapies in chronic laminitis could be useful, and are worthy of further investigation.

In recent years, mesenchymal stromal cells (MSCs) have shown promise as a therapy in treating musculoskeletal diseases, and it is currently believed that their therapeutic effect is mainly related to the release of proteins and extracellular vesicles (EVs), known as secretome. In this work, three batches of canine MSC-secretome were prepared by standardized processes according to the current standard ISO9001 and formulated as a freeze-dried powder named Lyosecretome. The final products were characterized in protein and lipid content, EV size distribution and tested to ensure the microbiological safety required for intraarticular injection. Lyosecretome induced the proliferation of adipose tissue-derived canine MSCs, tenocytes, and chondrocytes in a dose-dependent manner and showed anti-elastase activity, reaching 85% of inhibitory activity at a 20 mg/mL concentration. Finally, to evaluate the safety of the preparation, three patients affected by bilateral knee or elbow osteoarthritis were treated with two intra-articular injections (t = 0 and t = 40 days) of the allogeneic Lyosecretome (20 mg corresponding 2 × 10⁶ cell equivalents) resuspended in hyaluronic acid in one joint and placebo (mannitol resuspended in hyaluronic acid) in the other joint. To establish the safety of the treatment, the follow-up included a questionnaire addressed to the owner and orthopaedic examinations to assess lameness grade, pain score, functional disability score and range of motion up to day 80 post-treatment. Overall, the collected data suggest that intra-articular injection of allogeneic Lyosecretome is safe and does not induce a clinically significant local or systemic adverse response.