EVALUATION OF PLATELET-RICH PLASMA (PRP) AS AN ALTERNATIVE TO FETAL CALF SERUM (FBS) IN THE EXPANSION OF HUMAN MYOBLASTS: AN IN VITRO PROTOCOL.
Axel TOLLANCE1,3, Diego MICHEL1, Alexandre PROLA2, Axelle BOUCHE1,2, Antoine TURZI3, Didier HANNOUCHE1,2, Thomas LAUMONIER1,2 and Sarah BERNDT3
- 1 Department of Orthopedic Surgery, Geneva University Hospitals & Faculty of Medicine, Geneva, Switzerland.
- 2 Department of Cell Physiology and Metabolism, Faculty of Medicine, Geneva, Switzerland.
- 3 Regen Lab SA, 1052 Le Mont-sur-Lausanne, Switzerland.
Key words: platelet-rich plasma, muscle stem cells, cell expansion, muscle regeneration.
INTRODUCTION
Muscle diseases, whether congenital, acquired or degenerative, represent a major public health challenge, affecting patients' quality of life and often leading to significant morbidity.
Muscle regeneration is a complex process that involves the participation of diverse cell populations, including muscle stem cells (MSCs), which play a central role in restoring muscle integrity and function after injury [...1, 2].
However, in pathological conditions such as Duchenne muscular dystrophy (DMD), muscle regeneration capacities are compromised, highlighting the need to develop new therapeutic approaches to treat these debilitating muscle diseases.
MUSCLE STEM CELLS AND THEIR ROLE IN MUSCLE REGENERATION
MSCs reside in a quiescent state between the muscle fiber and the basal lamina, ready to be activated when needed, such as during muscle injury.
These cells play a crucial role in postnatal muscle regeneration and growth, ensuring the integrity and functionality of muscle tissue throughout life. When damage occurs to muscle, whether as a result of trauma or disease, MSCs are activated to meet the demand for repair.
This process involves activation of MSCs followed by proliferation of progenitor cells, known as myoblasts, which can then differentiate into mature muscle cells and integrate muscle tissue to restore its functionality [...].3].
In pathological conditions such as DMD, these regenerative processes are compromised due to MSC dysfunction.
In DMD, a mutation in the dystrophin gene causes progressive muscle degeneration, leading to reduced muscle function and an inability to effectively regenerate damaged muscle tissue.
MSCs in DMD-affected muscles show altered proliferative capacity and diminished differentiation potential, contributing to disease progression and muscle deterioration.
THERAPIES BASED ON MUSCLE STEM CELLS
MSC-based therapies have attracted considerable interest as potential strategies for muscle repair. Traumatic muscle injuries, such as muscle ruptures or tears, could benefit from muscle stem cell-based therapies to accelerate the healing process and restore normal muscle function [4, 5]. By injecting muscle stem cells directly into the injured area, it is possible to promote tissue regeneration and reduce scar formation, which could improve clinical outcomes for patients suffering such injuries.
However, despite their promising potential, several challenges remain before muscle stem cell-based therapies become commonplace in the clinic.
These challenges include the need to develop efficient methods for isolating and culturing muscle stem cells in sufficient quantities, as well as understanding the underlying mechanisms of their differentiation and integration into damaged muscle tissue.
A major concern lies in the selection and expansion of suitable cells in sufficient quantities for effective therapeutic application [...].6].
In this respect, the identification of new cell sources, such as human muscle reserve cells (HMRC), represents a significant advance.
These cells share many of the characteristics of quiescent MSCs and offer promising potential for muscle cell therapy.
EXPLANATION OF THE MODEL IN VITRO USED
To better understand the behavior of muscle cells and evaluate the efficacy of new therapies, we have used a model in vitro based on primary human stem cells.
We first extracted mononuclear cells from a muscle biopsy, specifically selected muscle stem cells by flow cytometry, then let them proliferate as myoblasts.
Once confluent, myoblasts can be differentiated in a medium low in growth factor. Around 70 % myoblasts fuse to form large polynucleated cells called myotubes, while the remaining cells remain mononucleated and acquire stem cell characteristics.
These cells are called CMRH and are the counterpart of the in vitro stem cells.
They share many of the characteristics of MSCs, such as their quiescent state, the expression of the transcription factor Pax7, and their ability to be activated after stimulation [...].7].
Figure 1. A. Schematic representation of the regeneration process in vivo. (1) Following injury, adapted signals activate muscle stem cells (MSCs) and induce their migration to the site of injury. These activated MSC re-enter the cell cycle and are called myoblasts. (2) Myoblasts at the site of injury proliferate to an appropriate number, enabling restoration of the damaged muscle. (3) Myoblasts differentiate, stop their cell cycle and enter the post-mitotic phase. (4) Finally, they fuse with each other or with the damaged fiber to restore the entire muscle. (5) At the same time, during myoblast activation/proliferation, a subpopulation remains quiescent and replenishes the MSC stock for future regeneration.
B. Schematic representation of our model in vitro and representative photo of a culture of myotubes and MuRC after 48 hours of differentiation
ALTERNATIVES TO FETAL CALF SERUM: PLATELET-RICH PLASMA (PRP)
Another important consideration concerns the cell culture conditions used for muscle cell expansion in the laboratory.
Traditionally, fetal bovine serum (FBS) has been widely used as a source of nutrients and growth factors for cell culture.
However, the use of FBS has its drawbacks, including risks of contamination, immunogenicity and ethical concerns linked to its animal origin [...8, 9, 10, 11].
To alleviate these problems, alternatives to FBS are being actively sought. Platelet-rich plasma (PRP) is one such potential alternative.
PRP is a biological product derived from blood, rich in growth factors contained in the alpha granules of platelets.
When activated, PRP releases these growth factors, promoting tissue regeneration.
The various PRP preparations include leukocyte-rich PRP (LR-PRP) and leukocyte-poor PRP (LP-PRP), as well as platelet lysate, which are derivatives of PRP [12, 13, 14, 15, 16].
Preliminary studies have shown that PRP, whether of autologous or allogeneic origin, can promote cell proliferation. in vitro.
These beneficial effects are attributed to the growth factors contained in PRP, such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β) and vascular endothelial growth factor (VEGF) [17, 18, 19, 20, 21].
However, further research is needed to fully assess their efficacy and safety, as well as to resolve the practical challenges associated with their widespread use in a clinical setting.
COMBINING PRP WITH HYALURONIC ACID
Hyaluronic acid (HA) is an essential component of the extracellular matrix that plays a crucial role in various biological processes, including cell culture. in vitro.
Its use offers several significant advantages in cell cultures, providing an environment conducive to cell growth, differentiation and functionality [...22].
One of the main advantages of using hyaluronic acid in cell cultures in vitro is its ability to stimulate cell proliferation.
By providing a three-dimensional substrate and support structure, hyaluronic acid creates a microenvironment favorable to cell growth, which can increase the proliferation rate of cells in culture [...23].
In addition, hyaluronic acid helps maintain cell viability by providing adequate hydration and regulating osmotic balance in cell cultures.
It acts as a water reservoir, retaining the moisture needed to keep cells healthy and functional throughout the culture process. in vitro.
Another major advantage of using hyaluronic acid is its ability to improve cell adhesion.
By providing binding sites for specific cell receptors, it promotes cell attachment to the culture surface, which is essential for their proper survival, proliferation and differentiation [...24].
Finally, hyaluronic acid may play a role in modulating cell differentiation by influencing cell-matrix interactions and cell signaling.
Studies have shown that its presence in cell cultures can promote cell differentiation in certain cell types, such as mesenchymal stem cells, chondrocytes and adipocytes.
FUTURE STUDIES: ASSESSING THE EFFECT OF PRP AND PRP-HA
Thus, in this study, we plan to investigate two human blood derivatives (non-activated allogeneic human PRP [PRP] or non-activated allogeneic human PRP combined with hyaluronic acid [PRP-HA]), used as FBS substitutes for expansion in vitro of primary human myoblasts prior to myogenic differentiation.
We will evaluate the proliferation rate in vitrothe expression of various cell surface markers, their capacity for myogenic differentiation and the level of expression of the transcription factor Pax7 in generated human muscle reserve cells. in vitro.
We want to test whether PRP or PRP-HA can effectively replace xenogeneic FBS in the expansion in vitro of human myoblasts, and if these culture conditions do not alter their inflammatory phenotype.
In addition, we will test whether human myoblasts, cultured in growth medium supplemented with PRP or PRP-HA, generate a higher percentage of Pax7High reserve cells compared with FBS-treated myoblasts.
Our preliminary results strongly suggest that non-activated allogeneic human PRP or PRP-HA are effective and suitable alternatives to FBS for the generation of human muscle reserve cells in a deeper quiescent state.
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