Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Abstract

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions -namely, patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor's own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Evaluating Feasibility of Human Tissue Engineered Respiratory Epithelium Construct as a Potential Model for Tracheal Mucosal Reconstruction

“…Plasma clot is often used for delivering stromal cells to the target site (i.e., bone defect) in clinical practice [55]. However, for tissue engineering purposes and as an alternate approach, using plasma from citrate-anticoagulated blood combined with calcium chloride as the plasma-clotting agent, is commonly practiced for the preparation of plasma gel.…”

3D fiber deposited polymeric scaffolds for external auditory canal wall

“…HMSC cultures were established as reported in our previous studies [27]. Briefly, the aspirate was diluted 1:3 in sterile saline and layered on Lymphoprep density gradient.…”

“…Due to the large scaffold size, 12 × 10 6 cells were seeded in each sample to obtain a complete cell colonization. To ensure a uniform seeding, the following three strategies were performed which availed themselves of multiple seeding shots (20 µL each) with cells suspended either in gelatin (filtered 2% aqueous solution) or in human pooled plasma, the latter crosslinked using a 7 mM calcium chloride solution (0.4 µL per µL of plasma) for 20 min [27]:…”

“…shots distributed along the body of the scaffold, which ultimately led to twelve million hMSCs seeded in each sample. Three seeding strategies were tested, including cells suspended in a gelatin aqueous solution with two different time schedules, or in pooled plasma, the latter transformed right after into a clot by crosslinking with calcium chloride to minimize cell loss [27]. Using a viable dye in between each seeding time-point permitted the empty areas to be localized, to which the second and third seeding shots were applied.…”

RUNX1 Plays an Important Role in Mediating BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells Line C3H10T1/2, Murine Multi-Lineage Cells Lines C2C12 and MEFs

“…When stimulated with lineage-specific induction media, MSCs differentiate toward osteoblastic, chondrogenic, myogenic, and adipogenic phenotypes [ 6 , 7 , 8 ]. With widespread source and characteristic of multipotent differentiation, MSCs have been widely used in bone tissue engineering in clinical setting [ 9 , 10 , 11 ]. Bone morphogenetic proteins (BMPs), which are members of the transforming growth factor-β (TGF-β) superfamily, are now acknowledged to be involved in regulating embryonic development and differentiation as well as cellular function [ 12 , 13 ].…”