Peripheral blood mononuclear cells contribute to myogenesis in a 3D bioengineered system of bone marrow mesenchymal stem cells and myoblasts

Abstract

In this work, a 3D environment obtained using fibrin scaffold and two cell populations, such as bone marrow-derived mesenchymal stem cells (BM-MSCs), and primary skeletal muscle cells (SkMs), was assembled. Peripheral blood mononuclear cells (PBMCs) fraction obtained after blood filtration with HemaTrate® filter was then added to the 3D culture system to explore their influence on myogenesis. The best cell ratio into a 3D fibrin hydrogel was 1:1 (BM-MSCs plus SkMs:PBMCs) when cultured in a perfusion bioreactor; indeed, excellent viability and myogenic event induction were observed. Myogenic genes were significantly overexpressed when cultured with PBMCs, such as MyoD1 of 118-fold at day 14 and Desmin 6-fold at day 21. Desmin and Myosin Heavy Chain were also detected at protein level by immunostaining along the culture. Moreover, the presence of PBMCs in 3D culture induced a significant downregulation of pro-inflammatory cytokine gene expression, such as IL6. This smart biomimetic environment can be an excellent tool for investigation of cellular crosstalk and PBMC influence on myogenic processes.

Macrophages and Urokinase Plasminogen Activator Receptor System in Multiple Myeloma: Case Series and Literature Review

“…BM was obtained from four healthy controls (M/F, 3/1; mean age, 39 years old; range, 22–55 years old). Briefly, total BM aspirate was seeded at a concentration of 50,000 total nucleated cells/cm 2 in T75 plastic flasks in minimum essential medium alpha (α-MEM) supplemented with 1% Glutagro TM (Corning, Manassas, VA, USA), 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Pen/Strep), and were incubated at 37 °C, 5% CO 2 , and 95% relative humidity, as previously described [ 78 , 79 ]. After 72 h, nonadherent cells were removed, while adherent cells were kept in culture until day 21.…”

“…All samples were run with the same PMT voltages, and a minimum of 30,000 events were recorded. Kaluza software (v.2.1, Beckman Coulter) was used for post-acquisition compensation and analysis, as previously described [ 78 , 79 ]. Cells were first identified using linear parameters (forward scatter area (FSC-A) vs. side scatter area (SSC-A)), and double cells were excluded (area vs. height, FSC-A vs. FSC-H) ( Figure 2 A).…”