Multipotent Mesenchymal Stromal Stem Cell Expansion by Plating Whole Bone Marrow at a Low Cellular Density: A More Advantageous Method for Clinical Use

Abstract

Mesenchymal stem cells (MSCs) are a promising source for cell therapy due to their pluripotency and immunomodulant proprieties. As the identification of “optimal” conditions is important to identify a standard procedure for clinical use. Percoll, Ficoll and whole bone marrow directly plated were tested from the same sample as separation methods. The cells were seeded at the following densities: 100 000, 10 000, 1000, 100, 10 cells/cm2. After reaching confluence, the cells were detached, pooled and re-plated at 1000, 500, 100, and 10 cells/cm2. Statistical analyses were performed. Cumulative Population Doublings (PD) did not show significant differences for the separation methods and seeding densities but only for the plating density. Some small quantity samples plated in T25 flasks at plating densities of 10 and 100 cells/cm2 did not produce any expansion. However, directly plated whole bone marrow resulted in a more advantageous method in terms of CFU-F number, cellular growth and minimal manipulation. No differences were observed in terms of gross morphology, differentiation potential or immunophenotype. These data suggest that plating whole bone marrow at a low cellular density may represent a good procedure for MSC expansion for clinical use.

Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions

“…These data are in accordance (only for HPL) with that reported in the literature about GMPcompliant isolation and expansion of BM-MSC (59)(60)(61). Moreover, we previously reported that, by plating whole BM at a low cellular density, it was possible to expand high numbers of MSCs for clinical use (30). In the present study, we further showed that, independently from the culture condition, the number of CFU-F/10 6 cells was significantly greater when the whole BM cells were plated at a low density, compared with cells plated at a high density.…”

Validation of analytical methods in compliance with good manufacturing practice: a practical approach

“…Briefly, whole bone marrow (wBM) was seeded at a density of 100,000/cm 2 in Mesenchymal Stem Cell Growth Medium (MesenCult® Proliferation Kit; Human, Stemcell technologies, Vancouver, BC, Canada) containing 10% of fetal bovine serum (FBS) in 75 or 150 cm 2  T-flasks and maintained at 37°C with an atmosphere of 5% CO 2 . After 5 days, the non-adherent cells were removed and re-feed every 3–4 days; at confluence, they were detached, and re-plated at different densities for one to four passages [9]. …”

The exciting prospects of new therapies with mesenchymal stromal cells

“…Apparently for these reasons, high density cultures have been used in most clinical trials. Recently, there has been a renewed interest in the properties of low density cultures of MSCs [38–40] and a current large NIH-sponsored clinical trial in patients with ARDS is employing MSCs expanded at low densities [41]. The issue of how best to isolate and expand MSCs in culture is still unresolved.…”