Isolation of Mesenchymal Stem Cells from Human Bone and Long-Term Cultivation Under Physiologic Oxygen Conditions

Abstract

Bone-derived stroma cells contain a rare subpopulation, which exhibits enhanced stemness characteristics. Therefore, this particular cell type is often attributed the mesenchymal stem cell (MSC). Due to their high proliferation potential, multipotential differentiation capacity, and immunosuppressive properties, MSCs are now widely appreciated for cell therapeutic applications in a multitude of clinical aspects. In line with this, maintenance of MSC stemness during isolation and culture expansion is considered pivot. Here, we provide step-by-step protocols which allow selection for, and in vitro propagation of high quality MSC from human bone.

Effects of hypoxia on proliferation and osteogenic differentiation of periodontal ligament stem cells: an in vitro and in vivo study

“…Recently, hypoxia has been widely explored to determine the effects on stem cell differentiation (Zhang et al, 2002;Klepsch et al, 2013). A previous study reported varying results for this analysis (Shav-Tal et al, 2001).…”

Intrinsically 32P‐Labeled Diamond Nanoparticles for In Vivo Imaging and Quantification of Their Biodistribution in Chicken Embryos

“…Additionally, MSCs represent a very suitable model for risk assessment as their response to radiation has been widely studied . Nanodiamond concentrations of 0.02, 0.2, 1, or 2 mg mL −1 were added to proliferating cells and incubated for either 24 or 72 h to assess cell viability (Figure a,b) . Only at the very high concentration of 2 mg mL −1 significantly increased death rates were revealed after 72 h. Note that an ND suspension of this concentration would have a specific activity of about 6 kBq mL −1 , assuming NDs were doped with 32 P as described above.…”

Hard Tissue Augmentation of Aged Bone by Means of a Tin-Free PLLA-PCL Co-Polymer Exhibiting in vivo Anergy and Long-Term Structural Stability

“…Human bone marrow-derived mesenchymal stem cells (MSC) were isolated, characterized, differentiated, and expanded as described previously [15]. The procedure yielded also good results when applied to isolate sheep cells.…”

“…Prior to applying the explanted cells in animal experiments the primary ovine bone marrow-derived MSC were evaluated in culture. The cells revealed a spindle-like morphology with long processes and showed indistinguishable cell proliferation and colony formation when cultivated at 3% or 20% O 2 , yet exhibited slower rates when compared to human bone marrow-derived MSC [15, 16]. Colony formation was determined by seeding cells in 58 cm 2 cell culture dishes at a density of 10 cells/cm 2 .…”

“…Ficoll-Paque TM PLUS was prepared and the aspirate layered on top with a volume ratio of 1: 1. All other steps were performed closely following the published standard protocol used to culture expand human MSC (Klepsch et al [15]). Briefly, isolated cells were seeded at a density of 200,000/cm 2 in minimal essential medium containing GlutaMAX TM -I (Gibco #41090) supplemented with 20% fetal calf serum (Invitrogen #10499044), 100 units mL –1 penicillin, and 100 µg mL –1 streptomycin (Difco #5854-59) at 37°C.…”