The protocol for the isolation and cryopreservation of osteoclast precursors from mouse bone marrow and spleen - PubMed (original) (raw)
The protocol for the isolation and cryopreservation of osteoclast precursors from mouse bone marrow and spleen
Iris Boraschi-Diaz et al. Cytotechnology. 2016 Jan.
Abstract
Osteoclasts are responsible for physiological bone remodeling as well as pathological bone destruction in osteoporosis, periodontitis and rheumatoid arthritis, and thus represent a pharmacological target for drug development. We aimed to characterize and compare the cytokine-induced osteoclastogenesis of bone marrow and spleen precursors. Established protocols used to generate osteoclasts from bone marrow were modified to examine osteoclastogenesis of the spleen cells of healthy mice. Osteoclast formation was successfully induced from spleen precursors using receptor activator of nuclear factor κB ligand (50 ng/ml) and macrophage colony stimulating factor (50 ng/ml). Compared to bone marrow cultures, differentiation from spleen required a longer cultivation time (9 days for spleen, as compared to 5 days for marrow cultures) and a higher plating density of non-adherent cells (75,000/cm(2) for spleen, as compared to 50,000/cm(2) for bone marrow). Osteoclasts generated from spleen precursors expressed osteoclast marker genes calcitonin receptor, cathepsin K and matrix metalloproteinase 9 and were capable of resorbing hydroxyapatite. The differentiation capacity of spleen and bone marrow precursors was comparable for BALB/c, C57BL/6 and FVB mice. We also developed and tested a cryopreservation protocol for the osteoclast precursors. While 70-80 % of cells were lost during the first week of freezing, during the subsequent 5 weeks the losses were within 2-5 % per week. Osteoclastogenesis from the recovered bone marrow precursors was successful up to 5 weeks after freezing. Spleen precursors retained their osteoclastogenic capacity for 1 week after freezing, but not thereafter. The described protocol is useful for the studies of genetically modified animals as well as for screening new osteoclast-targeting therapeutics.
Keywords: Bone marrow; Cryopreservation; Osteoclastogenesis; Protocol; Spleen.
Figures
Fig. 1
Osteoclastogenesis from bone marrow- and spleen-derived precursors. Bone marrow (BMC) and spleen cells (SC) from FVB mice were cultured overnight with MCSF (25 ng/ml). The following day, non-adherent precursors were plated at 50,000 cells/cm2 for bone marrow and 75,000 cell/cm2 for spleen cells and treated with MCSF (50 ng/ml) and RANKL (50 ng/ml) for 3–9 days for bone marrow and 3–12 days for spleen cells. Samples were fixed, and stained for TRAP. a, b Representative images of untreated cultures (left) and RANKL-treated cultures (right) generated from bone marrow precursors on day 5 (a) and from spleen precursors on day 9 (b). c, d Changes in average numbers of osteoclasts with time in cultures from bone marrow (c) and spleen (d) precursors. Data are mean ± SE, n = 5–25 experiments
Fig. 2
Optimization of cell culture conditions. a Non-adherent bone marrow or spleen precursors from FVB were plated at the indicated densities, treated with MCSF (50 ng/ml) and RANKL (50 ng/ml) for 5 days for the bone marrow and 9 days for the spleen cells, fixed, and stained for TRAP. b Bone marrow and spleen precursors from FVB mice were treated for 5 and 9 days, respectively, with MCSF (50 ng/ml) and RANKL (0, 25, 50 and 100 ng/ml), fixed, and stained for TRAP. c, d Bone marrow cells (BMC) were plated at 50,000 cells/cm2 (c) and spleen cells (SC) at 75,000 cells/cm2 (d), and treated with MCSF (50 ng/ml) and RANKL (50 ng/ml). The medium was changed either completely (90 %) or partially (50 %) at the indicated frequencies. Data are mean ± SE, n = 3–19 experiments, for a and b bone marrow and spleen groups were significantly different (p < 0.001) by two-way ANOVA
Fig. 3
Bone marrow- and spleen-derived osteoclasts express functional markers. Bone marrow and spleen precursors from FVB mice were treated with MCSF (50 ng/ml) and RANKL (50 ng/ml) for 5 days for bone marrow and 9 days for spleen cells. a–c Mature osteoclasts from FVB mice were transferred to calcium phosphate-coated plates, incubated for 48 h, and removed using 10 % sodium hypochlorite. a Representative images of resorption areas generated on calcium phosphate by bone marrow- and spleen-derived osteoclasts. b, c The average area of a single resorption pit (b) and average resorption area per 1 mm2 of substrate (c) generated by bone marrow- (BMC) and spleen- (SC) derived osteoclasts was assessed. Data are mean ± SE, n = 3–6 experiments. d mRNA was extracted and the expression of calcitonin receptor (CtR), cathepsin K (Cts K), and matrix metalloproteinase-9 (MMP9) was assessed, Data are mean ± SE, n = 3 experiments. *p < 0.05 as assessed by Student t test
Fig. 4
Comparison of osteoclastogenesis from different mouse strains. Osteoclast precursors were isolated from the bone marrow and spleen of BALB/c (grey), C57BL/6 (white) and FVB (black) mice. a The average number of osteoclasts generated from bone marrow or spleen precursors, which were plated at the indicated densities and treated with MCSF (50 ng/ml) and RANKL (50 ng/ml) for 5 days for the bone marrow and 9 days for the spleen cells. b The average number of osteoclasts generated from bone marrow or spleen precursors, which were treated for 5 and 9 days, respectively, with MCSF (50 ng/ml) and RANKL (0, 25, 50 and 100 ng/ml). c, d Bone marrow cells were plated at 50,000 cells/cm2 (c) and spleen cells at 75,000 cells/cm2 (d), and treated with MCSF (50 ng/ml) and RANKL (50 ng/ml). Changes in the average numbers of osteoclasts over time were assessed. For a–d, data are mean ± SE, n = 3–19 experiments
Fig. 5
Cryopreservation of osteoclast precursors. Bone marrow (BMC) and spleen cells (SC) from FVB mice were cultured overnight with MCSF (25 ng/ml). Non-adherent precursors were counted, washed, re-suspended in a solution containing 50 % FBS, 10 % DMSO and 40 % culture medium, placed at −80 °C for 24 h and then transferred to liquid nitrogen for 1–7 weeks. a, b The vials were thawed at 37 °C degrees in a water bath until 75 % of the vial was thawed, transferred to a 50 ml tube to which 9 ml of culture medium was added drop wise, centrifuged for 5 min at 1,000 rpm, re-suspended in 10–12 ml of culture medium and cultured in 75 mm2 flask with MCSF (50 ng/ml) for 24 h. a Non-adherent cells were collected and the number of viable cells was counted and normalized to the number of frozen cells. b Viable precursors were plated at 50,000 cells/cm2 for bone marrow and at 75,000 cells/cm2 for spleen precursors, treated with MCSF (50 ng/ml) and RANKL (50 ng/ml) during 5 and 9 days for bone marrow and spleen cells, respectively, and osteoclast numbers were assessed. For a, b data are mean ± SE, n = 3–11 experiments, bone marrow and spleen groups were significantly different (p < 0.001) by two-way ANOVA. c, d Representative images of osteoclasts generated from bone marrow (c) and spleen (d) precursors following cryopreservation
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