Cryopreserved Spontaneous Spheroids from Compact Bone-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

Abstract

Spontaneously formed spheroids from mouse compact bone-derived mesenchymal stromal cells (CB-MSCs) possess enhanced stemness and superior plasticity. In this study, the effect of cryopreservation on viability, stemness, and osteogenic differentiation capability of spontaneous CB-MSC spheroids were investigated. CB-MSCs were isolated from mouse femur and tibia. Spheroids were cryopreserved with various concentrations of dimethyl sulfoxide (DMSO). After thawing, the number of living and dead cells was measured. The expression levels of stem cell markers and osteogenic marker genes were analyzed. The cryopreserved and noncryopreserved spheroids were transplanted in mice with a beta-tricalcium phosphate as a scaffold to evaluate the in vivo bone-forming capability. The percentage of living cells was highest when 5% DMSO was used as a cryoprotectant, confirmed by the number of dead cells. The expression of stem cell marker genes and osteogenic differentiation capability were maintained after cryopreservation with 5% DMSO. The cryopreserved spontaneous CB-MSC spheroids showed remarkable new bone formation in vivo, identical to that of the noncryopreserved spheroids even without osteogenic induction. The cryopreserved spontaneous CB-MSC spheroids retained stemness and osteogenic differentiation capability and highlight the utility of spontaneous CB-MSC spheroids as ready-to-use tissue-engineered products for bone tissue engineering.

Keywords: MSCs; compact bone-derived mesenchymal stromal cells; cryopreservation; osteogenic differentiation capability; spontaneous spheroid.

FIG. 1.

Characterization of the spontaneous CB-MSC spheroid. Phase-contrast photomicrographs showing ALP staining of monolayer cultured cells (A) and spheroid (B). A high concentration of ALP-positive cells was observed in the middle of spontaneous CB-MSCs spheroid. Spontaneous CB-MSCs spheroids were positive for pluripotency markers: Nanog, Sox2, Oct4, and SSEA1 demonstrated (C). Scale bar: 100 μm (A); 50 μm (B, C). ALP, alkaline phosphatase; CB-MSCs, compact bone-derived mesenchymal stromal cells.

FIG. 2.

Effect of the cryoprotectant concentrations on cell viability after a freeze and thaw cycle. Different concentrations of DMSO (1%, 5%, 10%, 15%, and 20%) were assessed, with stem-C used as a conventional cryoprotectant for comparison. The percentages of viable cells were measured using the WST-8 assay (A). The highest percentage of viable cells was observed in the 5% DMSO group. The percentages of dead cells were measured using the LDH assay (B). The lowest percentages of dead cells were observed in the 5% DMSO group. Cell growth was measured at 3, 24, and 72 h after thawing (C). The highest cell growth was observed in the 5% DMSO group. Immunofluorescence for Ki-67 and TUNEL staining was performed (D). Scale bar: 50 μm. The percentage of TUNEL-positive cells was measured (E). The lowest number of apoptotic cells was observed in the 5% DMSO group. Data are presented as mean ± SD. n = 5. ***p < 0.001. DMSO, dimethyl sulfoxide; LDH; SD, standard deviation; TUNEL; WST.

FIG. 3.

Effect of spheroid cryopreservation on stemness. The results from qRT-PCR for MSC markers (A), pluripotency markers (B), and osteogenic markers (C) are shown. There were no significant differences in expression of MSC markers (CD29, Cd44, CD105, and Casp3 (SCA-1)), pluripotency markers (Fut4 (SSEA1), Sox2, Oct4, Nanog, and Klf4), and osteogenic differentiation markers (OCN, OPN, Col1a1, BSP, and Osterix) between the fresh and cryopreserved 5% DMSO groups. Data are expressed as the mean ± SEM, n = 3. MSC, mesenchymal stromal cells; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; SEM, standard error of the mean.

FIG. 4.

Osteogenic differentiation capability before and after cryopreservation. ALP staining and ALP activity for spontaneous CB-MSC spheroids without osteogenic induction (A). No positive cells were observed. ALP activity was low and no significant difference was observed between the fresh and the cryopreserved spheroids (5% DMSO). After 7 days of osteogenic induction, the cells from both fresh and 5% DMSO groups showed ALP-positive cells. The staining was almost identical between the fresh and 5% DMSO groups. ALP activity was measured after osteogenic induction, with no significant differences between the fresh and the 5% DMSO groups (B). BSP, Osterix, and DMP1 expression levels were measured after osteogenic induction, with no significant difference between the fresh and the 5% DMSO groups (C). Scale bar: 100 μm. Data are expressed as the mean ± SD, n = 3.

FIG. 5.

The in vivo bone-forming capability after cryopreservation. The bone formation was observed using HE staining of the sections from transplants. Fresh and cryopreserved (5% DMSO) spheroids were transplanted after cell seeding without osteogenic induction. Remarkably new bone formation was observed in the transplants from both the fresh and cryopreserved spheroids (A). Next, the spheroids from the fresh and 5% DMSO groups were seeded onto a scaffold and transplanted after 7 days of osteogenic induction. Abundant new bone formation was observed in both the fresh and 5% DMSO groups (B). Morphometric analyses showed no significant difference in the samples without osteogenic induction in the new bone area between the transplants from the fresh and cryopreserved groups (C). The area of new bone was also identical between the fresh and cryopreserved groups when cultured in the osteogenic induction medium for 7 days (D). Interestingly, the in vivo bone-forming capability was unaffected by osteogenic induction, with comparable new bone areas from the noninduced and induced groups. Data are expressed as the mean ± SD, n = 7. Scale bar: 200 μm (magnified images: 50 μm). HE, hematoxylin and eosin.