Vitreous cryopreservation and thawing of adipose-derived stem cells/demineralized bone matri

doi:10.3969/j.issn.2095-4344.0464

Abstract

Abstract:

BACKGROUND: Short-term (1 week) vitreous cryopreservation avoiding the formation of ice crystals has been achieved in preserving tissue-engineered bone composed of adipose-derived stem cells (ADSCs)/demineralized bone matrix (DBM) compound through adjusting particular composition of cryopreservation fluid. However, whether vitreous cryopreservation can be utilized to cryopreserve tissue-engineered bone for long term (12 weeks) and maintain cellular viability and osteogenic function after rewarming remains unclear.
OBJECTIVE: To investigate the effects of vitreous cryopreservation on viability and osteogenic function of ADSCs for short-term (1 week) and long-term (12 weeks) cryopreservation.
METHODS: ADSCs were isolated from New Zealand rabbits and expended to passage 3. Cells at passage 3 were seeded onto DBM derived from porcine trabecular bone and followed by 1 week osteogenic induction. The tissue-engineered bone was transferred to freezing vials of 2 mL containing vitreous cryopreservation fluid and then directly quenched into liquid nitrogen. The composition of cryopreservation fluid was 30% dimethyl sulfoxide, 70% low glucose-Dulbecco’s modified Eagle medium (L-DMEM), 0.8 mol/L trehalose. Following vitrification for 1 week or 12 weeks, the composite of ADSCs/DBM was removed and thawed. After rewarming, ADSCs viability were viewed under confocal laser microscope by staining viable cells with the green fluorescent dye Calcein AM and the red fluorescent dye Propidium iodide at days 1, 3, 7, 11 and 13. The number of cells seeded onto the DBM was assayed by Hochest33258 at days 1, 3, 5, 7, 9, 11 and 13. Meanwhile, alkaline phosphatase (ALP) activity was also assayed by PNP microplate method at days 1, 4, 7, 10, 14 and 21. Osteogenic gene expression including Runx2, OCN, ALP, COL-1 was detected by real- time PCR at days 1, 4, 7, 10, 14 and 21.
RESULTS AND CONCLUSION: After cryopreservation of 1 week or 12 weeks, it was found that more red-staining live cells was observed at 1 day post-rewarming by live/dead double staining, and the green-staining live cells increased at 3 days. By Hoechst 33258 assay, it was found that the cell number decreased at 1 and 3 days post-rewarming, compared with pre-cryopreservation. However, a constant increase in the cell number was observed beginning at 3 days, reaching the pre-cryopreservation level at 5 days post-rewarming. By PNP microplate method, it was found that ALP activity reduced at 1 and 4days post-rewarming, but compared with the level of pre-cryopreservation there were no significant difference. However, a constant increase in ALP activity was detected since 4 days. By real-time PCR, osteogenic gene expression including Runx2, OCN, ALP, COL-1 reduced at days 1 and 4, but compared with the level of pre-cryopreservation there was no significant difference. However, a constant increase in the osteogenic gene expression was since 4 days. The cell viability and osteogenic function were observed without significant difference at each time point after rewarming of cells that had undergone vitreous cryopreservation for 1 or 12 weeks. Preliminary findings indicate that vitreous cryopreservation can maintain cellular viability and osteogenic function of tissue-engineered bone. Cryopreservation time (1 and 12 weeks) has no significant effect on the cell viability and osteogenic function of the tissue-engineered bone after rewarming.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Adipose Tissue, Mesenchymal Stem Cells, Decalcification Technique, Femur, Cryopreservation, Tissue Engineering

CLC Number:

R394.2

Liu Lei, Yong Qi, Li Na, Yang Duo, Zhang Guo-ying, Cui Lei. Vitreous cryopreservation and thawing of adipose-derived stem cells/demineralized bone matri[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(9): 1357-1363.

Figures/Tables 2

2.1 脂肪干细胞形态以及成骨、成脂分化能力原代培养24 h后，少量细胞贴壁生长，可见较多红细胞悬浮，培养3 d半量换液，可见大量细胞贴壁生长(图1A)。传代培养第3代后，细胞仍呈梭形，放射状排列，呈成纤维细胞样特征，增殖能力旺盛(图1B)。第3代脂肪干细胞成骨诱导2周后行碱性磷酸酶染色和茜素红染色，诱导组细胞内可见蓝染的碱性磷酸酶颗粒(图1C)，以及橘黄色的钙结节沉积(图1D)；成脂诱导2周后行油红O染色，结果显示细胞内有红染的脂滴形成(图1E)。 2.2 玻璃化冻存前细胞在脱钙骨上的增殖情况共聚焦显微镜观察，第3天时有大量的绿染活细胞黏附于支架材料上，第7天时细胞增殖明显，绿染的活细胞形态清晰可见，几乎无红染的死细胞。Hochest33258检测结果发现成骨诱导组和非成骨诱导组细胞数目均在第7天达到高峰随后进入平台期，诱导组和非诱导组保持相似的生长规律。结果证明脱钙骨有很好的组织相容性，可以保证细胞在支架材料上存活，由于第7天细胞数目达到高峰，选择诱导7 d后进行玻璃化冻存，见图2。 2.3 冻存前碱性磷酸酶活力细胞支架材料复合物经成骨诱导后随着时间延长碱性磷酸酶表达逐渐升高，14 d达到高峰随后逐渐降低，而非诱导组碱性磷酸酶活力无明显变化，第4天以后诱导组碱性磷酸酶活力与非诱导组比较差异有显著性意义(P < 0.05)，见图3。 2.4 复苏后细胞在支架材料的增殖情况玻璃化冻存1周或者12周，共聚焦显微镜观察，复苏后第1天仍然可见较多绿染的活细胞，红染的死细胞数目较冻存前多，随着时间延长红染的死细胞逐渐减少，绿染的活细胞逐渐增多。Hochest33258细胞定量结果表明，与冻存前(成骨诱导1周)的细胞数相比，复苏后第1天细胞数目降低12.8%和13.3%，复苏后第3天细胞数目分别降低30.2%和31.2%，复苏3 d后细胞数目开始持续增加，第5天细胞数目到达冻存前的83.3%和82.2%，达到冻存前水平。玻璃化冻存1周与12周比较，复苏后在各个检测时间点的细胞数目无明显差异，见图4。 2.5 复苏后碱性磷酸酶活力玻璃化冻存1周或者12周，复苏后1，4 d，碱性磷酸酶活力较冻存前降低，但是与冻存前差异无显著性意义(P > 0.05)，4 d后碱性磷酸酶活力持续升高。玻璃化冻存1周与12周比较，碱性磷酸酶活力差异无显著性意义(P > 0.05)，见图5。 2.6 复苏后成骨相关基因ALP、Runx-2、Col-1、OCN表达玻璃化冻存1周或者12周，复苏后1 d和4 d各成骨基因表达量较冻存前降低，但是与冻存前相比差异无显著性意义(P > 0.05)，4 d后各成骨基因表达量持续升高。冻存1周与12周比较，各个检测时间点成骨基因表达量差异无显著性意义(P > 0.05)，见图6。"

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