Optimization of repeated freeze-thaw and ultrasonication for collection of lysate of adipose-derived stem cells

doi:10.3969/j.issn.2095-4344.2017.17.002

Abstract

Abstract:

BACKGROUND: It has been believed mesenchymal stem cells (MSCs) play a role in treatment through paracrine mechanism. Various side effects such as embolism, tumorigenesis and immunological reaction caused by direct injection of MSCs can be avoided by extracting MSC lysate. However, there is a larger difference in current collection methods and standards of MSC lysate.
OBJECTIVE: To compare repeated freeze-thaw and ultrasonication for the collection of lysate of MSCs.
METHODS: Adipose-derived mesenchymal stem cells (ADMSCs) were isolated from the abdominal subcutaneous fat of healthy individuals, and purified with adherence screening method, followed by in vitro amplification using fetal bovine serum medium. The common surface makers of these cells were tested by flow cytometry (1×10⁹, 2×10⁹, 4×10⁹/L). Repeated freeze-thaw and ultrasonication were employed for cell cytoclasis at three different densities respectively in saline and double distilled water, and a comprehensive comparison was performed on cytoclasis rate and the content of protein in cell lysate between the two methods.
RESULTS AND CONCLUSION: (1) ADMSCs obtained from in vitro isolated human adipose tissue grew in a swirl or radial pattern with a homogenous size and neat arrangement. CD44, CD90, CD105 and other commonly used surface markers were highly expressed. (2) The study for optimization of lysate collection revealed that the higher cell density implicated a longer time for cell wall disruption and cytoclasis, as well as significantly increased cytoclasis rate. (3) BCA protein assay showed that the highest content of protein was obtained in saline solvent using ultrasonication method. Comprehensive analysis on the results leads to a conclusion that ultrasonication method with saline as the solvent is the optimized method for extraction of ADMSCs lysate, and the cell concentration of less than 4×10⁹/L is recommended.

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: Sonication, Freezing, Tissue Engineering

CLC Number:

R394.2

Wang Jun-yi, Jin Yin-peng, Li Hong-chao, Meng Ling-yu, Li Li, Wang Xiao-jin, Zhou Rong, Chen Cheng-wei, Fu Qing-chun, Cheng Ming-liang. Optimization of repeated freeze-thaw and ultrasonication for collection of lysate of adipose-derived stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(17): 2631-2637.

Figures/Tables 2

Optimization of the lysate collection from ADMSCs Ultrasonication method The cytoclasis curve of ultrasonication demonstrated that in distilled water groups (Figure 2A), the cytoclasis rate continuously increased with the time for cytoclasis in the cells with three different densities, with over 95% at 1 minute for the cells with density c, and 98.25% in 10 minutes for those with the density a. Similar results were also observed in the saline groups (Figure 2B). These findings indicate that in the same solute, a higher cell density is associated with longer cytoclasis time and lower time-cytoclasis rate, and the cytoclasis rate is significantly increased with the time for cytoclasis. Comparison between groups with different solutes showed a quicker cytoclasis process in distilled water groups than in the saline groups for all the three density cells. For example, it took approximately 10 minutes for the cells with density b to be completely disrupted (> 98%) in distilled water, in contrast to 13 minutes in saline. This shows that the cytoclasis rate of ultrosonication method is higher in distilled water than in saline for the cells with a same density. The protein concentration curve obtained by BCA method (Figure 2C, D) revealed that the protein concentration of the lysate in both of the two solutes showed an elevated trend with the time of ultrasonication, with a significantly higher protein concentration measured in saline groups than in distilled water groups at each time point. The cells had a good protein activity when a complete cytoclasis was just achieved. Meanwhile, the protein concentrations were also compared in the cells with different densities. Shown on the cytoclasis curve, the protein concentration in saline groups at the time point of cytoclasis was respectively 0.573, 0.277 and 0.114 mg/mL for the cells with three different densities, all of which were higher than those in distilled water groups. Repeated freeze-thaw method The cytoclasis curve with repeated freeze-thaw method (Figure 3A, B) showed that in distilled water, a cytoclasis rate of at least 95% was achieved after the first freeze-thaw for all the cells with three densities, and a complete disruption was obtained after the second freeze-thaw process. In contrast, in saline groups only a cytoclasis rate of 14% was obtained after the first freeze-thaw for cells with density a, and 87.88% for cells with the density c. The cytoclasis rate gradually increased with the progression of the freeze-thaw process. These results imply that repeated freeze-thaw method shares a same principle with ultrasonication for cytoclasis. Comparison between different solute groups revealed that the cytoclasis rate in freeze-thaw method was significantly higher in distilled water than in saline. Protein concentration curve derived from BCA method (Figure 3C, D) showed that the number of freeze-thawing processes was associated with the increased protein concentration of lysate in both of the solutes. Comparison between the solutes revealed the protein concentration at the timing point of complete cell disruption was higher in saline groups than in distilled water groups."

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