超声破碎及反复冻融收集脂肪间充质干细胞裂解液的最优条件

doi:10.3969/j.issn.2095-4344.2017.17.002

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (17): 2631-2637.doi: 10.3969/j.issn.2095-4344.2017.17.002

• 脂肪干细胞 adipose-derived stem cells • 上一篇下一篇

超声破碎及反复冻融收集脂肪间充质干细胞裂解液的最优条件

王俊怡¹，金银鹏²，李洪超¹，孟凌玉²，李莉²，王晓今²，周荣²，陈成伟²，傅青春²，程明亮³

¹贵州医科大学临床医学院，贵州省贵阳市 550004；²解放军第八五医院，上海肝病研究中心，上海市 200235；³贵州医科大学附属医院感染科，贵州省贵阳市 550004

修回日期:2017-05-03 出版日期:2017-06-18 发布日期:2017-06-29
通讯作者: 傅青春，硕士，主任医师，解放军第八五医院，上海肝病研究中心，上海市 200235；程明亮，主任医师，贵州医科大学附属医院感染科，贵州省贵阳市 550004
作者简介:王俊怡，女，1992年生，湖北省襄阳市人，汉族，贵州医科大学临床医学院在读硕士，主要从事传染病学研究。共同第一作者：金银鹏，男，1988年生，重庆市人，汉族，硕士，主要从事干细胞研究。
基金资助:
南京军区医学创新重大课题(14ZX01)；中国肝炎防治基金—天晴肝病研究基金项目(TQGB20150104)

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

Wang Jun-yi¹, Jin Yin-peng², Li Hong-chao¹, Meng Ling-yu², Li Li², Wang Xiao-jin², Zhou Rong², Chen Cheng-wei², Fu Qing-chun², Cheng Ming-liang³

¹Clinidal Medical College of Guizhou Medical University, Gujyang 550004, Guizhou Province, China; ²Shanghai Liver Diseases Research Center, the 85th Hospital of PLA, Shanghai 200235, China; ³Department of Infection, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China

Revised:2017-05-03 Online:2017-06-18 Published:2017-06-29
Contact: Fu Qing-chun, Master, Chief physician, Shanghai Liver Diseases Research Center, the 85th Hospital of PLA, Shanghai 200235, China; Cheng Ming-liang, Chief physician, Department of Infection, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China
About author:Wang Jun-yi, Studying for master’s degree, Clinical Medical College of Guizhou Medical University, Guiyang 550004, Guizhou Province, China Jin Yin-peng, Master, Shanghai Liver Diseases Research Center, the 85th Hospital of PLA, Shanghai 200235, China Wang Jun-yi and Jin Yin-peng contributed equally to this work.
Supported by:
the Medical Innovation Project of the Nanjing Military Region, No. 14ZX01; China Hepatitis Prevention and Treatment Foundation-Tian Qing Liver Research Fund Project, No. TQGB20150104

摘要/Abstract

摘要：

文章快速阅读：

文题释义：
超声细胞破碎法和反复冻融法：是目前常用作细胞裂解液获取的两种最常见的实验室方法，也是本篇中主要研究对比的两种方法。前者是将细胞置于超声细胞破碎机中利用声波破碎细胞；后者则是利用反复冷冻与融化时细胞中形成了冰晶及剩余液体中盐浓度的增高可以使细胞破裂。
脂肪干细胞：是指从脂肪组织中分离得到的一种间充质干细胞，不但具有跨胚层多向分化潜能，在不同的培养条件下可以分化成肌肉、软骨、脂肪组织、神经组织或肝脏组织，而且具备取材方便、来源广泛、增殖能力强、免疫原性低等优点，近年来成为了干细胞治疗研究的热点。

摘要
背景：旁分泌在间充质干细胞治疗方面起主要作用。提取间充质干细胞裂解液，可以避免直接间充质干细胞本身所致栓塞、肿瘤以及免疫排斥反应等问题，然而目前提取间充质干细胞裂解液的方法存在差异。
目的：比较超声破碎及反复冻融方法获得间充质干细胞裂解液的差异。
方法：①从健康的人腹部皮下脂肪中分离出脂肪间充质干细胞，经贴壁筛选法纯化细胞，胎牛血清培养基体外培养扩增，并采用流式细胞检测仪鉴定其常用表面标记；②采用反复冻融及超声细胞破碎2种破壁方法，将1×10⁹ L^-1，2×10⁹ L^-1，4×10⁹ L^-1 3种不同密度的细胞分别置于生理盐水与双蒸水中进行破碎，比较细胞破碎率及裂解液蛋白含量的差异。
结果与结论：①通过体外分离人体脂肪组织，可获得脂肪间充质干细胞；②细胞裂解时，细胞浓度越高，破壁所需时间越长，而破碎时间越长，则细胞破碎率越大；③生理盐水中超声细胞破碎法得到的蛋白含量较其他条件高；④结果提示在生理盐水环境下使用超声方法提取脂肪间充质干细胞裂解液尤为有效，且推荐细胞浓度为4×10⁹ L^-1以内。

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
ORCID: 0000-0002-4329-2854(Wang Jun-yi)

关键词: 干细胞, 脂肪干细胞, 间充质干细胞, 脂肪间充质干细胞, 超声, 反复冻融, 细胞裂解液, 细胞治疗, 细胞外膜泡

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

中图分类号:

R394.2

王俊怡，金银鹏，李洪超，孟凌玉，李莉，王晓今，周荣，陈成伟，傅青春，程明亮. 超声破碎及反复冻融收集脂肪间充质干细胞裂解液的最优条件[J]. 中国组织工程研究, 2017, 21(17): 2631-2637.

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.

图/表（结果） 2

Morphology and identification of ADMSCs

In the process of cultivation and amplification of primary ADMSCs, P0 generation cells grew in whirling or radial pattern from initial fibroid form to P3 generation with uniform size and close arrangement (Figure 1). Flow cytometry results showed CD73 (100%), CD44 (100%), CD90 (100%) and CD105 (99.7%) were positive over 99% cell surface, while cells were negative for CD34, CD45 and CD19 accounting for 96%.

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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引言

Mesenchymal stem cells (MSCs), a kind of adult stem cells with self-regeneration function and multi-directional differentiation potential, can be isolated from various tissue sources such as bone marrow, umbilical cord and fat, and bear similar phenotypic characteristics^[1]. MSCs can be induced in vitro to differentiate into osteocytes, chondrocytes and adipocytes^[2-3], as well as cardiomyocytes, endothelial cells and nerve cells. Due to its universal sources, strong ability of in vitro proliferation, multi-directional differentiation potential and the role as cell replacement in the management of various diseases, the research over the past few years on MSCs has covered a wide range of fields including liver diseases, cardiovascular diseases and neurological diseases^[4-7], and even clinical MSC transplantations which have been conducted by several research groups^[8-9].

As exploration goes deeper, large numbers of studies and clinical trials on the effects of cell therapy based on MSC are actually limited, and many potential risks such as tumorigenesis, embolism and immunological reaction have been found^[10-16]. With the better understanding of MSCs, it is believed that the paracrine mechanism plays an even more crucial role compared with the self-differentiation ability of MSCs, as MSCs are usually cleared quickly within a short period after being injected into the body, whereas it can promote the secretion of a number of cytokines through paracrine mechanism, which will subsequently facilitate the regeneration of new tissues via multiple mechanisms including activating endogenous stem cells, regulating immune system and reducing inflammatory reactions, restraining apoptosis of the damaged cells as well as promoting the formation of new blood vessels^[17-20]. Soluble cytokines are mostly secreted from extracellular vesicles (EVs) of MSCs, and exosomes, which recently are the highlight of the research, are considered to be the major way used by MSCs to carry out the paracrine mechanism^[21-22]. Currently, some research teams have shown their interest in stem cell lysate used for disease treatment, such as wound healing, management of acute liver failure as well as inhibitory effect on the growth of cancer. MSCs are believed to function not just as the cell replacement, but more importantly as a structure with its control functioning via paracrine effect^[23-25].

By screening through the study results of our team and other research teams, we found that a variety of methods demanding varied conditions have been used to collect MSC lysate, with ultrasonication and repeated freeze-thaw as the two methods most frequently used. However, there is no report regarding the comparison of the two ways for lysate collection^{[11, 14, 26-29]}. So, this study aimed to compare and optimize the methods for lysate extraction by a comprehensive evaluation on cytoclasis rate, the protein content in lysate and optimum number (time) of operations, so as to extract the cell lysate with higher efficiency and better perform the subsequent research of animal experiments.

材料方法

Design

A randomized controlled cytology study.

Setting and time

It took 1 month to complete of this experiment in the School of Life Sciences of Tongji University in Shanghai, China.

Materials

Adipose tissue (provided by the 85^th Hospital of PLA) was taken from abdominal subcutaneous fat of five healthy young men. The study protocol was approved by the Ethics Committee of the 85^th Hospital of PLA with ethics approval No. 2013/18. The research follows the international and national guidelines in according with the Declaration of Helsinki and the relevant set of ethical principles. All patients (or parents, guardians) provided written informed consent for publication of associated data and accompanying images. The article complies with CONSORT/STROBE reporting guideline.

Methods

Isolation and culture of human adipose-derived MSCs (ADMSCs)

Healthy human fat specimens were collected, isolated and cultured as described previously^[18,24]. The adipose tissues were washed thrice with PBS buffer and cut into 1 mm³ small pieces, and then digested with 0.15% collagenase (Gibco) for 45 minutes. After 5 minutes of centrifugation at 1 200 r/min, the cell precipitation was collected and F12/DMEM medium (Biological Industries) containing 10% fetal bovine serum (FBS) (Gibco) was added for culture in a 5% CO₂ saturated humidity incubator at 37 ℃ (Thermo Scientific)^[19]. The experiment was carried out using the third generation of cells. Passage 3 cells with higher cell activity and form stabilization were suitable for various experimental tests.

Flow cytometry was used for detecting molecular marker on surface of ADMSCs. Recovery of passage 3 hASCs cells: 3 tubes of 500 μL single cell suspension of passage 3 ADMSCs digested with 0.25% pancreatic enzyme were added with each 2 μM of anti CD44-FITC, CD90-FITC, CD73-FITC, CD19-FITC, CD34-FITC, CD45-FITC and CD105-PE (eBioscience) monoclonal antibody, respectively. They were cleaned thrice by PBS after incubating at 37 ℃ for 30 minutes.

Collection of ADSC lysate

Considering the rate for cytoclasis varies in different osmotic concentration (such as in saline solution and distilled water) and hyposmotic solution (distilled water) generally accelerate the swelling and rupture of cells, both of the solvent groups (saline and distilled water) were established for the two collection methods.

Ultrasonication method

The ADMSCs with three different densities (density a: 4×10⁹/L, density b: 2×10⁹/L and density c: 1×10⁹/L) was respectively divided into saline groups and distilled water groups. Specifically, 4×10⁶, 2×10⁶ and 1×10⁶ cells were prepared in turn, and each was dissolved in 1 mL saline or distilled water for experimental use. Ultrasound crusher (Misonix, USA) was set as follows: amplitude, 20; water bath temperature, 4 ℃; total observing, 33 minutes; trypan blue staining (Solarbio, Beijing, China) was used before and after cytoclasis, and the cytoclasis rate was microscopically calculated, with four parallel counts at each time point. In consideration of rapid dying of cells dissolved in distilled water (it was found in preliminary experiment that all c group cells dissolved in distilled water without any treatment died within approximately 12 minutes), the entire experiment need to be finished within 10 minutes immediately to detect living cells as much as possible.

The ADMSCs obtained at different time points were then centrifuged at 10 000×g for 5 minutes and the supernatant was collected for determination of the protein concentration. After BCA reagent (Solarbio) was added, the ADMSCs were placed in a 37 ℃ incubator for 15 minutes for absorbance reading at 562 nm using the microplate reader (SpectraMax M5; Molecular Devices, USA) and for the calculation of the protein concentration.

Repeated freeze-thaw method

The ADMSCs with three different densities in saline groups and double-distilled water groups were repeatedly frozen and thawed for several times. The freeze-thaw process was initiated with 30 minutes of freezing process at a -80 ℃ refrigerator till completely frozen, followed by a thawing process under room temperature (25 ℃). Trypan blue staining was employed before and after cytoclasis, and the cytoclasis rate was microscopically calculated, with four parallel counts at each time points. The lysate was collected at the freeze-thaw process for determination of protein concentration as previously described.

Cytoclasis rate and the content of protein in cell lysate

Eventually, cytoclasis curves and BCA curves for both of the two methods were compared to obtain the optimal method for lysate collection based on the comprehensive analysis of the experimental results. BCA specific detection method: Standard curve was made as specified in BCA kit (Solarbio). Lysate extracted with various disruption methods in various conditions was added into 96-well plate, and then it was placed in a 37 ℃ incubator for 15-30 minutes after BCA reagent was added. Last, its absorbance was read at 562 nm using the microplate reader (Molecular Devices, MD, USA) and protein concentration (μg/mL) was worked out according to the standard curve. Each pole was set two spared holes.

Main outcome measures

Cytoclasis rate and the content of protein in cell lysate.

Statistical analysis

Experimental data were analyzed using SPSS 19.0 (IBM Corp., Armonk, NY, USA), and the mean±SEM was used for the quantitative indicators. Statistical analysis was performed using independent-sample T test.

讨论

In the present study, two most commonly used lysate collection methods, repeated freeze-thaw and ultrasonication, were systematically compared based on the comprehensive analysis of the cytoclasis curve and protein concentration curve, aiming to identify a way for MSC lysate collection with better convenience and efficiency by optimizing the test conditions so that a better collection method can be used in the future research concerning disease treatment using animal models.

Three cell density subgroups were established respectively for the two methods to identify the association between cell density and methods and time for cytoclasis, as well as to objectively evaluate the difference between the two methods. Considering the disruption of cell membranes is related to the osmotic pressure of the solute, and solution with a lower osmolality will have an accelerated cytoclastic process, different solutes, saline and distilled water, were also introduced for test grouping so as to observe the potential difference in protein concentration in different solute groups at the timing point of complete cytoclasis. But the final emphasis of this paper is to provide convenience for future animal experiments or clinical experiments; therefore, it is presumed that prior to the animal experiment we will add NaCl required for preparation of normal saline that is made into histocompatibility solution, even if finally protein level from cells dissolving in distilled water is higher than that in normal saline.

Similar findings were observed in the cytoclasis curves of ultrasonication and repeated freeze-thaw for cells with different densities in the same solute, that is, higher cell density is usually associated with longer time for cell disruption and lower time-cytoclasis rate, and the cytoclasis rate is gradually increased with the time for cytoclasis.

Comparison between the protein concentration curves of disrupted cells with the same density in different solutes revealed that the protein concentration of the lysate at the complete cytoclasis was always higher in saline than in distilled water using either of ultrasonication or repeated freeze-thaw, particularly in the former method. It is likely that the hypoosmolality formed by distilled water leads to the swelling of the cells and a subsequent damage to part of the protein's primary structure, resulting in a loss of total protein concentration. Although a higher cytoclasis rate was obtained in distilled water, a hypoosmotic solute, we still exclude the selection of distilled water as the solute for cytoclasis as both of the comprehensive cytoclasis rate and the protein content have to be considered as the parameters for cytoclasis optimization.

Through analysis of the cytoclasis curve for cells with the same density in saline, we found that compared with the repeated freeze-thaw, ultrasonication is time-saving and has a protein concentration curve continuously above that of repeated freeze-thaw, indicating that the content of effective components collected by ultrasonication is higher than that by repeated freeze-thaw method. On the basis of a comprehensive evaluation, we believe that ultrasonication is an appropriate method and condition for lysate collection in an isosmotic solution.

In addition, it is thought that the increasing trend of protein concentration curve is caused by excessive precipitation of structural proteins due to organelle membrane rupture induced by long-term mechanical treatment, and prolonged treatment will also compromise the activity of the effective proteins in lysates such as a variety of cytokines. Therefore, we suggest a 15-minute limit in ultrasonication or two or three freeze-thaw sessions for cells at a density of less than 4×10⁹/L.

This study for the first time compared the most commonly used mechanical cytoclasis methods in scientific research (ultrasonication and repeated freeze-thaw) to optimize the method for MSC lysate collection, which provides a more convenient and efficient way in the future works. As described in some studies^{[25, 30-32]}, a new tool, Lysis Buffer Kit, could be used for cytoclasis. However, with a nature of chemical cytoclasis, this method has to employ chemical agents to promote the disruption of the cell membrane. So, it is not taken into consideration till convincing reports are given to demonstrate whether the chemical components will impose any influence on the collected cell contents (such as the stability of proteins and nucleic acids).

Moreover, the present study focused on cytoclasis rate curve and total protein content rather than other parameters for further optimization of lysate collection such as the stability of specific protein components and nucleic acids. Hence, in the further study, some effective component(s) in the cells can be used as more specific parameter(s) for a deeper exploration on the methodology for lysate collection, which is very necessary.

In this study, we optimized successfully methods and conditions of extracting MSC lysate and concluded that: ultrasonication method is time-saving and efficient for cells in normal saline; for cells with a density of 4×10⁹/L, the ultrasonication method is advised to be controlled within 15 minutes, while extraction with the freeze-thaw method should be 2-3 times as appropriate.

超声破碎及反复冻融收集脂肪间充质干细胞裂解液的最优条件

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

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