幼猫眼外肌成肌细胞原代培养和鉴定

doi:10.3969/j.issn.2095-4344.2014.51.002

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (51): 8206-8211.doi: 10.3969/j.issn.2095-4344.2014.51.002

• 肌肉肌腱韧带组织构建 tissue construction of the muscle, tendon and ligament • 上一篇下一篇

幼猫眼外肌成肌细胞原代培养和鉴定

刘涛¹，刘桂香¹，尹娅楠¹，周娟²

¹青岛大学附属医院眼科，山东省青岛市 266003；²青海大学附属医院内分泌科，青海省西宁市 810001

出版日期:2014-12-10 发布日期:2014-12-10
通讯作者: 刘桂香，博士，教授，青岛大学附属医院眼科，山东省青岛市 266003
作者简介:刘涛，男，1988年生，汉族，山东省济宁市人，青岛大学在读硕士，主要从事斜视、弱视及小儿眼科方面的研究。
基金资助:
山东省自然科学基金资助项目(ZR2013HM014)

Primary culture and identification of kitten extraocular muscle myoblasts

Liu Tao¹, Liu Gui-xiang¹, Yin Ya-nan¹, Zhou Juan²

1 Department of Ophthalmology, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China
2 Department of Endocrinology, Affiliated Hospital of Qinghai University, Xining 810001, Qinghai Province, China

Online:2014-12-10 Published:2014-12-10
Contact: Liu Gui-xiang, M.D., Professor, Department of Ophthalmology, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China
About author:Liu Tao, Studying for master’s degree, Department of Ophthalmology, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China
Supported by:
the Natural Science Foundation of Shandong Province, No. ZR2013HM014

摘要/Abstract

摘要：

背景：制作斜视模型需要双眼前移、具有一定双眼视功能的高级哺乳类动物(如猫科)，体外分离培养纯度高、活力强、生物特性均一的幼猫眼外肌成肌细胞，对组织工程及探讨药物治疗斜视可行性显得至关重要。

目的：探索幼猫眼外肌成肌细胞原代培养方法。

方法：利用两步酶消化法联合差速贴壁法获取幼猫眼外肌成肌细胞，进行体外培养，对获得的细胞进行形态学研究，以免疫组织化学方法进行鉴定。

结果与结论：在生长培养基条件下，成肌细胞增殖旺盛；在分化培养基条件下，成肌细胞分化良好，可融合成肌管，且表达结蛋白。提示利用两步酶消化法联合差速贴壁法成功培养幼猫眼外肌成肌细胞，该方法实用性强，所得成肌细胞纯度高，为进一步观察成肌细胞生长特性，并深入探讨药物治疗斜视可行性奠定了基础。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

关键词: 组织构建, 组织工程, 斜视, 眼外肌, 成肌细胞, 原代培养, 鉴定, 幼猫, 山东省自然科学基金

Abstract:

BACKGROUND: Strabismus models need to be made in senior mammals with certain binocular visual function and eyes located forward, such as Felidae. Myoblasts of cat extraocular muscle via in vitro isolation and cultivation are with characteristics including high purity, strong vitality, and uniform biological characteristics, which are critical for tissue engineering and feasibility of drug therapy for strabismus.

OBJECTIVE: To explore an experimental method for primary culture of kitten extraocular muscle myoblast.

METHODS: Myoblasts from kitten extraocular muscle were obtained by the two-step enzymatic digestion method and pre-plating technique, and then passaged following primary culture in vitro. The cells were counted and observed by light microscopy combined with immunocytochemical identification.

RESULTS AND CONCLUSION: Myoblasts grew well under the growth medium and differentiated well under the differential medium, which could be fused into myotube and express Desmin. The two-step enzymatic digestion method conveniently permits the purification and proliferation of myoblasts. These results provide the basis for future studies to further observe the growth characteristics of myoblasts and discuss the feasibility of drug treatment of strabismus.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: strabismus, myoblasts, desmin, muscle contraction

中图分类号:

R318

刘涛，刘桂香，尹娅楠，周娟. 幼猫眼外肌成肌细胞原代培养和鉴定[J]. 中国组织工程研究, 2014, 18(51): 8206-8211.

Liu Tao, Liu Gui-xiang, Yin Ya-nan, Zhou Juan. Primary culture and identification of kitten extraocular muscle myoblasts[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(51): 8206-8211.

图/表（结果） 4

Separation and culture in vitro of myoblasts

Myoblasts from the extraocular muscle were treated by two-step combined differential attachment (pancreatic enzyme and type II collagenase), and the cell survival rate was (91.0±2.1)% via trypan blue staining. After observation, cells cultured separately were greatly different from the myoblasts in the late phase, and would not merge into a myotube. The isolated original generation of myoblasts was in spherical shape under inverted microscope with strong refraction (Figure 1A). The cells began to be adhered to the wall after 12 hours of culture (Figure 1B), became fusiform or spindle shape gradually after 24 hours, and completely adhered after 72 hours (Figure 1C). The myoblasts were arranged parallel to the long axis. When myoblasts completely adhered and grew for a period of time, many myoblasts which were close with each other began to fuse. If cell fusion rate was more than 80% still without digestion and generation of the cells, myoblasts would appear to have widespread and irreversible fusion. On the 72^nd hour of differentiation culture, a great mass of myotube cell formed (Figure 1D). After 30 minutes of subculture, the generated cells began to adhere to the wall and fully attached after 12 hours (Figures 1E, F).

Identification and purity results of myoblasts

Desmin filaments of myoblast antibody were stained. The cytoplasm was dyed brown or brownish red with one or two light-dyed cell nuclei. If more than 90% of the cells presented positive reaction, the cultured cells were extraocular muscle cells, as shown in Figure 2. After 8 days of primary culture, myoblasts were given immunochemical staining and then positive cells were counted. The cell purification rate was (95.0±3.8)%.

Proliferation ability of myoblasts

Myoblasts seeded onto 24-well plates were adhered to the wall quickly, and completely adhered after 12 hours with a larger growth rate. The cells entered the logarithmic phase on the 2^nd day, while the growth slowed significantly on the 6^th day due to contact inhibition. The cell growth curve is shown in Figure 3.

Differentiation capability of myoblasts

The myoblasts were added with differentiation medium and fused in large amount. The fusion rate was increased significantly at 24 and 48 hours and reached the peak at 72 hours. After that, the variation tended to be stable, as shown in Figure 4. The myoblasts were arranged parallelly and mutually fused into myotubes. Multiple nuclei were arranged in the middle of the myotube (Figure 1D). Myotube formation is a sign of skeletal satellite cell differentiation^[7].

参考文献

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[4]Fujimaki S, Machida M, Hidaka R, et al. Intrinsic ability of adult stem cell in skeletal muscle: an effective and replenishable resource to the establishment of pluripotent stem cells. Stem Cells Int. 2013;2013:420164.
[5]Yin H, Price F, Rudnicki MA. Satellite cells and the muscle stem cell niche. Physiol Rev. 2013;93(1):23-67.
[6]The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.
[7]Zhou F, Qin YW, Jing Q, et al. Primary culture of adult rat skeletal myoblasts. Zhonghua Laonian Xinnaoxueguan Bing Zazhi. 2001;3(1):43-46.
[8]Wu H, Ren Y, Li S, et al. In vitro culture and induced differentiation of sheep skeletal muscle satellite cells. Cell Biol Int. 2012;36(6):579-587.
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[13]Wei Y, Li Y, Chen C, et al. Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture. Exp Cell Res. 2011;317(7):1016-1027.
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[17]Gao P, Zhu XT, Yang YC, et al. The effects of glycyl-glutamine on the proliferation of in vitro cultivated porcine skeletal muscle satellite cells and medium IGF-1 level. Huanan Nongye Daxue Xuebao. 2005;26(2):100-102.
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引言

Strabismus not only affects the development and formation of binocular vision function, but also destroys the established visual performance, resulting in a series of perceptual disorders^[1]. Treatment for concomitant strabismus at present is still given priority to surgical treatment, which corrects eye positions by changing the end position or the length of the extraocular muscle. For each patient, in addition to the certain operation risks, the long-term effect is not stable due to individual differences. Postoperative recurrence rate is increased gradually as time goes on and many patients require reoperation^[2].

Ideal animal modeling is a key part in the study on etiology, pathogenesis and treatment of concomitant strabismus. Cat’s eyes are located in the right ahead of the face, with certain convergence capacity. Strong contraction force of medial rectus provides necessary conditions for convergence and adjustment. And the distribution, starting point, end point, direction and adjacency of cat’s extraocular muscle and orbital nerves are highly similar to the homonymic structures of human. These suggest that cats can be made into an appropriate model of strabismus. Extraocular muscles contain muscle satellite cells. Isolated muscle satellite cells that are cultured in vitro are collectively known as myoblasts. Skeletal myoblast is a kind of muscle-derived stem cell, which plays an important role in the growth, repair and regeneration of skeletal muscles^[3-5]. Research on growth, proliferation and gene expression changes of myoblasts cultured in vitro provides the basis and thought for the feasibility of looking for drugs to treat strabismus. This study aimed to establish an effective system for isolation, culture and identification of kitten extraocular myoblasts, thus laying a foundation for further experiments.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

材料方法

Design

A single sample observation.

Time and setting

The experiment was conducted in the Biochemical Laboratory of Qingdao University from August to December 2013.

Materials

Animals

We selected 12 domestic cats of either gender, which were, in sensitive period (4-6 weeks), given suitable feed under appropriate environment. All the disposals were in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued from the Ministry of Science and Technology of the People’s Republic of China in 2006^[6].

Methods

Primary culture of kitten extraocular muscle myoblast

Healthy kittens, aged 6-8 weeks, were anesthetized with ketamine and scarified through air embolism. After washed by soapy water for three times (20 minutes), the kittens were immersed in beakers with 75% ethanol in volume fraction for disinfection (30 minutes). Then the kittens were wiped with sterile gauze and placed on aseptic operating table. After repeated flushing of conjunctival sac with gentamicin and sterile draping, the intact eyeballs were separated carefully with medial rectus behind the eyeball as much as possible. The separated eyeballs were placed in a sterile culture dish, and risen with PBS (the entire eyeball including extraocular muscle) in clean bench. Medial rectus muscle that was isolated as much as possible was flushed with PBS repeatedly and cut into small pieces (< 1 mm³) in a disinfected Petri dish and then put in a 15 mL centrifuge tube. The specimen was digested by 5 mL of type II collagenase (1 g/L) for

30 minutes, during which pipetted for three times, and then centrifuged at 2 000 r/min for 3 minutes, and the supernatant was abandoned. The specimen was digested by 5 mL of trypsin (2.5 g/L) for 40 minutes, during which pipetted for three times, and 5 mL of growth medium (DMEM/F-12 culture, 20% fetal bovine serum, 100 U/mL penicillin-streptomycin solution) was used to terminate digestion. Sterile pipette was used for repeatedly blowing, and then 200 stainless steel mesh sieve was used for filtration; the filtrate was centrifuged for 3 minutes (3 000 r/min) and the supernatant was abandoned. The growth medium was re-suspended and placed in untreated culture bottle, which stood in an incubator at 37 ℃ with CO₂ volume fraction of 0.05 for 30 minutes. One drop of cell suspension and one drop of 0.2% trypan blue solution were mixed for cell counting. The percentage of unstained living cell to the total cell was calculated and the cell vitality was identified. The cell suspension was inoculated in an untreated culture bottle in which 4 mL of growth medium was added, and the cells were cultured in the incubator at 37 ℃ with CO₂ volume fraction of 0.05. When the cells were adherent to the wall of flask, free cells were removed by changing liquid. When cell cultured was confluent to 80%, the culture liquid was removed and 2 mL of 0.25% trypsin digestive juice was added. The cells were cultured in water bath at 37 ℃ for 1-2 minutes, when the cells shrank to round, they were made fall off from the bottle wall by gently patting, and the growth medium was added to terminate the digestion. The cell suspension was moved into the 15 mL centrifuge tube to be centrifuged at 3 000 r/min for 3 minutes, then the supernatant was removed and the growth medium was added. After pipetting, cells were passaged based on the cell density of 1:2. Each bottle was supplanted by culture medium to 4 mL, and cultured in the incubator at 37 ℃ and 5% CO₂, with liquid change of every 3 days. The myoblasts were observed under inverted microscope for the shape, dynamic change, refraction and adherence time, and the images were taken on cell growth situation at different phases.

Identification and purity of kitten extraocular muscle myoblasts

The third generation of myoblasts was taken for climbing. When the myoblasts were completely adhered to the cover glass wall, paraformaldehyde (40 g/L) was used for fixation. Endogenous peroxidase interference was removed by 3% hydrogen peroxide, and then the cells were added with 100 μL Desmin primary antibody to culture at 37 ℃ for 60 minutes and then added with 100 μL of secondary antibody to culture at 37 ℃ for 30 minutes. DAB kit was used for develop for 10 minutes, hematoxylin for staining, glycerin gelatin for cementing and microscope for observation. Brown or brownish-red particles in cytoplasm referred to positive staining, with fibroblasts as the negative control (myoblasts: positive, in brown or brownish red; fibroblasts: negative, in blue). After immunochemical staining of the cultured cells, five view fields were randomly selected under microscope (×200). The proportion of Desmin positive cells on all the cells within the field of vision was recorded. Purification rate=number of stained positive cells/total number of cells, and the average value was the percentage of myoblast purification.

Proliferative ability of myoblasts

The sub-cultured third generation of cells was digested with 0.25% trypsin and seeded into the 24-well culture plate at the density of 1×10⁵/well. The cells were added with growth medium (containing 20% fetal bovine serum) and cultured in the incubator at 37 ℃ with the CO₂ volume fraction of 0.05. The cells were taken from three random holes of each plate daily to be digested and counted. The average value was taken, with cultivation days as the x-coordinate and cell average as the y-coordinate to draw myoblast growth curve. Culture medium of the rest cells was changed every 3 days.

Differentiation capability of myoblasts

The sub-cultured third generation of cells was taken. When the cells grew to the beginning of the fusion, the medium was changed for differentiation medium (DMEM/F-12 culture medium containing 2% fetal bovine serum, 100 U/mL penicillin-streptomycin solution). On the 5^th day of differentiation, cell morphology and growth situation were observed under inverted phase contrast microscope. From the day of differentiation culture, cell fusion rate was determined (five 200-fold view fields were randomly taken to calculate the ratio of the number of myotube nuclei to the total number of nuclei in each view filed).

Main outcome measures

Morphology and cell survival rate of myoblasts from the extraocular muscle of kittens, the growth and differentiation conditions, and the purity and staining of the cultured cells.

Statistical analysis

SPSS 17.0 (SPSS, USA) was used by the first author for statistical processing. The measuring data was presented as mean±SD, and analyzed by t-test.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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讨论

In this experiment, we improved the method of Wu et al ^[8]. Mechanical method combined with two-step enzyme digestion method is used to cut the muscle tissue into pieces. Type II collagen enzyme is used to separate single muscle fibers, while trypsin is used to release skeletal satellite cells from the area between basement membrane and myolemma. For different kinds of animals, differences in myolemma and composition proportion lead to different enzyme digestion time. Too long digestion time can damage the myoblasts while too short digestion time can reduce the acquisition of myoblasts^[9-10]. Good controlled enzyme digestion time, therefore, is crucial. Experimental results in this study show that the best digestion time of the extraocular muscle of kittens is 30 minutes by type II collagenase and 40 minutes by trypsin, and the cells are pipetted for three times in each period.

The key of skeletal myoblast primitive culture is to improve the purity of cells. The skeletal tissue contains a lot of impure cells given priority to fibroblasts and red blood cells. Fibroblasts have the characteristics of strong differentiation potential and rapid proliferation in vitro ^[11], which may affect the purity of myoblasts. Red blood cells do not stick to the wall, so they can be removed by liquid replacement. Since fibroblasts are easier to attach to the wall, He and co-workers adopted^[12]differential attachment combined with Percoll gradient centrifugation for purification of satellite cells, to get high-purity skeleton satellite cells. Differential attachment was adopted in this experiment. The digested cell suspension was mixed with growth medium to pre-culture in the CO₂incubator for a period of time. Repeated experiments found that after 30 minutes of pre-culture, most of the fibroblasts were already attached to the wall, while myoblasts were almost free, when the cells in culture liquid were basically purified myoblasts. Wei et al^[13]used flow cytometry to screen the cells and performed in vitro culture in order to get high-purity cells. In addition, Wang et al ^[14] also can get highly purified myoblasts group via cloning culture. Rada et al ^[15-16]used magnetic cell sorting to combine specific monoclonal antibody with immune magnetic beads, which were combined with the cells. Then the cells were placed in filtration column of the magnetic field for adsorption. After that, the magnetic field was removed and the purified cells were obtained. But these methods are complex to operate with high cost, so they are difficult to be widely used in experimental studies. Moreover, selection of culture medium is also very important for the purification of cells during in vitro culture. Compared to DMEM medium, DMEM/F-12 medium has relatively more nutrients and it contains glutamine in addition, which has significant promotion effects on cell proliferation during in vitro culture^[17]. Rando and Blau^[18] found during in vitro culture of primary rat myoblasts in DMEM medium, fibroblasts showed a better growth trend than myoblasts and became dominant cells on the 8^th day; while using F-10 medium, myoblasts grew faster and eventually became the dominant cells. In this study, the F-12 medium was used which is similar to F-10 medium and the myoblasts from kitten extraocular muscles had good growth, proliferation and differentiation in vitro, with the purity of more than 95% on the 8^th day.

Literature reports that myoblasts have poor attachment, so if untreated glass bottle or disposable plastic culture bottle is used, the cells will die without attachment^[19]. This experiment adopted culture bottles with or without treatment of polylysine respectively to culture cells. The results showed that myoblasts in treated culture bottle were earlier in both the beginning of attachment and complete attachment than those in untreated culture bottle. Myoblasts in both of the two kinds of culture bottles showed good attachment and strong proliferation, and the vast majority of myoblasts did not die for not sticking to the wall. Whether the pretreatment of culture bottle impacts the cell proliferation and differentiation characteristics still needs further research.

Skeletal myoblasts are a kind of muscle-derived stem cells. Its functional protein, desmin filaments, is special for the skeletal muscle^[20-21]. Cellular immunochemical staining is used for monoclonal antibodies of desmin filaments, and more than 90% of the cells show positive results, proving the cultured cells are skeletal muscle cells, namely the extraocular muscle cells.

By drawing the myoblast growth curve and observing its proliferation characteristics, this study found that the kitten extraocular myoblast cultured in vitro could extend up to 9-10 generations, but with the increase of number of generation, the ability of cell proliferation was gradually declined, thus a large number of flat and irregular cells appeared with aging characteristics. Referencing to Menasché’s views^[22], therefore, kitten extraocular myoblasts used for subsequent experimental study are better within fifth generations. High-density cell culture can promote cell differentiation, but to facilitate to observe cell fusion in subsequent experiments and further compare cell differentiation ability, objective and easy differentiation cultivation method must be used, so as to avoid a wide range of cell integration and differentiation in a short time. Under the culture condition in vitro, the speed of cell differentiation can be controlled by reducing animal serum content in culture medium. Our experimental findings indicate that with DMEM/F-12 medium containing 2% fetal bovine serum in volume fraction as differentiation medium, myoblasts from the extraocular muscle have smooth differentiation and good growth condition.

Literatures^[23-27] have reported the separation and purification methods of myoblasts. We have successfully conducted the primary culture of myoblasts from kitten extraocular muscle, observed their growth, proliferation and differentiation characteristics, and confirmed that the cultured cells were extraocular myoblasts through the cell immunochemical tests. This experiment lays a foundation for further observation on the growth characteristics of myoblasts as well as morphological and functional changes of the skeletal muscle, and further exploration on the feasibility of the drug treatment for strabismus is required.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

幼猫眼外肌成肌细胞原代培养和鉴定

Primary culture and identification of kitten extraocular muscle myoblasts

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