球形多孔微载体支持下高密度培养人肝细胞L-02的定时形态变化

doi:10.3969/j.issn.2095-4344.2015.12.023

中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (12): 1924-1930.doi: 10.3969/j.issn.2095-4344.2015.12.023

• 材料力学及表面改性 material mechanics and surface modification • 上一篇下一篇

球形多孔微载体支持下高密度培养人肝细胞L-02的定时形态变化

张瑞，刘明

内蒙古医科大学附属医院普外科，内蒙古自治区呼和浩特市 010050

修回日期:2015-02-17 出版日期:2015-03-19 发布日期:2015-03-19
通讯作者: 刘明，主任医师。内蒙古医科大学附属医院普外科，内蒙古自治区呼和浩特市 010050
作者简介:张瑞，男，汉族，1972年生，内蒙古自治区包头市人，上海交通大学医学院附属瑞金医院毕业，主任医师，医学博士。
基金资助:
国家高新技术研究发展计划“863”项目(2007AA02Z487 )；国家自然科学基金项目(30672043，30772105，20074031)；内蒙古科技厅面上项目(2012MS1124)，内蒙古自治区高等学校科学研究项目(NJSZY11128)

Timed morphological changes of human hepatocytes L-02 cultured at high density by the support of spherical porous chitosan microcarriers

Zhang Rui, Liu Ming

Department of General Surgery, First Affiliated Hospital, Inner Mongolia Medical University, Hohhot 010050, Inner Mongolia Autonomous Region, China

Revised:2015-02-17 Online:2015-03-19 Published:2015-03-19
Contact: Liu Ming, Chief physician, Department of General Surgery, First Affiliated Hospital, Inner Mongolia Medical University, Hohhot 010050, Inner Mongolia Autonomous Region, China
About author:Zhang Rui, M.D., Chief physician, Department of General Surgery, First Affiliated Hospital, Inner Mongolia Medical University, Hohhot 010050, Inner Mongolia Autonomous Region, China
Supported by:
the National High Technology Research and Development Program of China (863 Program), No. 2007AA02Z487; the National Natural Science Foundation of China, No. 30672043, 30772105, 20074031; the General Program of the Science and Technology Department of Inner Mongolia Autonomous Region, No. 2012MS1124; the Scientific Research Program in High Education of Inner Mongolia Autonomous Region, No. NJSZY11128

摘要/Abstract

摘要：

背景：微载体培养技术作为一项体外高浓度细胞培养技术，近年来已在肝细胞的体外培养中得到应用。
目的：对壳聚糖球形多孔微载体培养的人肝细胞L-02进行定时的形态学观察。
方法：以自制的壳聚糖球形多孔微载体样本为支架来培养人肝细胞L-02设为实验组；无壳聚糖球形多孔微载体支持下人肝细胞的培养设为对照组。对两组细胞进行定时的细胞计数，对实验组进行形态学观察，包括倒置相差生物显微镜观察和扫描电子显微镜观察。
结果与结论：两组培养的细胞数量均呈现前3 d增长，在培养第3天细胞数量达到最高值；实验组3个样本培养的细胞数明显高于对照组无微载体培养的细胞数量(P < 0.05)，实验组各样本之间细胞数差异无显著性意义(P > 0.05)。倒置相差生物显微镜下动态观察，可见前3 d微载体表面黏附生长的肝细胞则逐渐增多，培养第3天可见大部分微载体表面有许多肝细胞黏附成团，总的存活率均在90%以上，且肝细胞保持着良好的形态学结构；扫描电子显微镜观察，微载体表面、切面和内部均可看到有许多球状肝细胞紧密黏附。结果说明，以自制的壳聚糖球形多孔微载体作为一种支架，在体外三维环境下可以进行高浓度细胞培养。

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

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关键词: 生物材料, 材料相容性, 微载体, 三维, 壳聚糖, 人工肝脏, 肝细胞培养, 形态学观察, 基础生物医学, 组织工程, 国家自然科学基金

Abstract:

BACKGROUND: Recently, microcarrier culture as a high concentration cell culture technology has been used in hepatocyte culture in vitro.
OBJECTIVE: To periodically observe the morphological changes of human hepatocyte L-02 cultured by the spherical porous chitosan microcarrier.
METHODS: The self-made spherical porous chitosan microcarrier sample as a support to culture human hepatocytes L-02 acted as experimental group; non-spherical porous chitosan microcarrier as a support to culture human hepatocytes L-02 as control group. Cells in two groups underwent cell count at regular time, and the morphological changes were observed in the experimental group, including inverted phase contrast biological microscope observation and scanning electron microscope observation.
RESULTS AND CONCLUSION: The quantity of cultured cells in the two groups was increased in the first 3 days and reached the peak at the 3rd day. The cell quantity in the experiment group was obviously higher than that in the control group (P < 0.05). There was no significant difference in the three samples of the experimental group (P > 0.05). The quantity of hepatocytes adhered to the microcarrier surface was gradually increased in the former 3 days under the inverted phase contrast biological microscope. There were lots of cell clusters on the surface of a greater part of microcarriers, with the total survival rate of more than 90%, and hepatocytes kept a good morphological structure. Under the scanning electron microscope, lots of hepatocytes adhered tightly to each other on the surface and section of microcarrier as well as inside the microcarrier. It is indicated that the self-made spherical porous chitosan microcarrier as a support in three-dimensional environment can undergo cell culture with high concentration.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

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Key words: Tissue Engineering, Biocompatible Materials, Liver, Drug Carriers

中图分类号:

R318

张瑞，刘明. 球形多孔微载体支持下高密度培养人肝细胞L-02的定时形态变化[J]. 中国组织工程研究, 2015, 19(12): 1924-1930.

Zhang Rui, Liu Ming. Timed morphological changes of human hepatocytes L-02 cultured at high density by the support of spherical porous chitosan microcarriers[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(12): 1924-1930.

图/表（结果） 1

Cell counting of cultured human hepatocytes L-02

The number of cells in the two groups showed an increasing trend in the first 3 days of culture and reached peak at day 3, followed by a downward trend at days 4, 5 days. The largest increase in the cell amount was visible within 2-3 days. The cell number in the experimental group was significantly higher than that in the control group from beginning to end (P < 0.05), but there was no difference among different samples in the experimental group (P > 0.05). Experimental results showed that compared with the control group, the spherical porous chitosan microcarrier as a scaffold was better to generate a larger number of hepatocytes (Figure 1).

Dynamic observation results of human hepatocytes L-02 under the inverted phase contrast microscope (Figure 2)

Seen in Figure 2, in the experimental group, a few of scattered hepatocytes were adhered to the microcarriers at days 1-5 of culture under the inverted phase contrast microscope, and the vast majority of cells still suspended in the culture media. At the 1^st day of culture, many human hepatocytes L-02 showed adherent growth on microcarrier samples 1, 2, 3. At the 2^nd day, the cells adherent to the microcarriers were increased gradually in number, and most of the cells became a little oblate from the spherical shape, to form the clear cell contact with nuclear clearly visible. At the 3^rd day, there were many cell masses on the surface of most of the microcarriers, adherent cells were in spherical or irregular polygonal shape that was similar to the morphology of epithelioid cells, merged into pieces and spread over the surface of the microcarriers, on which, villous protuberances changed slightly. Most of hepatocytes were directly or indirectly firmly adhesive to the microcarriers. On the locally enlarged microcarriers, clusters or projections formed on the surface of microcarriers were caused by a large amount of adherent cells which were mostly seen in a viable state. At the 4^th and 5^th days, the morphological changes proceeded, but hepatocyte necrosis, loss and floating were increase locally on the microcarriers and in the media. These above-mentioned morphological manifestations were observed from human hepatocytes L-02 cultured on the microcarrier 2 as an example.

In addition, human hepatocytes cultured on the microcarrier samples 1, 2, 3 in the experimental group and in the control group for 1, 2, 3, 4, 5 days were sampled for overall survival assessment, including the cells adherent to the microcarriers and suspending in the culture medium). The overall survival rates were all over 90%, and hepatocytes maintained a good morphological structure.

Ultrastructural changes of cultured human hepatocytes L-02

Based on the cell counting and microscopic observations at different periods, cultured human hepatocytes L-02 showed a good number and growth condition at day 3 of culture. Therefore, at the 3^rdday of culture, on the clean bench, two or three samples from each of the three kinds of microcarriers were taken with a small sterile spoon followed by fixation, graded dehydration, drying, positioning and metal-spraying, and then the scanning electron microscope was used to observe the cell growth on these samples. There were sheets in mutual integration, unevenly covering the general surface of microcarrier samples 1, 2, 3, and villous projections could be seen faintly on the surface as well as the exposed surface pores. After local surface enlargement, there were many hepatocytes that were spherical and firmly adhered to the surface and pores of microcarriers. The number of hepatocytes on each microcarrier was varied and distributed unevenly, and the cell size was 20-30 μm. The hepatocyte surface was covered with microvilli, and there was a close interconnection between the cells. The spherical hepatocytes were interconnected by microvilli into a group, and local projection changes were visible on most of the cells. The above-mentioned results were observed from the human hepatocytes L-02 cultured on the microcarrier sample 3 as an example (Figure 3).

Under the electron microscope, there were uneven and mutually fused sheets and microvilli covering the surface of microcarriers 1, 2, 3, and a lot of spherical hepatocytes were closely adherent to the pores inside the microcarriers. But the number of hepatocytes on each microcarrier was varied and distributed unevenly, and the cell size was 20-30 μm. The hepatocyte surface was full of microvilli. Some hepatocytes were interconnected by microvilli into a group or even locally aggregated in heaps that were more seen. These above-mentioned morphological manifestations were observed from human hepatocytes L-02 cultured on the microcarrier 2 as an example (Figure 4).

参考文献

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引言

Bioartificial liver support system is an in vitro liver support device for the treatment of liver failure, and its efficacy is superior to other liver support systems. In recent years, relevant research has made great progress^[1-4]. High-density and high-activity culture of hepatocytes is the core problem of bioartificial liver support system. Thus, to provide an in vitro three-dimensional growth environment similar to the in vivo environment can promote the growth and differentiation of hepatocytes, which requires a high-density, high-activity culture method.

In recent years, the high-density hepatocyte culture methods in bioartificial liver research include hollow fiber tube culture, collagen fixed culture, spherical aggregation method and microencapsulated culture, but these methods have a certain degree of deficiency, thereby constraining the development of bioartificial liver studies^[5-7]. Microcarrier culture method is currently considered to be an effective method for high-density culture of animal cells, which has also been widely used in bioengineering field. Macroporous or porous microcarriers developed in recent years have many advantages, but they also differ from the high-density culture method required for bioartificial livers^[4,8-10]. “Real” porous microcarriers are full of pores both inside and outside, and these pores are interconnected with similar pore sizes. The pores, which the cells can free access to, are suitable for high-activity cell growth. Because of increased surface area, these pores are more conductive to the adhesion of cells. Porous structure is more conducive to the transport of nutrients and growth of cells. But so far, there are rarely reports on the “real” porous microcarriers, and further research and development is required. Thus, porous microcarriers have great potential in the future tissue culture.

Currently, the three-dimensional cell culture has been widely used in basic biology and biomedical research, and it is mainly characterized as a good structure that can directly reflect the structure-function relationship in comparison with monolayer and two-dimensional cultures. In addition, cell morphology and microenvironment in the three-dimensional cell culture are closer to the in vivo status^[11-13]. In the study of hepatocyte transplantation and bioartificial liver, hepatocyte culture is still the key, and how to easily obtain a sufficient number of hepatocytes with good activity and well-functioning has become the most important issue. Microcarrier culture technique as an in vitro high-density cell culture technology has been applied in the in vitro culture of hepatocytes^[10^,^14-16]. This study aimed to prepare a preliminary exploration and research of spherical porous chitosan microcarriers that acted as three-dimensional scaffolds for in vitro culture of hepatocytes, thereby providing a theoretical basis and experimental evidence for high-density, high-activity, large-scale culture of hepatocytes used in bioartificial liver and hepatocyte transplantation.

Based on the relevant literature and previous work^[17-22], the method of cell culture at a“relatively static” state was adopted in this study. Human hepatocytes L-02 were cultured three-dimensionally on the self-made spherical porous microcarriers (samples 1, 2, 3), in order to avoid the shear stress, stimulated microgravity or material exchange that exist in the other three-dimensional culture methods. Meanwhile, the morphology of cultured human hepatocytes L-02 was observed at regular intervals, including cell counting, inverted phase contrast microscope observation and scanning electron microscope observation.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
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材料方法

Design

An experimental observation of hepatocyte cultures.

Time and setting

The experiment was done at the laboratory of the Organ Transplant Center of Ruijian Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai Institute of Digestive Surgery) from September 2007 to May 2009.

Materials

Normal human hepatocytes L-02 were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences; spherical porous microcarriers (samples 1, 2, 3) for small-size three-dimensional culture were made in the laboratory.

Experimental methods

Culture of human hepatocytes L-02

The cells cultured on spherical porous chitosan microcarriers served as experimental group, while the cells cultured on spherical porous microcarriers without chitosans as controls. Small-size culture at “relatively quiescent” was used, and the main steps were as follows: Silicification of culture plates→ sterilization and preparation prior to usage of microcarriers → balance before human hepatocyte inoculation → small-size inoculation of human hepatocytes L-02→ “relatively” static culture of human hepatocytes L-02.

Human hepatocyte L-02 morphology

Cell counting: at 1-5 days of culture, samples 1, 2, 3 of L-02 cell media (1 mL) cultured with three kinds of microcarriers and the culture medium (1 mL) in the control group were taken on the clean bench for centrifugation at 1000 r/min for 5 minutes, then the supernatant was removed and the equal volume of 1 mL/L crystal violet-citric acid solution was added followed by paraffin sealing. The samples were placed in a 37 ℃ incubator for 1 hour or more, mixed again, and pipetted every 1 hour, and stored at room temperature for 3 hours. After that, the right amount of liquid close to the liquid level was extracted with a straw and placed onto the blood count plate to calculate purple-dyed nuclei.

Dynamic observation under inverted phase contrast microscope: at 1-5 days of cell culture, samples from the experimental group were taken to observe the adhesion and growth of L-02 cells on microcarriers 1, 2, 3, and photoed.

Scanning electron microscopic observation: at day 3 of cell culture, on the clean bench, two or three samples from each of microcarriers 1, 2, 3 in the experimental group were taken randomly with a small spoon, fixed with 2% glutaradehyde for 8 hours, fixed with 10 g/L osmium tetroxide for 30 minutes, rinsed with PBS (pH 7.4), dehydrated with 30%, 50%, 70%, 80%, 90%, 100% ethanol in order (each for 10 minutes), and transferred to iso-amyl acetate in room temperature for 2 hours. After dried in a critical dryer, microcarrier samples 1, 2, 3 were gently placed onto glass sheets covered with double-sided tape using tweezers. Under vacuum conditions, gold ions were sputtered, and scanning electron microscope was used to observe cell growth that was then photoed. In addition, another one sample from each of three kinds of microcarriers was taken randomly and placed onto the glass sheets covered with double-sided tape using tweezers following dying in the critical dyer. Under the vacuum conditions, gold ions were sputtered, and scanning electron microscope was used to observe cell growth that was then photoed.

Main outcome measures

Cell counting was done at regular intervals in the two groups, and cell morphology was observed in the experimental group including the inverted phase contrast microscope and scanning electron microscopy observations.

Statistical analysis

Data were processed by the first author with SSPS 12.0 statistical software, and numerical values were in line with normal distribution and expressed as mean±SD. Intergroup comparison was performed by F test and t test based on repeated measure data, and a value of P < 0.05 was considered significant.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

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讨论

Experimental findings from this study have demonstrated that the microcarrier culture method at a “relatively static” state is feasible for high-efficiency cell culture. This study was a preliminary study on the spherical porous chitosan microcarrier and hepatocyte culture, aiming to explore the feasibility and conditions of this self-made microcarrier for cell adhesion and culture. Therefore, based on the actual demands, the small-size laboratory culture method at a “relatively static” status was used in this study. The 6-well culture plates were used to control the microcarriers and inoculation concentration of hepatocytes in the small-size culture. Within the first 16 hours, the culture plates were gently shaken horizontally at different periods, and after 16 hours, the culture plates were no more shaken, and subject to static cultivation. The number of cultured human hepatocytes L-02 was significantly higher in the experimental group than the control group (P < 0.05). Microcarrier culture of human hepatocytes is a high-concentration culture method used widely in recent years^{[20, 23-25]}, including static culture and suspension culture. How to choose an appropriate culture method is based on actual demands, culture scale size, and selection of culture system. Thus, the results from this study provide new ideas and methods for a high-density culture of human hepatocytes.

In addition, the microcarrier culture is a very flexible and more sensitive culture technique. Although the technology is not very complicated, the preparatory work is crucial to cell culture^[26]. Before cell culture, microcarriers and instruments used in the experiment were sterilized or operated aseptically in strict accordance with the experimental requirements, the 6-well plates were silicified, and human hepatocytes L-02 were also balanced before inoculation. No cell contamination occurred during the experiment. Within the first 16 hours of culture, hepatocytes were mostly in suspension, which were more adherent to the microcarriers and less free. It can ensure the sterility of microcarrier-cultured hepatocytes, greatly increase the chance of cells in contact with the microcarriers, and also improve the high-density and high-efficiency microcarrier culture of human hepatocytes L-02. The experimental results also proved that the number of cultured hepatocytes on spherical porous microcarriers samples was higher than that in the control group without microcarriers at different periods (P < 0.05). Therefore, the preparatory work for microcarrier culture of human hepatocytes is also very important for the quality and quantity of cultured cells.

Currently, there are many factors affecting the microcarriers culture of human hepatocytes, and how to avoid and overcome these problems is very critical. In the microcarrier culture, there is a larger surface area per unit volume of the cells, and the slight vibration can make the microcarrier suspension, so that the concentration of cultured hepatocytes increases significantly. Microcarriers provide a support for hepatocytes, and on the microcarriers, the physiology and function of hepatocytes are close to normal. However, there are still a number of factors directly affecting the adhesion and culture of hepatocytes on the microcarriers^[27-29]: (1) microcarrier concentration and cell seeding density; (2) stirring speed; (3) affinity of microcarrier materials; (4) other factors, such as the silicification of culture plates, selection of microcarrier concentration and cell seeding density, pre-inoculation balance of cells, preparation of medium compositions and culture medium replacement. In this study, self-made spherical porous chitosan microcarriers were used for laboratory small-size cell culture: 15 mg microcarrier sample was added per well containing 5 mL culture solution, which is in accordance with the reported suitable microcarriers concentration of 3-5 g/L. Human hepatocytes were seeded at a density of 2×10⁸/L per well, which is also within the range of (1-4)×10⁸/L that has been reported at home and abroad. Experimental results show that there are no remarkable influences on the growth space and physiological activity of cultured hepatocytes. Within the first 16 hours of culture, the culture plates were gently shaken horizontally at different periods, and the majority of hepatoctyes were attached to the microcarriers, and no hepatocytes were taken off from the microcarriers and exposed to overlarge shear damage. Chitosan used for microcarrier preparation is a kind of glycosaminoglycan, containing a positively charged amino group in the molecule, and it has been widely used in many fields because of its non-toxic effects and excellent bioaffinity. Under dynamic observation of hepatocyte growth, human hepatocytes L-02 were seen to be quickly and closely attached to the microcarrier samples, indicating the chitosan materials used have a strong adhesion ability and affinity. Therefore, the self-made spherical porous chitosan microcarriers can avoid the impact of the above factors, cultured on which the most of hepatocytes have normal physiological morphology and functions.

Compared with other solid microcarriers reported in literatures, the self-made spherical porous microcarriers have a greater number of advantages. As reported, solid microcarriers are mainly used to culture human hepatocytes, including macroporous, non-porous and porous solid microcarriers^{[25, 27, 29-30]}. Similarities between the solid and self-made spherical porous microcarriers are as follows: hepatocytes are spherical, microvilli are visible on the surface, and hepatocytes are aggregated into a group. But differing from the solid microcarriers, the self-made spherical porous microcarriers are characterized that the number of hepatocytes is increased, the microcarrier surface is covered with normally distributed hepatocytes and hepatocyte masses, there are many hepatocyte inside the microcarries with no microvilli, and cultured hepatocytes become smooth and regular. Based on cell counting, these solid microcarriers that is different from the self-made microcarriers in morphology have been also confirmed to obtain more hepatocytes than the monolayer, two-dimensional culture techniques and non-microcarrier culture method. Compared with the solid microcarriers, the self-made spherical porous microcarriers have the following advantages: more hepatocytes can be adherent to the internal pores so that the seeding area can be enlarged several times to times to greatly enhance the three-dimensional space and nutrients transmission path for hepatocyte growth and to significantly increase the amount and concentration of cultured hepatocytes; hepatocytes growing within the porous are protected and exposed to small shear damage, and cultured hepatocytes are closer to the normal physiological morphology, grow vigorously, and have good function and metabolic activity.

Therefore, the self-made spherical porous chitosan microcarriers as a scaffold for high-concentration cell culture provide a theoretical and experimental basis for further microcarrier culture to support a sufficient number of hepatocytes with good function in hepatocyte transplantation and bioartificial liver research.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程
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球形多孔微载体支持下高密度培养人肝细胞L-02的定时形态变化

Timed morphological changes of human hepatocytes L-02 cultured at high density by the support of spherical porous chitosan microcarriers

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