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

doi:10.3969/j.issn.2095-4344.2015.12.023

Abstract

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

全文链接：

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

CLC Number:

R318

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.

Figures/Tables 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 1st day of culture, many human hepatocytes L-02 showed adherent growth on microcarrier samples 1, 2, 3. At the 2nd 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 3rd 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 4th and 5th 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 3rd day 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)."

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