| Acute liver failure (ALF) is a severe clinical syndrome characterized by critical condition of the patient, fast progression, unfavorable prognosis and a high mortality, whose only effective treatment by far is orthotopic liver transplantation (OLT). However, due to a series of factors including the lack of donor organ, the high cost for covering preoperational period, sophisticated operational procedure and the long-term usage of immunosuppressive agents, patients usually die of quickly aggravated condition while waiting for donors, which significantly restricts the popularization of liver transplantation. Under such a background, bioartificial liver support system (BALSS) gradually becomes another important treatment for liver failure for its advantages of clearing various kinds of harmful substances while supplying necessary substances for patients, improving the internal environment, substituting certain functions of the failing liver, and creating regenerating conditions for the liver. As a bridge towards liver transplantation and a lower mortality, BALSS turns into the second choice of option for liver failure after liver transplantation.Bioartificial liver (BAL) relies mostly on the biological functions of its core, hepatic cells in the reactor, to support patients with liver failure. Generally, hepatic cells require serum to grow. In ordinary media, the majority of hepatic cells cannot reproduce without serum, because serum provides hormones needed for cell growth and reproduction, growth factors, transfer proteins and other nutrients to maintain the healthy growth and to promote growth and reproduction. During BAL treatment, however, serum residue in the reactor easily triggers allergic reaction when in direct contact with the blood of patients of experimental animals, resulting in unfavorable therapeutic effect or even death of experimental animals. Therefore, it is of vital importance to clear serum residue in BAL, which is also an immediate problem to solve in BAL application.Meanwhile, studies revealed that in cell culture serum has many other disadvantages. First, bioactivity and factor difference contained in serum with different batches would result in unfavorable reproducibility of the product and results, thus lots of extra works would be contributed to validation. Second, serum from unstable sources could contain unknown components that inhibit cell growth, which is adverse to isolation and purification of target products like vaccine and monoclonal antibody. Last but not least, there exists the risk of contamination with exogenous virus and pathogenic factors that serum could be infected by virus and mycoplasma.As a result, many scientists started to carry out researches on serum-free medium. This medium replaces serum with substances with defined components, not only satisfying the needs to cell culture but also avoiding disadvantages caused by serum usage. Consequently, serum-free medium became popular and serum-free techniques of cell culture gained attention from researchers.In sum, this research focuses on serum-free culture of hepatic cells. In terms of amount, vitality, function and other aspects, we try to achieve the same effect in C3A cell line culture in serum-free medium as in serum medium in order to eliminate all sorts of disadvantages of serum-free medium as well as to lay a foundation for the application of hepatic cells and BAL in clinical treatment. To be specific, this research includes the following two parts: Ⅰ. A serum-free medium suitable for hepatic cells growth. Ⅱ. The comparative study of C3A cell line culture in four different serum-free media.ObjectiveTo develop a serum-free medium suitable for hepatic cells (C3A) growth in order to lay the foundation for hepatic cells and BAL in clinical treatment. MethodsThere were three groups in the experiment according to different media, the serum-free medium group, the complete medium group and the basic medium group. We cultured C3A cells by gradually decreasing serum concentration so that cells adapted themselves to the change in media and adjusted themselves to serum-free medium. After the cells stabilized, we observed their growing status, growth curve and vitality curve. We also used automatic biochemical analyzer to measure the ALT leakage and urea amount in the supernatant; we measured albumin secretion through radioimmunoassay; and we detected the variation of genetic level by real-time qPCR.Results:In all three groups, cells grew vigorously, morphologically normal, with translucent and abundant cytoplasm and favorable adherence. The growth began24hours after inoculation and continued with time, then decreased gradually after reaching a peak. The experimental group and the negative control group reached the peak in the5th day after inoculation, with maximum amount of cells (2.98±0.032)×106and (1.90±0.091)×106respectively. Then the growth began to fall. But the positive control group reached its peak in the7th day after inoculation with a maximum amount of cells (3.50±0.060)×106. Cells in the experimental group and the positive control group had similar growth rate. Their density during the culture period was not significantly different in statistics except for the6th and the7th day(p=0.000, p=0.000) after inoculation (p>0.05),but that of the experimental group was significantly higher than the density of the negative control group from the3rd to the8th day after inoculation (p=0.000).From the5th day on, there was no statistically significant difference in cell vitality between serum-free medium group and complete medium group (P=0.429)but the vitality of these two group is significantly higher than that of the basic medium group (p<0.001).From the2rd day on, the urea synthesis level of the serum-free medium group was significantly higher than that of the other two groups (p<0.001)while the level of the complete medium group was higher that than of the basic medium group (p<0.001).There was also statistically significant difference in albumin secretion among the three group (p<0.001),but the secretion amount of these groups was close to each other, which indicates that serum-free medium is capable of maintaining cell functions. Besides, real-time qPCR demonstrated that there was no statistically significant difference in gene expression of UGT, GST, AFP and ALB between serum-free medium group and complete medium group (P=0.640,0.075,0.504,1.000) but the gene expression of CK18, CK19and HNF4a decreased (P<0.001).ConclusionWe developed a serum-free medium suitable for hepatic cell (C3A) growth, which can achieve the same effect as traditional serum media in terms of cell amount, vitality, function and other aspects, thus we lay a foundation for the application hepatic cells and BAL in clinical treatment.ObjectiveThis research aims to find out the serum-free medium which is most suitable for hepatic cell growth and can to the greatest extent yield the function of hepatic cells by comparing the effect of hepatic cell (C3A) culture in four different serum-free media thus to lay a foundation for the culture, screen and application of BAL seed cell as well as the application of BAL in clinical treatment.MethodsThere were six groups in the experiment according to different media, group A with HepatoZYME-SFM medium, group B with William’s Medium E, group C with DMEM/F12medium containing ITS, EGF, nicotinamide, Asc2P, dexamethasone, L-proline and so on, group D with self-developed serum-free medium, group E, the positive control group, with complete medium and group F, the negative control group, with basic medium. We cultured C3A cells by gradually decreasing serum concentration so that cells adapted themselves to the change in media and adjusted themselves to serum-free medium. After the cells stabilized, we observed their growing status, growth curve and vitality curve. We also used automatic biochemical analyzer to measure the ALT leakage and urea amount in the supernatant; we measured albumin secretion through radioimmunoassay; and we detected the variation of genetic level by real-time qPCR.ResultsIn all six groups, cells grew vigorously. By observing the growth curve we found that the growth began24hours after inoculation and continued with time, then decreased gradually after reaching a peak. Group A, B, C, D, E and F reached the peak in the5th day after inoculation, with maximum amount of cells (1.40±0.060)×106,(2.48±0.041)×106,(3.01±0.050)×106,(2.98±0.032)×106and (1.90±0.091)×106respectively. Then the growth began to fall. But group E reached its peak in the7th day after inoculation with a maximum amount of cells (3.50±0.060) X106. Group C maintained a relatively high growth rate in the early stage of culture whose cell amount apparently outnumbered other groups (p＜0.001). In the4th day, the cell density of group E exceeded that of group C, and there was significantly different in statistics between the two groups (p=0.000). The low density of group A became evident since the3rd day and it remained being lower than other groups (P<0.001). Group B and F had similar cell density which is of mediate level. The density of group D and E was not significantly different in statistics except for the6th and the7th day after inoculation (p>0.05). As to cell vitality, group E remained being the highest in the rank whose vitality was significantly higher than other five groups during the first three days (p<0.001) while the vitality of group B and F turned out to be lower than other groups since the5th day (group B vs A, C, D, E, p=0.005,0.007,0.000,0.001). Group A situated in the middle of the rank. The vitality of group C and D was relatively high but there was no statistically significant difference between these two (p>0.05). In terms of cell function, ALT leakage dropped first and then increased. The leakage amount of group F raised significantly from the3rd day, higher than other five groups with a maximum amount of80.000±7.321IU/L (P<0.001), resulting in early development of cell damage. Meanwhile the leakage amount of group A increased greatly from the6th day, higher than the rest four groups (P<0.001), with a maximum amount of53.167±2.483IU/L. Group B, C, D and E had similar amount in leakage which was relatively low. But urea detection revealed that urea secretion amount of group D remained highest except for the1st day, higher than other five groups (p<0.001). And that of group E was significantly higher than that of group A, B, C and F during culture (p<0.001), in which the amount of group C was higher than that of group A, B and F (p<0.001) but lower than that of group D and E. therefore, the urea secretion amount of group A, B and F remained low. As to albumin observation, though there was statistically significant difference in secretion amount between group D and E (p<0.001), the actual amount was relatively high and close to each other. Meanwhile, the secretion amount of group A and C was relatively low and that of group B was moderate.In addition, real-time qPCR demonstrated that the There were no statistically significant difference between each groups and E group.(p=0.774,0.866,0.197,0.877,0.293) Apart from group A (p=0.000,0.000), the rest four groups were not statistically different from complete medium group in GST and ALB expression (p>0.05). Group A, B, C, D and E had similar AFP expression (p>0.05). And the AFP expression of group F was significantly lower than that of the complete medium group (p=0.041). The HNF4α expression of group A and D was lower than that of the complete medium group (p<0.001ConclusionThe serum-free medium, developed by our research group, with independent intellectual property right, can replace serum medium in hepatic cell culture. Through various kinds of mechanisms, this serum-free medium guarantees favorable cell growth in terms of morphology, density, vitality and function as achieved by traditional serum medium. The new medium is most suitable for hepatic cell (C3A) as it yields the greatest cell function. Satisfying therapeutic needs of BAL, serum-free medium lay a foundation for the culture, screen and application of BAL seed cells as well as the clinical application of BAL. |
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