Discussion on Serum Technique and Cryopreservation Technique in the Culture of Liver Sinusoidal Endothelial Cells

Yue PENG Aolei SU Tiejian ZHAO Peng LIU Qing WANG Zhihao SHANG

Abstract Hepatic sinusoidal endothelial cells are a kind of highly differentiated cells in hepatic sinusoids， which play an important role in the occurrence and development of liver fibrosis. Hepatic sinusoidal endothelial cells are often used in the study of liver fibrosis. In the process of culturing liver sinusoidal endothelial cells， the treatment of serum for culture and cryopreservation of cells are relatively complicated and error-prone. If the technical details of serum treatment and cell cryopreservation are not handled properly， it will have a more obvious effect on the effect of hepatic sinusoidal endothelial cell culture. We have gained experience in the theoretical study of liver fibrosis and the operation of cell culture experiments， and this paper summarized the details of liver sinusoidal endothelial cell culture such as the problems of serum preservation， thawing， precipitates and heat inactivation， as well as methods and precautions for cell cryopreservation.

Key words Liver sinusoidal endothelial cell; Cell culture; Serum; Cell cryopreservation

The hepatic sinusoid is a barrier for the exchange of material between blood and hepatocytes， and is one of the most important and most permeable substance exchange structures in the human body. Hepatic sinusoidal endothelial cells are the main cells that constitute the hepatic sinusoids， and their most characteristic structure is that they have fenestrae and a basement membrane[1]. Fenestrae play a role of screening and barrier in the process of substance exchange between blood and liver cells. In the pathological condition of liver fibrosis， there is a continuous basement membrane between hepatic sinusoidal endothelial cells and hepatocytes， accompanied by the disappearance of fenestrae and the activation of hepatic stellate cells. These changes are called "sinusoid capillarization"[2-3]. Studies have shown that， sinusoidal endothelial cells play an important role in the activation process of the core cells in hepatic fibrosis——hepatic stellate cells[4-6]. When liver fibrosis and liver damage occur in the body， liver sinusoidal endothelial cells can become an efficient pro-inflammatory cell， and secrete a series of cytokines and chemokines， which mediate the acceleration of liver inflammation and make the liver immune function change[7-9]. Research on the structure and function of hepatic sinusoidal endothelial cells has become a new hot spot for the intervention of liver fibrosis lesions[10]. Our research group has focused on liver fibrosis in recent years， and has made in-depth research on the structure， function， and culture technology of hepatic sinusoidal endothelial cells. In the theoretical study and experimental operation of the cell culture technology， we found that some technical problems in the treatment of cultured serum and cell cryopreservation methods are complicated and error-prone. If these details are not handled properly， it will obviously affect the activity and proliferation capacity of the cultured hepatic sinusoidal endothelial cells， which will affect the final results of liver fibrosis experiments. This paper summarized and discussed these technical details.

Treatment of Serum for Culture

The composition and content of the synthetic cell culture media used in recent years have been relatively complicated， but they still cannot fully meet the needs of cell growth in vitro， especially the proliferation of liver sinusoidal endothelial cells with high nutritional requirements. Therefore， a certain proportion of natural media must be added to the synthetic media to supplement the nutritional components. At present， the natural medium is mostly selected from fetal bovine serum or calf serum， and the addition ratio varies from a few percent to several tens of percent[11]. The role of serum in cell culture is very important， especially for hepatic sinusoidal endothelial cells with high nutritional requirements[12]， and the added serum must be of high quality and sufficient amount.

Storage method and thawing method of serum for culture

Serum used for the culture of hepatic sinusoidal endothelial cells must be stored in an environment of -20 to -70 ℃. If stored at 4 ℃， the time cannot exceed 15 d. The serum cannot be placed in an environment at 37 ℃ for too long. If it is placed at 37 ℃ for more than 24 h， the serum will become cloudy to naked eyes， and many unstable proteins and other components in the serum will also be damaged， affecting the serum quality， which in turn affects the effect of cell culture. If a bottle of serum cannot be used up in the primary culture medium， the serum can be pre-packed into sterile 50 ml centrifuge tubes in 40 ml portions， and the centrifuge tubes are taken out according to the amount used each time. And the serum in each tube taken out should be used up. It should be noted that when the serum is frozen， the volume will increase by about 10%， so when the serum is filled in centrifuge tubes， sufficient space must be reserved for the expansion volume， otherwise the serum may be squeezed out of the container and become contaminated， resulting in waste; or burst occurs when the centrifuge tube is frozen.

When thawing and using serum for the preparation of hepatic sinusoidal endothelial cell culture medium， the stepwise thawing method should be adopted[13]， so as not to damage the quality of serum. Specifically， after taken out from -20 ℃ or -70 ℃， the serum is put in a refrigerator at 4 ℃ for 24 h， and then moved to room temperature. After the serum is melted， it is repacked. It must be noted that during the room temperature thawing process， the container must be shaken regularly， evenly， and slightly （be careful not to cause air bubbles and damage the quality of the serum）， so that the temperature and ingredients in the container are always uniform， and the occurrence of precipitation is reduced. The serum should not be directly thawed at 37 ℃ rapidly after taken from –20 ℃， because if the temperature changes too much， it is easy to cause protein coagulation and production of a large amount of precipitation， which seriously affects the quality of the medium.

Generally， the serum provided by the manufacturer is sterile serum， which can be used directly without further sterile filtration. If it is found that there is obvious precipitate in the serum， the correct way of handling is to add the serum to a medium and mix it with the medium for filtration. In a word， the serum should not be filtered directly.

Precipitates in serum

Type of precipitate

Floccule： The precipitates look like cloud or cotton. There are many reasons for the formation of flocs in the serum， but the more common reasons are the denaturation of lipoproteins in the serum and the fibrin occurred in the thawed serum[14]. If the amount of these flocculated precipitates is not too large， it generally does not affect the quality of the serum itself. However， if we want to reduce these flocculent precipitates， a centrifuge can be used to centrifuge （3 000 rpm， 5 min） the serum to remove them. The serum supernatant obtained after centrifugation can be added to the medium and filtered together. It is not recommended to directly filter the serum to remove these flocculated precipitates， because these precipitates that have basically no effect on cell culture will quickly block the filter membrane.

"Little black spots" observed under a microscope： Normally， in heat-treated serum， the formation of precipitates will increase significantly. Some precipitates look like some "black spots" under the microscope. Experimenters often mistakenly believe that the serum is contaminated. If such serum is placed at 37 ℃ to culture these "microorganisms"， it can be found that the amount of the precipitates is obviously increased， which is more likely to be mistaken for the proliferation of microorganisms. However， if these "small black spots" are detected by the culture medium of bacteria， no pollution will be observed. It is generally believed that there is nanobacteria in the serum[15]. Generally speaking， these "small black spots" will not affect the growth of cells. However， if the quality of the serum is suspected， we can immediately stop using it and replace with another batch of serum

Ways to avoid serum precipitate

（1） When thawing serum， we should follow the stepwise thawing method （from -20 to 4 ℃， and then to room temperature）. If the temperature changes too much when the serum is thawed （such as from -20 ℃ to 37 ℃）， it is very easy to produce a large amount of precipitates.

（2） When thawing serum （especially during the thawing step at 37 ℃）， it should always be shaken evenly to make the temperature and composition uniform， which can reduce the occurrence of precipitates.

（3） The serum should not be placed at 37 ℃ for too long. If it is left at 37 ℃ for too long， the serum will become cloudy， and many unstable components in the serum will be damaged， which will affect the quality of the serum.

（4） Heat inactivation of serum is very easy to cause an increase in precipitates. If not necessary， this step can be omitted. If the serum must be thermally inactivated， the principle of inactivation at 56 ℃ for 30 min should be followed， and the serum should be shaken regularly， uniformly， and with small amplitudes during the inactivation process. Excessively high inactivation temperature， too long time， or uneven shaking will cause a significant increase in precipitates.

（5） It has been found from hepatic sinusoidal endothelial cell culture experiments that fetal bovine serum stored in a refrigerator at 4 ℃ sometimes precipitates after 24 h. The reason is that these serums are not pre-aged. When it is stored at 2-8 ℃， various proteins and lipoproteins in the serum （such as cold agglutinin， fibrinogen， vitronectin， etc.） may aggregate and form precipitate or visible turbidity[16]. It is found in cell culture practice that this phenomenon has no significant effect on the quality of serum. We recommend storing fetal bovine serum in an environment below -20 ℃. And it should be taken out and used at one time to avoid repeated freezing and thawing.

Heat inactivation of serum

Heat inactivation refers to heating serum that has been completely thawed at 56 ℃ for 30 min. The purpose of this heat treatment is to deactivate the complement components in the serum and inactivate the complement system. The use of heat-inactivated serum is recommended only in immunological studies or when culturing ES cells， insect cells， and smooth muscle cells. Cell culture experiments have shown that even heat-inactivated serum that is properly processed is not necessary for most cells. Such treated serum has only a slight promotion on cell growth， or it can be said to have no effect at all[17]. On the contrary， the high temperature treatment often affects the quality of serum and causes a decrease in cell growth rate. Moreover， for heat-treated serum， the formation of precipitates will increase significantly. These precipitates look like many "small black spots" （nanobacteria） under an inverted microscope. Therefore， in order to maximize the preservation of serum quality， unless heat treatment is required by the experiment， it is generally not recommended for this treatment of serum. Hepatic sinusoidal endothelial cell culture experiments do not require this step.

Yue PENG et al. Discussion on Serum Technique and Cryopreservation Technique in the Culture of Liver Sinusoidal Endothelial Cells

About Cryopreservation of Cells

In the experiment of hepatic sinusoidal endothelial cell culture， the acquisition of cells is uncertain. Once successful， a large number of cells will be cultured， usually exceeding the research needs. So many cells will be wasted. The successful and long-term cryopreservation of the successfully cultured cells can enable them to obtain the maximum application in scientific research and clinical applications. However， at present， there is no standard internationally recognized method for cryopreservation[18].

The ultimate goal of any cryopreservation method is to avoid the formation of intracellular ice crystals that can damage cell substructures to the greatest extent， thereby maintaining cell activity， adhesion capacity， and metabolic activity after thawing[19]. Whether the cryopreservation of hepatic sinusoidal endothelial cells is correct depends on the speed of cryopreservation， the concentration of cryopreserved cells， the type of cryopreservation agent， the final concentration of cryopreservation agent and other factors.

Method and steps for freezing storage of hepatic sinusoidal endothelial cells

Preparation for freezing

One day before cryopreservation， half or full amount of culture medium must be replaced to allow cells to obtain sufficient nutrients before cryopreservation. The growth of cells should be observed before cryopreservation. It is better to take cells in logarithmic growth phase for freezing.

Preparation of cryopreservation solution （prepared before use）： Dimethyl sulfoxide with a final concentration of 8% is added to freshly prepared medium， followed by mixing well， giving a suitable cryopreservation solution for hepatic sinusoidal endothelial cells. The solution is cooled at room temperature and should be used in time.

Freezing operation

When freezing hepatic sinusoidal endothelial cells， the standard methods of cell subculture should be followed. Cells in the logarithmic growth phase in culture are collected， and a small amount of cell suspension （about 0.1 ml） is taken to count the cell concentration and calculate the cell viability before freezing.

The cell suspension is centrifuged to remove supernatant. An appropriate amount of cryopreservation solution is added to adjust the concentration of hepatic sinusoidal endothelial cells to 1-5×106 cells/ml. After mixing evenly， aliquot portions of the obtained cell suspension are subpackaged into the cryopreservation tubes that have been marked completely， each tube containing 1 ml， and a small amount of the cell suspension is taken for contamination detection.

Two methods commonly used for cryopreservation of hepatic sinusoidal endothelial cells

Cryopreservation method one （traditional method）： The cryopreservation tubes are placed in an environment at 4 ℃ for 30-60 min， moved to -20 ℃ for 30 min and then to -80 ℃ for 16-18 h （or overnight） and finally stored in a liquid nitrogen tank for long-term storage.

It should be noted that the cryopreservation tubes should not be placed at -20 ℃ for more than 1 h to prevent the ice crystals in the cells from becoming too large and causing massive death of hepatic sinusoidal endothelial cells[20]. The -20 ℃ step can also be skipped， and the cells can be put directly in a -80 ℃ refrigerator. Experiments have shown that the effect of such operation is only a slight decrease in cell survival rate.

Cryopreservation method two （program cooling）： The cryopreservation tubes are put into a controllable constant-speed cooling machine with a set program， so that the temperature drops by 1-3 ℃ per minute， until it drops below -80 ℃ （generally reduced to -120 ℃）. Then， the cryopreservation tubes are moved to liquid nitrogen tank for long-term storage. It is reported that this method is most suitable for cryopreservation of suspension cells and hybridoma cells[21]. We found in experiments that this method is also suitable for the cryopreservation of hepatic sinusoidal endothelial cells， and the survival rate of the stored cells is not significantly different from the traditional method.

Precautions for freezing cells

For cryopreservation of hepatic sinusoidal endothelial cells， the state when the growth conditions are good and the survival rate is high should be selected. The cells are collected at a cell density of about 80%-90% and frozen.

Before cryopreservation， whether hepatic sinusoidal endothelial cells still retain their unique properties should be checked （one or two days before freezing， the cells should be tested for the existence of antibodies and other substances）.

About cryoprotectant

Dimethyl sulfoxide is used as a cell cryoprotectant in most cell experiments， and hepatic sinusoidal endothelial cells are no exception[22]. The dimethyl sulfoxide used for cryopreservation should be of reagent grade， sterile and colorless （filtered with 0.22 microporous membrane， or directly purchased sterile products）. When dimethyl sulfoxide is opened for the first time， it should be divided into small volumes of 5-10 ml immediately and stored in the dark at 4 ℃. The reagent should not be defrosted multiple times after use. Repeated freezing and thawing will cause the dimethyl sulfoxide structure to crack， releasing harmful substances to damage the frozen cells， and will increase the chance of cell contamination.

Glycerol added during freezing should also be of reagent grade. After sterilized with high-pressure steam， the glycerol is stored at 4 ℃ in the dark. It should be used within one year after opening， because harmful components can be produced after long-term storage， and will be toxic to frozen sinusoidal endothelial cells.

The most commonly used cryopreservation fluid for hepatic sinusoidal endothelial cells is prepared with 8% dimethyl sulfoxide and 90%-95% fresh culture medium for the culture of hepatic sinusoidal endothelial cells.

When preparing the cryopreservation solution， it must be noted that dimethyl sulfoxide will release a large amount of heat energy during dilution， so it is forbidden to directly added dimethyl sulfoxide into the culture where cells grow， so as to avoid cell damage caused by the heat energy generated by the dilution. The cryopreservation solution must be prepared in another container before use. After cooling， it is added to the cells for cryopreservation.

Should the cryoprotectant dimethyl sulfoxide be removed immediately after the cells are thawed and recovered？ This problem depends on the type of cell. Except for a few cells that are specifically sensitive to dimethyl sulfoxide， most of them do not require the step of removing dimethyl sulfoxide immediately after thawing[23]. Therefore， after thawing the hepatic sinusoidal endothelial cells， they should be directly placed in a culture bottle containing 10-15 ml of fresh culture medium for culture， and the fresh culture medium can be replaced every other day to remove dimethyl sulfoxide. Such an operation can largely avoid the problem that cells cannot grow or adhere after thawing.

If it is necessary to remove the cryoprotectant of hepatic sinusoidal endothelial cells under special experimental requirements， the thawed cell suspension can be added to a centrifuge tube containing 5-10 ml of culture medium and centrifuged （1 000 rpm， 5 min）. After the centrifugation is completed， the supernatant is removed， fresh medium is added， and the mixture is mixed evenly， and it can be put into a CO2 incubator for culture.

Conclusions

Hepatic sinusoidal endothelial cells undergo significant changes in structure and function during the occurrence and development of liver fibrosis[24]. Their roles and mechanisms of action are very complex and have a wide range of action[25]. Therefore， the research on this cell is of great significance for preventing and reversing the process of liver fibrosis. Therefore， the research on hepatic sinusoidal endothelial cells is of great significance for preventing and reversing the process of liver fibrosis. In this article， we summarized and elaborated the practical experience gained in the theoretical study of liver fibrosis and the experimental operation of liver sinusoidal endothelial cell culture， and described the preservation， thawing， precipitation and heat inactivation of serum， as well as the methods and precautions for cryopreservation of liver sinusoidal endothelial cells in detail. Because these technical problems are often encountered during the culture of hepatic sinusoidal endothelial cells， they are also more complicated and more error-prone. If these details are not handled properly， it will obviously affect the activity and proliferation capacity of the cultured hepatic sinusoidal endothelial cells， which will affect the final results of liver fibrosis experiments.

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