Large-Scale Production and Cultivation of Hepatocytes on Biosilon Microcarriers

Artificial Organs 14(6):421-428, Raven Press, Ltd., New York 0 1990 International Society for Artificial Organs

Large-Scale Production and Cultivation of Hepatocytes on B io silon Microcarriers

Alexander Shnyra, Alexander Bocharov, Natalia Bochkova, and Valentin Spirov

Department of Cellular Biology, All- Union Cardiology Research Center, Academy of Medical Sciences of the U.S .S .R. , Moscow, U.S.S.R.

Abstract: A method for large-scale production of hepatocytes on microcarriers have been developed for the purpose of bioartificial liver support system. Hepatocytes obtained by collagenase treatment of rat liver were effi- ciently attached and spread on a microcarrier surface in the presence of 0,-saturated perfluorodecalin. In order to compare the metabolic activities of hepatocytes under long-term cultivation on microcarriers with those of cells under conventional monolayer culture, some liver- specific functions were investigated. Microcarrier-attached hepatocytes cultured in the absence of serum for 8 days synthesized and secreted albumin and fibronectin.

Primary hepatocyte cultures prepared from rat liver by the collagenase perfusion technique can maintain certain specific functions for several days and are suitable for different metabolic studies (1). Under in vitro conditions, hepatocytes retain the ability to synthesize some plasma proteins (2,3) and metabolize various agents and toxins (4). In this regard, cultured hepatocytes have wide applica- tions for studying molecular mechanisms underly- ing various metabolic reactions in the cells (5).

The ability of transplanted hepatocytes to provide artificial liver support for recipients with fulminant hepatic failure has been recently reported (6,7). Special attention has been paid to the culture of hepatocytes on microcarriers, since liver cells attached to microcarrier surfaces can correct liver insufficiency where hepatocyte suspensions have no positive effects (8). Furthermore, hepatocytes cultured on microcarriers can be used in bioreactors of artificial liver support systems based on plasma-

~-

Received June 1990. Address correspondence and reprint requests to Dr. A. Shnyra

at Department of Clinical Bacteriology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden.

Moreover, hepatocytes on microcarriers retained the ability to conjugate bilirubin for 4-5 days. With respect to these specific metabolic properties, microcarrier- attached hepatocytes were comparable to those from routine dish culture. These results suggest that this method developed for large-scale production of hepatocytes on microcarriers will allow one to obtain metabolically ac- tive cells suitable for extracorporeal liver support systems. Key Words: Hepatocytes-Microcamers-Protein synthesis-Bilirubin metabolism-Artificial liver support systems.

pheresis or hemoperfusion techniques (9). How- ever, current methods of hepatocyte attachment and cultivation on microcarriers are laborious and offer only a limited number of the cells (10,ll).

The aim of this work was to describe the new method and some principles of large-scale production of hepatocytes on Biosilon microcarriers, which are based on conventional microcarrier techniques. The approaches necessary to achieve the optimal conditions for hepatocyte attachment and long-term cultivation on microcarriers are presented. To compare the properties of the cells on microcarriers (three-dimensional system) with those of routine monolayer culture (two-dimensional systems), investigations of hepatocyte synthesis and secretion of some plasma proteins as well as their ability to conjugate bilirubin have been carried out.

MATERIALS AND METHODS

Animals Wistar male rats weighing 20&250 g were used.

The animals received standard laboratory diet ad libitum.

42 I

422 A . SHNYRA ET AL.

Reagents Collagenase (specific activity of 131 U/mg) was

purchased from Wortington (Freehold, NJ, U.S.A.); dexamethasone, insulin, bovine albumin, bilirubin, and horseradish peroxidase were from Sigma (St. Louis, MO, U.S.A.). William’s E medium and 1 M HEPES solution were obtained from Flow Laboratories (Rickmansworth, U.K.); Hank’s balanced salt solution (HBSS) without Ca2+ was from Gibco (Paisley, Scotland); all electrophoresis reagents were from Bio-Rad (Richmond, CA, U.S.A.).

Hepatocyte isolation procedure Hepatocytes were isolated from male Wistar rats

by the two-step collagenase perfusion technique as described previously (12). Livers were washed in situ via the portal vein with warmed (37°C) Ca2’- and Mg2+-free HBSS at a flow rate of 40 mYmin for 10-12 min and then perfused with 0.05% collagenase in the same solution supplemented with 5 mM CaCl, and 50 mM HEPES. The reperfusion with collagenase solution lasted 20-25 min at 37°C pH 7.5. After complete dispersion of the liver cells, the suspension was passed through a 50 p m nylon filter to remove tissue debris and cellular aggregates. The resultant cell suspension was centrifuged at 50 x g for 3 min. The supernatant was discarded and hepatocytes were washed three times with HBSS (8°C). The final cell suspension contained at least 95% hepatocytes. The viability of the cells was 80-90% as assessed by the Trypan blue (Sigma, 4 mg/ml) exclusion test. The number of cells in suspension was estimated by microscopy using a he- mocytometer . Finally, hepatocytes were suspended in the William’s E medium supplemented with lo-’ M dexamethasone and 8 pg/ml of insulin.

Cultivation of hepatocytes in a two-dimensional system

Sixty-millimeter Petri dishes (Nunc, Roskilde, Denmark) were coated with human fibronectin (5 Fg/cm2). The cells were inoculated at a density of lo5 cellslcm’ and cultured in a CO, incubator. After 2-3 h of incubation, unattached cells were washed out with warmed (37°C) HBSS and 5 ml of fresh culture medium was added to each plate. Subse- quently, culture medium was changed every day.

Hepatocyte cultivation in a three-dimensional system

Hepatocytes were cultured on Biosilon microcarriers (Nunc) coated with human fibronectin ( 5 p,g/cm2). For attachment of the cells to the microcarriers, 25 ml of perfluorodecalin (Aldrich-

Chemie, Steinheim, F.R.G.) oxygenated by bubbling with sterile o,, 1.5 x 10’ hepatocytes, 5 ml of microcarriers, and 50 ml of culture medium were inoculated into a 250 ml flask (Techne, Oxford, U.K.) and incubated in a CO, humidifier at 37°C with constant stirring at 5 rpm. After 2 h of incubation, unattached cells, perfluorodecalin, and culture medium were removed and 150 ml of fresh culture medium was added. The following cultivation was carried out at 25 rpm. The culture medium was changed every day.

Determination of lactate dehydrogenase (LDH) The activity of LDH in culture medium was de-

termined on the “Spectrum” High Performance Di- agnostic System (Abbot, North Chicago, IL, USA) using a Boehringer Mannheim test combination kit.

Determination of protein Hepatocyte proteins were disrupted by sonica-

tion and hydrolysis was carried out in the presence of 0.1 M NaOH. Following neutralization of NaOH with 0.1 M HCl, the cellular protein content was determined by the Bradford method using the Bio- Rad Protein Assay Kit and bovine serum albumin as standard.

Purification of rat serum proteins and preparation of antibodies

Rat serum albumin and rat fibronectin were pun- fied as described previously (12). The purity of these protein preparations was at least 90% as assessed by SDS-polyacrylamide gel electrophoresis. Antisera to these proteins were generated in rab- bits. Specific antibodies were purified from these sera by affinity chromatography on either albumin- Sepharose and fibronectin-Sepharose sorbents and employed for ELISA of the proteins in hepatocyte- conditioned culture medium.

Enzyme-linked immunosorbent assay (ELISA) The ELISAs were performed in 96-wells Titertek

PVC Immunoassay plates (Flow Laboratories, U.K.). The wells were coated with 100 p,l of antialbumin or antifibronectin affinity-purified antibodies at a concentration of 10 pg/ml in phosphate- buffered saline containing 0.1% Tween-20 (PBST). After overnight incubation at 4“C, the plates were washed three times with 200 p1 of the PBST and different dilutions of hepatocyte-conditioned media were added and incubated at 37°C for 1 h. For quan- titative determinations, simultaneous incubation with known concentrations of purified albumin and fibronectin was carried out. Subsequently, the plates were washed thoroughly with PBST and a conjugate of corresponding antialbumin or antifi-

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HEPATOCYTES ON BIOSILON MICROCARRIERS 423

bronectin antibodies with horseradish peroxidase was added. After 1 h of incubation at 37°C and subsequent washing with PBST, 100 pl of substrate solution (0.1 M citrate buffer, pH 5.5, contained 0.4 mdml of o-phenilendiamine and 0.012% hydrogen peroxide) was added. At the end of 15 min of incubation at room temperature, the reaction was stopped with 4 N H,SO, (50 pYwel1). The optical density was measured at 492 nm in a Titertek mul- tiscan micro-ELISA spectrophotometer (Flow Lab- oratories, U.K.). In each assay, some wells after incubation with William’s E medium were used for estimation of background absorbance value. A plot of the logarithm of the optical density at 492 nm vs. the logarithm of the concentration of the protein standards was done and the absorbance of test samples was compared to this curve.

Bilirubin conjugation by hepatocyte cultures The unconjugated bilirubin was prepared accord-

ing to the following procedure: 100 mg of bilirubin was dissolved in 4 ml of 0.1 M NaOH and then mixed with 7 ml of bovine serum albumin in William’s E medium at a concentration of 40 mg/ml. The stock bilirubin solution in bovine albumin base was filtered and used for investigation of the bilirubin conjugation rate in hepatocyte cultures. For cell culture assays, the stock solution was diluted in William’s E medium with supplements to a final concentration 175 pg of bilirubidml and was added to the hepatocyte cultures. At intervals, the culture medium was collected and production of conjugated bilirubin was determined as described (13).

Scanning electron microscopy (SEM) of hepatocytes on microcarriers

Preparations for SEM were washed three times with HBSS and the cells were fixed with 2.5% glu- taraldehyde in 0.1 M cacodylate buffer, pH 7.3 and 0.1 M sucrose at 37°C. Fixed cultures of hepatocytes on microcarriers were dehydrated in a graded series of ethanol solutions and further dehydration by critical point-drying. A thin layer of gold was deposited on the cell surface by a sputtering system (JFC- 1100, JEOL, Tokyo, Japan). The samples were examined in a JEOL scanning electron micro- scope (JMS-35).

RESULTS

Attachment and cultivation of hepatocytes on Biosilon microcarriers

The large culture surface area that the microcarrier system provides in small volume demands inoculation of hepatocytes at a high seeding density

for formation of a cellular monolayer. However, spontaneous cell sedimentation during the static period of cell cultivation leads to a sharp decrease in oxygen supply and, as a result, depression of hepatocyte metabolism. Thus, the maintenance of a stable oxygen level in the medium plays a key role in successful attachment and spreading of hepatocytes on microcarriers.

In the method developed, the initial stages of hepatocyte cultivation on microcarriers were carried out in the presence of oxygenated perfluorodecalin (Fig. 1). This compound has a high affinity for oxygen, is water immiscible, and is nontoxic for the cells. These properties enabled adequate oxygen supply to cells and allowed rapid removal of this agent from the culture medium. In culture vessels, perfluorodecalin forms a level boundary surface with the culture medium. Upon slight stirring (5 rpm), hepatocytes obtained by collagenase diges- tion of liver and fibronectin-coated microcarriers were evenly distributed on this surface. Thus, the oxygen supply sufficient for support of hepatocyte metabolism was achieved. Under these conditions, the cells rapidly attached to microcarriers and spread.

For determination of the plating efficiency of primary hepatocyte cultures on microcarriers, the cells were seeded at various densities. Figure 2 shows the average number of hepatocytes spread out from six cultures of the cells. Inoculation of 1.2 x lo5 cells/cm2 provided efficient hepatocyte monolayer formation on microcarriers. Since 5-7 x 10’ hepatocytes could be isolated from a single rat liver, our procedure allowed at least 3.0-3.5 x 10’ cells to be cultured on 20-25 ml of Biosilon microcarriers.

After a 2 h cultivation, approximately 5040% of inoculated cells were attached to Biosilon microcarriers (Fig. 3). The viability of microcamer-attached hepatocytes was at least 80%, as assessed by Try- pan blue exclusion. During 24 h in culture, hepatocytes flattened and acquired the epithelial morphol- ogy that was preserved during 7-8 days.

Assessment of detachment and mechanical damage to hepatocytes on microcarriers

Both seeding conditions and mechanical inter- ventions during hepatocyte cultivation on microcarriers may impair hepatocyte functions and even lead to cellular death. These processes may be assessed by accumulation of some cytoplasmic en- zymes released into culture medium due to alter- ations in permeability of the cellular membrane. In this investigation, the release of the cytoplasmic enzyme, lactate dehydrogenase (LDH), by hepato-

Artif Organs, Vol. 14, No. 6 , 1990


HEPATOCYTES

nealment of liver with collagenase

Separation of hepatosytes by mtrifugation at 50 x g

washing 3-times withmedium

Microcarriers

(MtlJremednrm (5oml)

Perfluorodecalin 0 2 saturated (25 ml)

C u l t u r e conditions:

s m g : 5 rpm for 2 h

cytes cultured on microcarriers vs. those of the cells cultured on the routine two-dimensional system was compared (Fig. 4). Both cultures showed an increase in LDH activity at the beginning of cultivation that may be attributed to cell damage during the isolation procedure. On subsequent cultivation of hepatocytes, the LDH activity in the culture medium decreased. However, hepatocytes cultured on microcarriers released greater amounts of LDH compared with routinely cultured cells. This may be due to peculiarities of cultivation in a three-dimensional system, such as damage to cells resulting from microcarrier collisions and the impact of microcarrier particles against the walls of the culture vessels. Increased LDH release by cultured hepatocytes was also observed after 8-9 days of cultivation, probably as a result of degradation and death of the primary culture of cells. Nevertheless, after 5-7 days of cultivation in a three-dimensional system, hepatocytes did not detach from microcarriers, as assessed by determination of cellular protein content per area unit of microcarriers (Fig. 5) . Vi- ability of hepatocytes on microcarriers was at least 80% for 7 days, as assessed by Trypan blue exclusion (data not shown).

FIG. 1. The procedure of hepatocyte attachment and cultivation on Biosilon microcarriers. The diagram shows the optimal ratio of hepatocytes in suspension and microcarriers coated for 1 h at 37°C with 5 p,g/ml of fibronectin. Perfluorodecalin was saturated for 15 min by bubbling (1 bubble/s) with sterile oxygen. The culture medium and perfluorodecalin were warmed at 37°C before inoculation of isolated hepatocytes.

% \ B 41 a 8

Y

1 0 1 0 0 2 0 0 3 0 0 4 0 0 5

-3 2 Inoculatecld(xl0 /cm J

0

FIG. 2. Hepatocyte plating efficiency on fibronectin-coated microcarriers. Hepatocytes were obtained as outlined in the Materials and Methods. Cells were seeded at various concentration to the standard volume of Biosilon microcarriers. The number of attached hepatocytes after 2 h culture was determined according to the following procedure: Microcarrier- attached hepatocytes were washed from nonattached cells and were disrupted with 0.1 M NaOH and sonication. After hydrolysis, total cellular protein was estimated as described in the Materials and Methods section. A plot of the number of isolated hepatocytes in suspension vs. protein content was used as a standard curve to determine the number of cultured cells. The data are presented as mean 2 SEM of four separate experiments.

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HEPATOCYTES ON BIOSILON MICROCARRIERS 425

FIG. 3. Scanning electron microscopy of 2 h culture of hepatocytes on Biosilon microcarriers.

Hepatocyte synthesis of some plasma proteins For assessment of adequacy of culture conditions

for realization of diverse cellular functions in a course of cultivation, the restoration of the synthetic activities of hepatocytes in vitro have been studied by measuring albumin and fibronectin production by the cells. Observations show that stable albumin synthesis was maintained by both cultures of hepatocytes during 5 days (Fig. 6 ) . The synthesis rate was about 10&120 pg of albumin/106 cells/24 h. Upon subsequent cultivation, albumin production decreased, probably due to degradation and death of the cells. Unlike albumin, fibronectin production by hepatocytes in vitro increases during the course of cultivation and reaches a maximum at 10-14 p.g

2 3 4 5 6 7 8 9 Time (days)

FIG. 4. Lactate dehydrogenase activity in conditioned culture medium. LDH activity released by cultured hepatocytes was estimated as outlined in the Materials and Methods section. The number of cultured cells was determined as described in the legend to Fig. 2. Values are mean +- SEM from three separate cell cultures.

0 2 4 6 8 1 0 Time (days)

FIG. 5. Cellular protein content of microcarrier-attached hepatocytes under cultivation. Total hepatocyte protein was determined as described in the Materials and Methods section. Results are given as mean f SEM of three to five experiments.

0 2 4 6 8 1 0 Time (days)

0 2 4 6 8 1 0 T h e (days)

FIG. 6. Albumin synthesis and secretion by hepatocyte cultures. Albumin content in conditioned culture medium was estimated by the ELlSA technique as described in the Mate- rials and Methods section. Cell number was determined as given in the legend to Fig. 2. Each point represents the mean ? SEM of four experiments.

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of fibronectin/106 cells/24 h after 4-6 days in culture (Fig. 7). The subsequent cultivation of hepatocytes caused a decrease in fibronectin synthesis. The sim- ilarity of the values and patterns of albumin and fibronectin production under two- and three-dimensional cultivation suggests that the technique developed for attachment and seeding of hepatocytes on microcarriers has no effect on protein synthesis by cultured hepatocytes.

Bilirubin conjugation in cultured hepatocytes Bilirubin, the major product of heme metabolism,

is excreted by hepatocytes in bile after microsomal esterification with glucuronic acid. In view of the importance of these processes for characterization of metabolic integrity of hepatocytes in vitro, the conjugation of bilirubin has been studied in our experiments for the estimation of the effects of developed culture conditions on maintenance of this 4 1 function.

Figure 8 illustrates the rate of bilirubin conjugation in hepatocytes cultured in three- or two-dimensional systems. A constant rats of conjugated derivative formation was retained for 4 days and was similar in both techniques. Hepatocytes in routine culture produced approximately 15 Fg of conjugated bilirubin/106 cells/h and for the cells cultured on microcarriers this parameter was 25 p.g/lO6cellslh.

DISCUSSION

Successful hepatocyte transplantation in the treatment of fulminant hepatic failure has recently been demonstrated (6-8). The bioartificial liver sup-

- 0 2 4 6 8 1 0

Time (days1 FIG. 7. Fibronectin synthesis and secretion by hepatocyte cultures. Fibronectin concentration in the culture medium was determined by ELSA. Cell number was determined as presented in the legend to Fig. 2. The data are given as mean 2 SEM of four experiments.

FIG. 8. Bilirubin conjugation by hepatocyte cultures. Rate of bilirubin conjugation by the cells in culture was measured as described in the Materials and Methods section. Number of cultured hepatocytes was determined as presented in the legend to Fig. 2. Data expressed as mean f SEM of three to five separate experiments.

port of hepatic function provided certain prospects for the development of novel approaches to the cor- rection of liver insufficiency using donor hepatocytes (14). In this regard, culture hepatocytes on microcarriers can be effectively used in bioreactors for on-line plasma or blood detoxification (9,151. However, the investigation into this field was ham- pered by the absence of techniques for efficient large-scale production of primary hepatocyte culture on microcarriers and limited information about the metabolic properties of the cells under three- dimensional conditions.

It has been shown that attachment and spreading of hepatocytes in culture are markedly increased on fibronectin-treated surfaces (16); thus, in this research, microcarriers coated with fibronectin have been used. Since the attachment and plating efficiency of primary hepatocyte culture depends on the oxygen tension in the culture medium in the developed technique of large-scale production of hepatocytes on microcarriers, the cultivation of cells was started in the presence of 0,-saturated perfluorodecalin. The use of perfluorodecalin to provide a sufficient oxygen supply at the beginning of cultivation is essential for rapid and efficient attachment of hepatocytes to microcarriers.

No differences in hepatocyte attachment to different commercially available microcarriers such as Biosilon (Nunc), Cytodex 3 (Pharmacia Fine Chem- icals, Uppsala, Sweden), Bio-carriers and Bio- beads S-X12 (Bio-Rad), and Cytospheres (Flow Laboratories, U.K.) were observed. However, the

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HEPATOC YTES ON BIOSILON MICROCARRIERS 427

Biosilon microcarriers show unique properties compared with others: They (I) do not agglutinate after treatment with fibronectin, (11) are nonporous, and (111) have suitable size, which allows good blood flow via a hemoperfusion column with microcarriers and do not cause clot formation in the hemoperfusion column (these results will be presented elsewhere).

To examine whether the developed attachment and culture conditions affected the metabolic integrity of hepatocytes, some metabolic characteristics of the cells cultured in two- and three-dimensional system were compared. With both techniques, no changes in the release of the cytoplasmic LDH were observed and no detachment of the cells from microcarriers occurred for 5-7 days of cultivation. Moreover, during this period, the capability of cultured hepatocytes to retain production of some proteins was assessed by the in vitro synthesis and secretion of albumin and fibronectin. In the organ- ism, the liver, especially hepatocytes, is the primary source of these proteins, which serve several important functions: albumin transports xenobiotics, bilirubin, metals, and hormones and maintains osmotic pressure in biological fluids (17), and fibronectin mediates the attachment of cells and serves as an opsonin, facilitating the functions of phagocytic cells (18). However, it is well known that hepatocytes in vitro have a tendency to lose their specific functions after a few days in culture (1). Many attempts have been made to prevent cellular degradation and death of the primary hepatocyte culture (19,20). The results of these investigations show that, among other approaches, the com- position of the culture medium and hormones are the important factors for maintenance of specific metabolism in cultured hepatocytes. Thus, changes in production of some proteins by cultured hepatocytes could be moderated by culture medium content (21). Albumin synthesis by cultured hepatocytes has been shown to be stimulated by dexamethasone as well as insulin, whose addition restored albumin mRNA and albumin synthesis in serum-free culture medium (22,23). Recently, it has also been demonstrated that glucocorticoids increase the production and secretion of fibronectin by cultured hepatocytes (24).

In accordance with the observations in the present study, William’s E medium supplemented with high concentrations of dexamethasone ( A4) and insulin (8 pg/ml) was used to prolong the survival of hepatocyte culture and promote retention of specific metabolic functions. The use of William’s E medium with hormonal supplements in-

stead of RPMI-1640 for hepatocyte cultures increases albumin production three- to fourfold (100- 120 &lo6 cells/24 h) and fibronectin secretion at least 1.5-fold (10-14 pg/106 cells/24 h) in comparison to recently reported results (25). In that study, it was also shown that hepatocytes on microcarriers retain the ability to synthesize fibrinogen and the character of this production was similar to fibronectin. Therefore, the results of the present study suggest that the developed conditions for attachment and cultivation of hepatocytes on microcarriers allow the maintenance of specific cell protein synthesis for at least 7 days. It should be mentioned that a decrease in protein production by hepatocytes pre- cedes the increased release of LDH. This indicates that disorders in protein synthetic integrity in hepatocytes are a relevant approach to the estimation of the degradation processes in primary hepatocyte cultures.

The liver and hepatocytes play a particularly important role in metabolism and degradation of various endogenous compounds, xenobiotics, and toxins. Conjugation of these compounds with glucuronic acid, which is catalyzed by UDP-glucuronyl transferase, is responsible for their neutralization and elimination. Retention of these specific reactions by cultured hepatocytes upon developed culture conditions has been studied by characterization of bilirubin conjugation. The results of this investigation indicate that the developed conditions for hepatocyte attachment to microcarriers have no effects on this specific hepatocyte function. Similar patterns and a stable rate of bilirubin conjugation have been obtained in both two- and three-dimensional hepatocyte systems for 4-5 days. The higher rate of bilirubin conjugation in hepatocytes cultured on microcarriers may result from constant stirring, which cancels the concentration gradient of bilirubin occurring in a two-dimensional system. Possi- bly, this facilitates diffusion of bilirubin across the hepatocyte membrane. On the other hand, the higher permeability of hepatocyte membranes in the three-dimensional system assessed by LDH release may be responsible for this difference.

CONCLUSIONS

A new method for large-scale production and long-term cultivation of hepatocytes on microcarriers has been described. The method allowed microcarrier-attached hepatocytes to have comparable properties to the cells under monolayer culture conditions in that they have no changes in metabolic integrity. One of the advantages of the technique is

Art$ Organs, Vol. 14, No. 6 , 1990


that 3.CL3.5 X lo8 cells (cultured on microcarriers) can be obtained from a single liver. The simplified procedure reduces the number of handling steps re- quired to produce a given quantity of cells by routine methods. Furthermore, it involves only the conventional microcarrier technique and equipment and therefore can be easily adopted by any laboratory for the purpose of developing bioartificial liver support systems.

Acknowledgment: This work w a s supported by Grant 118-88/89 of the Academy of Medical Sciences of the U.S.S.R. The authors thank Inne Fedina for skillful tech- nical assistance.

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Documents

Large-Scale Production and Cultivation of Hepatocytes on Biosilon Microcarriers