In vitro proliferation and long-term preservation of functional primary rat hepatocytes in cell fibers

Primary hepatocytes are essential cellular resources for drug screening and medical transplantation. While culture systems have already succeeded in reconstituting the biomimetic microenvironment of primary hepatocytes, acquiring additional capabilities to handle them easily as well as to expand them remains unmet needs. This paper describes a culture system for primary rat hepatocytes, based on cell fiber technology, that brings scalability and handleability. Cell fibers are cell-laden core–shell hydrogel microfibers; in the core regions, cells are embedded in extracellular matrix proteins, cultured three-dimensionally, and exposed to soluble growth factors in the culture medium via the hydrogel shells. By encapsulating primary rat hepatocytes within cell fibers, we first demonstrated their proliferation while maintaining their viability and their hepatic specific functions for up to thirty days of subsequent culture. We then demonstrated the efficiency of proliferating primary rat hepatocytes in cell fibers not only as cell-based sensors to detect drugs that damage hepatic functions and hepatocellular processes but also as transplants to improve the plasma albumin concentrations of congenital analbuminemia. Our culture system could therefore be included in innovative strategies and promising developments in applying primary hepatocytes to both pharmaceutical and medical fields.

Takeuchi a,b* [a] Institute of Industrial Science, The University of Tokyo, Japan.
[c] LIMMS/CNRS-IIS UMI 2820, Institute of Industrial Science, The University of Tokyo, Japan [d] Faculty of Teacher Education, Shumei University, Chiba, Japan.

Culture of primary rat hepatocytes in cell fibers
After formation of core-shell hydrogel microfibers encapsulating primary rat hepatocytes, they were incubated at 37 ºC for more than 15 minutes to set the ECM proteins at the core region into gels. Subsequently, primary rat hepatocytes in cell fibers were cultured in DMEM supplemented with 1% Glutamax, 10% heat-inactivated FBS, 100 IU mL -1 penicillin, 100 mg mL -1 streptomycin, insulin, 6.25 mg mL -1 transferrin, 6.25 ng mL -1 selenous acid (Lonza, Tokyo, Japan), and 1mM HEPES at 37 ºC under a water saturated 5% CO2 environment; this medium is the one that have been optimized to culture primary rat hepatocytes in cell fibers for long term ( Figure S1c). Media exchanges were performed every 48 hours.
When primary rat hepatocytes were cultured either using cell fibers or using collagen-coated 24-well plates, the morphologies of these primary cells were observed using an inverted fluorescence microscope (IX71; Olympus, Tokyo, Japan).
When they reached confluence, the cells were harvested by trypsin/EDTA (Gibco) treatment and were encapsulated into core-shell hydrogel microfibers using the same protocol as used for encapsulation of primary rat hepatocytes except for the condition regarding ECM proteins for the core solution: bovine type I collagen (AteloCell, IAC-50, Native collagen, KOKEN, Japan) alone.

Preparation of 3T3CM to stimulate primary rat hepatocytes for proliferation in vitro
3T3CM was used as the media supplement for primary rat hepatocytes to proliferate according to previous report. [31] The duration and the conditions of culturing NIH/3T3 cells before their encapsulation into cell fibers were determined based upon the findings in our preliminary experiments. Specifically, the amount of HGF in the conditioned medium is always higher when NIH/3T3 cells are cultured in cell fibers than when cultured in petri dishes ( Figure S2), and the HGF amount is highest after 5 days of culture when cells are cultured in cell fibers. Furthermore, the 3T3CM collected from in cell fibers has showed comparable effects on maintenance of both microscopic morphology of fiber-shaped hepatic cell aggregates as well as hepatocyte-specific functions, namely albumin secretion and urea synthesis, with the media supplemented with both recombinant mouse HGF and recombinant mouse EGF ( Figure S3). Based on the findings as described above, we decided to use the culture media supplemented with 3T3CM that had been cultured in cells fibers for 5 days, and in this study, to make primary rat hepatocytes to proliferate, the media supplemented with 3T3CM started to be used after 2 days of culture. Since the start of using 3T3CM, culture media were exchanged every 24 hours. *Medium 1 = William's medium supplemented with 1% Glutamax, 100 IU mL -1 penicillin, 100 mg mL -1 streptomycin, 6.25 mg mL -1 insulin, 6.25 mg mL -1 transferrin, 6.25 ng mL -1 selenous acid, 10 mM HEPES, 40 ng mL -1 dexamethasone, and 2% of Matrigel **Medium 2 = DMEM supplemented with 1% Glutamax, 10% heat-inactivated FBS, 100 IU mL -1 penicillin, 100 mg mL -1 streptomycin, 6.25 mg mL -1 insulin, 6.25 mg mL -1 transferrin, 6.25 ng mL -1 selenous acid, and 1mM HEPES.

Figure S2. Culture of NIH/3T3 cells either in cell fibers or in collagen-coated petri dishes to prepare NIH/3T3 cell conditioned medium.
(a-c) Representative dark-field images (n=4 cell fibers) of NIH/3T3 cells encapsulated in cell fibers after 2 days, 5 days and 7 days of culture. Scale bars; 75 µm. (d-e) Representative dark-field images (n=4 petri-dishes) of rat hepatocytes in collagen-coated petri-dishes after 2 days, 5 days and 7 days of culture. Scale bars; 75 µm. (g) The concentrations of mouse hepatocyte growth factor secreted by NIH/3T3 cells cultured either in cell fibers and in collagen-coated petri dishes over 7 days of culture (n=4 per data point).

Figure S3. Culture of primary rat hepatocytes in cell fibers for up to 14 days under various proliferation stimulations.
(a-l) Representative dark-field images (n=6 cell fibers) of rat hepatocytes encapsulated in cell fibers at the initial cell density of 2.5x10 7 cells mL -1 after 2, 4, 7, 14 days of culture either with 3T3CM, with recombinant mouse hepatocyte growth factor (HGF) alone (rmHGF alone), or with recombinant mouse HGF and recombinant mouse epidermal growth factor (EGF) (rmHGF and rmEGF). Scale bars; 75 µm. (m-n) Concentrations of albumin secreted (m) and urea synthesized (n) by primary rat hepatocytes cultured in cell fibers (n=3 at least per data point) over 14 days either with 3T3CM, with rmHGF alone, or with rmHGF and rmEGF. Figure S4. Concentration-reaction curves for acetaminophen and diclofenac relating to viability, albumin secretion, and urea synthesis of primary rat hepatocytes cultured either in cell fibers or in collagen-coated petri dishes after 4 days of culture. Figure S5. Concentration-reaction curves for acetaminophen and diclofenac relating to viability, albumin secretion, and urea synthesis of primary rat hepatocytes cultured either in cell fibers or in collagen-coated petri dishes after 7 days of culture. Figure S6. Concentration-reaction curves for acetaminophen and diclofenac relating to viability, albumin secretion, and urea synthesis of primary rat hepatocytes cultured in cell fibers after 14 days of culture. Figure S7. Concentration-reaction curves for acetaminophen and diclofenac relating to viability, albumin secretion, and urea synthesis of primary rat hepatocytes cultured in cell fibers after 30 days of culture. Figure S8. Transplantation of cell fibers encapsulating primary rat hepatocytes into the intra-mesenteric space of an analbumenic rat.
(a, b) Cell fibers encapsulating primary rat hepatocytes were rinsed with serum-free culture medium once. (c, f) The cell fibers were sucked into a 23G butterfly needle connected with a 20 mL syringe. After laparotomy of the rat, (d, e) small and large intestines were exposed, and (g-q ) the cell fibers were injected into intramesenteric space and placed along the portal vain. CrM vein, Crania Mesenteric vein; J-I, Jejuno-Ileum; Ca, Caecum; Co, Colon.   Data are presented as the mean ± standard deviation of at least four independent cell fibers and four collagen-coated wells from collagen-coated 24-well plates.  Table S3. 50% inhibitory effect (IC50 values) for acetaminophen and diclofenac relating to viability, albumin secretion, and urea synthesis of primary rat hepatocytes cultured either in cell fibers for 4 days, or in other types of systems reported previously. Data are presented as the mean ± standard deviation of at least four independent coreshell hydrogel microfibers in this study. Table S4. Inhibition rates of acetaminophen and diclofenac relating to viability, albumin secretion, and urea synthesis at their various concentrations using primary rat hepatocytes in cell fibers after 4, 7, 14, and 30 days of culture. P-value of each concentration of acetaminophen or diclofenac was assessed by using oneway analysis of variance (ANOVA). *Respective inhibition rates of viability, albumin secretion, or urea synthesis at the considered concentration point were significantly different from each other after 4, 7, 14, and 30 days of culture at the 0.05 level.