Intermittent Starvation Extends the Functional Lifetime of Primary Human Hepatocyte Cultures

Abstract

Primary human hepatocyte (PHH) cultures have become indispensable to mitigate the risk of adverse drug reactions in human patients. In contrast to dedifferentiating monocultures, coculture with nonparenchymal cells maintains PHH functions for 2-4 weeks. However, because the functional lifespan of PHHs in vivo is 200-400 days, it is desirable to further prolong PHH functions in vitro toward modeling chronic drug exposure and disease progression. Fasting has benefits on the longevity of organisms and the health of tissues such as the liver. We hypothesized that a culturing protocol that mimics dynamic fasting/starvation could activate starvation pathways and prolong PHH functional lifetime. To mimic starvation, serum and hormones were intermittently removed from the culture medium of micropatterned cocultures (MPCCs) containing PHHs organized onto collagen domains and surrounded by 3T3-J2 murine fibroblasts. A weekly 2-day starvation optimally prolonged PHH functional lifetime for 6+ weeks in MPCCs versus a decline after 3 weeks in nonstarved controls. The 2-day starvation also enhanced the functions of PHH monocultures for 2 weeks, suggesting direct effects on PHHs. In MPCCs, starvation activated 5' adenosine monophosphate-activated protein kinase (AMPK) and restricted fibroblast overgrowth onto PHH islands, thereby maintaining hepatic polarity. The effects of starvation on MPCCs were partially recapitulated by activating AMPK using metformin or growth arresting fibroblasts via mitomycin-C. Lastly, starved MPCCs demonstrated lower false positives for drug toxicity tests and higher drug-induced cytochrome-P450 activities versus nonstarved controls even after 5 weeks. In conclusion, intermittent serum/hormone starvation extends PHH functional lifetime toward enabling clinically relevant drug screening.

Keywords: AMPK; fasting; metformin; micropatterned coculture.

Figure 1.

Intermittent starvation maintains hepatocyte island area and morphology over time. (A) Schematic of the MPCC platform. Left to right: Collagen type-I is micropatterned using soft lithographic techniques. Hepatocytes attach to the collagen domains and unattached cells are washed off. The next day, 3T3-J2 fibroblasts are seeded in the surrounded areas. (B) Experimental timeline for intermittently starving cultures of serum and hormones (dexamethasone, glucagon, and insulin). (C) Phase contrast images of PHHs in MPCCs at 1 week prior to starvation and then at 3, 4, or 6 weeks following the weekly 2-day starvation protocol shown in panel 'B'. The nonstarved MPCC images are shown for comparison. White circles outline PHH islands. Graph on right quantifies relative PHH island area over time, normalized to PHH island area in 1-week-old MPCCs. Scale bars on images represent 400 µm. **p ≤ .01, ***p ≤ .001, and ****p ≤ .0001. Abbreviations: MPCC, micropatterned cocultures; PHH, primary human hepatocyte.

Figure 2.

Intermittent starvation maintains hepatocyte function and activates AMPK. (A) Albumin and (B) urea secretions, and (C) CYP3A4 enzyme activity in starved (as described in Figure 1B) and nonstarved cultures over time. (D) Functional bile canaliculi in PHHs within starved and nonstarved micropatterned cocultures (MPCCs) as assessed by the excretion of the CDF dye. Left: images of representative bile canaliculi in PHH islands within the 2 conditions. Right: relative area of excreted CDF in PHH islands within starved versus nonstarved MPCCs after 6 weeks of culture. Data are normalized to the nonstarved controls. (E) p-AMPK per mg of protein in MPCC lysates following 2 starvation periods. Scale bars on images represent 400 µm. *p < .05, **p ≤ .01, ***p ≤ .001, and ****p ≤ .0001. Abbreviations: p-AMPK, phosphorylated adenosine monophosphate-activated protein kinase; PHH, primary human hepatocyte.

Figure 3.

Metformin treatment recapitulates some of the functional benefits of intermittent starvation. (A) Albumin production over time, (B) urea synthesis over time, and (C) CYP3A4 enzyme activity after 6 weeks of culture in nonstarved, starved (as described in Figure 1B), and metformin-treated micropatterned cocultures (MPCCs). Metformin treatment followed the same schedule as starvation except that cultures were treated with metformin in serum/hormone-supplemented culture medium instead. (D) Phase contrast images of MPCCs under various treatments as described above after 6 weeks of culture. White circles outline PHH islands. Graph on the right quantifies relative PHH island area in MPCCs under various treatments after 6 weeks of culture. (E) Functional bile canaliculi in PHHs within MPCCs under various treatments as assessed by the excretion of the CDF dye. Left: images of representative bile canaliculi in PHH islands within the 3 conditions. Right: relative area of excreted CDF in PHH islands within the 3 conditions after 6 weeks of culture. Data are normalized to the nonstarved controls. Scale bars on images represent 400 µm. *p < .05, **p ≤ .01, ***p ≤ .001, and ****p ≤ .0001 relative to the nonstarved control. Abbreviation: PHH, primary human hepatocyte.

Figure 4.

Growth arresting fibroblasts maintains hepatocyte island integrity in a similar way as intermittent starvation. Micropatterned cocultures (MPCCs) were created using either proliferative fibroblasts for the nonstarved and starved (as described in Figure 1B) conditions or growth-arrested (GA) fibroblasts via mitomycin-C. (A) Fibroblast nuclei count between PHH islands in 6-week-old MPCCs (3 different models described above) as assessed via live cell imaging of nuclei using Hoechst 33342 staining. (B) Measurement of PHH island solidity over time and (C) representative phase contrast images of PHH island edges outlined in white in the 3 different models above over time. White arrows indicate fibroblasts invading the PHH island. The 1-week time-point shown in panel ‘B' is for the nonstarved MPCCs. Solidity is measured as island area/convex hull area. Scale bars on images represent 50 µm. *p < .05, **p ≤ .01, ***p ≤ .001, and ****p ≤ .0001 relative to the nonstarved control. Abbreviation: PHH, primary human hepatocyte.

Figure 5.

Growth arresting fibroblasts recapitulates some of the functional benefits of intermittent starvation. Micropatterned cocultures (MPCCs) were created using either proliferative fibroblasts for the nonstarved and starved (as described in Figure 1B) conditions or growth-arrested (GA) fibroblasts. (A) Albumin production over time, (B) urea synthesis over time, and (C) CYP3A4 enzyme activity after 6 weeks of culture in nonstarved MPCCs, starved MPCCs, and MPCCs with GA fibroblasts. (D) Phase contrast images of different MPCC models as described above after 6 weeks of culture. White circles outline PHH islands. Graph on the right quantifies relative PHH island area in different MPCC models after 6 weeks of culture. (E) Functional bile canaliculi in PHHs within different MPCC models after 6 weeks of culture as assessed by the excretion of the CDF dye. Left: images of representative bile canaliculi in PHH islands within the 3 MPCC models. Right: relative area of excreted CDF in PHH islands within the different MPCC models. Data are normalized to the nonstarved controls. Scale bars represent 400 µm. *p < .05, **p ≤ .01, ***p ≤ .001, and ****p ≤ .0001 relative to the nonstarved control. Note: The data and images for starved and nonstarved conditions shown in this figure are the same as those shown in Figure 3 for comparison with the GA condition. Abbreviation: PHH, primary human hepatocyte.

Figure 6.

Intermittent starvation prolongs micropatterned coculture (MPCC) utility for clinically relevant drug screening. (A) MPCCs were maintained either nonstarved or starved for 2 days every week for 4 weeks (as described in Figure 1B) and then treated with drugs. (B) IC₅₀ values for albumin (left) and urea (right) for starved and nonstarved cultures treated for 6 days with prototypical hepatotoxins (top) and nonhepatotoxins (bottom) at 25× and 100× C_max (C_max: maximum drug concentration measured in human plasma) for each drug. N.T. represents “not toxic” (ie, IC₅₀ value could not be interpolated). (C) Induction of CYP enzymes in starved and nonstarved MPCCs after 4 days of treatment with prototypical inducer drugs. Abbreviations: OME, omeprazole; PB, phenobarbital; RIF, rifampin.