METHOD OF CULTURING IMMORTALIZED HUMAN HEPATIC PROGENITORS OR CELLS
20220396773 · 2022-12-15
Assignee
- INSERM (Institut National de la Santé et de la Recherche Médicale) (Paris, FR)
- UNIVERSITÉ DE RENNES 1 (Rennes, FR)
- ÉCOLE DES HAUTES ÉTUDES EN SANTÉ PUBLIQUE (EHESP) (Rennes cedex, FR)
Inventors
- Georges BAFFET (Le Rheu, FR)
- Marie CUVELLIER (Rennes, FR)
- Frédéric EZAN (Rennes, FR)
- Sophie ROSE (Rennes, FR)
Cpc classification
C12N5/0671
CHEMISTRY; METALLURGY
International classification
Abstract
A method of obtaining 3D cell structures including differentiated human hepatic cells. The method includes: a first step of culturing stem cell-derived or immortalized human hepatic progenitors in a non-adherent culture vessel, preferably a low or ultra-low attachment culture vessel; a second step of transferring the stem cell-derived or immortalized human hepatic progenitors to a culture medium including methacrylated gelatin (GelMa), thereby embedding the stem cell-derived or immortalized human hepatic progenitors in a GelMa matrix; and a third step of covering the GelMa matrix with culture medium and culturing the stem cell-derived or immortalized human hepatic progenitors embedded in the GelMa matrix, thereby obtaining 3D cell structures including differentiated human hepatic cells. Also, methods for engineering an artificial liver model or an artificial liver organ, and for assessing in vitro the metabolism, toxicity and/or therapeutic effects of a compound.
Claims
1.-16. (canceled)
17. A method of obtaining 3D cell structures comprising differentiated human hepatic cells, said method comprising: a) culturing stem cell-derived or immortalized human hepatic progenitors in a non-adherent culture vessel; b) transferring the stem cell-derived or immortalized human hepatic progenitors to a culture medium comprising methacrylated gelatin (GelMa), thereby embedding the stem cell-derived or immortalized human hepatic progenitors in a GelMa matrix; and c) covering the GelMa matrix with culture medium and culturing the stem cell-derived or immortalized human hepatic progenitors embedded in the GelMa matrix; thereby obtaining 3D cell structures comprising differentiated human hepatic cells.
18. The method according to claim 17, wherein at step a) the non-adherent culture vessel is a low or ultra-low attachment culture vessel.
19. The method according to claim 17, wherein at step a) the stem cell-derived or immortalized human hepatic progenitors are cultured in a non-adherent culture vessel for a duration ranging from about 1 h to about 96 h.
20. The method according to claim 17, wherein at step a) the stem cell-derived or immortalized human hepatic progenitors are cultured in a non-adherent culture vessel for a duration ranging from about 5 h to about 72 h.
21. The method according to claim 17, wherein at step b) the stem cell-derived or immortalized human hepatic progenitors are transferred to a culture medium comprising methacrylated gelatin (GelMa) at a final concentration ranging from about 1% (w/v) to about 20% (w/v).
22. The method according to claim 17, wherein at step c) the stem cell-derived or immortalized human hepatic progenitors embedded in the GelMa matrix are cultured for at least seven days.
23. The method according to claim 17, wherein the culture medium comprises insulin and a glucocorticoid.
24. The method according to claim 17, further comprising: d) incubating the 3D cell structures comprising differentiated human hepatic cells embedded in the GelMa matrix with a collagenase or an enzyme mixture with collagenolytic activity, thereby isolating the 3D cell structures comprising differentiated human hepatic cells from the GelMa matrix.
25. The method according to claim 17, wherein the differentiated human hepatic cells are human hepatocytes or hepatocyte-like cells.
26. The method according to claim 17, wherein the stem cell-derived or immortalized human hepatic progenitors are hepatocellular carcinoma-derived and/or transformed human hepatic progenitors.
27. The method according to claim 17, wherein the stem cell-derived or immortalized human hepatic progenitors are undifferentiated HepaRG cells.
28. A spheroid comprising human hepatocytes or hepatocyte-like cells, wherein said spheroid is a polarized structure with multiple bile canaliculi and comprises a number of human hepatocytes or hepatocyte-like cells ranging from 10 to 100 and/or has a size ranging from 20 to 120 μm.
29. The spheroid of claim 28, wherein the spheroid is obtainable with a method comprising: a) culturing stem cell-derived or immortalized human hepatic progenitors in a non-adherent culture vessel; b) transferring the stem cell-derived or immortalized human hepatic progenitors to a culture medium comprising methacrylated gelatin (GelMa), thereby embedding the stem cell-derived or immortalized human hepatic progenitors in a GelMa matrix; and c) covering the GelMa matrix with culture medium and culturing the stem cell-derived or immortalized human hepatic progenitors embedded in the GelMa matrix; thereby obtaining 3D cell structures comprising differentiated human hepatic cells.
30. The spheroid of claim 28, wherein the spheroid is embedded in a GelMa matrix.
31. A method for engineering an artificial liver model or an artificial liver organ comprising differentiated human hepatic cells, the method comprising culturing stem cell-derived or immortalized human hepatic progenitors according to the method of claim 17 with at least another type of cells, wherein the stem cell-derived or immortalized human hepatic progenitors embedded in the GelMa matrix are contacted with the at least another type of cells.
32. The method according to claim 31, wherein said at least another type of cells is embedded in a matrix.
33. The method according to claim 31, wherein said at least another type of cells is another type of hepatic cells.
34. The method according to claim 31, wherein the stem cell-derived or immortalized human hepatic progenitors are cultured with stellate cells and/or endothelial cells.
35. An in vitro method for assessing the metabolism, toxicity and/or therapeutic effects of a compound, the method comprising: a) obtaining 3D cell structures comprising differentiated human hepatic cells according to the method of claim 17; b) contacting the 3D cell structures comprising differentiated human hepatic cells with a compound; and c) assessing the metabolism, toxicity and/or therapeutic effects of the compound on the 3D cell structures comprising differentiated human hepatic cells.
36. The in vitro method according to claim 35 wherein the differentiated human hepatic cells are human hepatocytes or hepatocyte-like cells.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
[0308] The present invention is further illustrated by the following examples.
Example 1
[0309] Materials and Methods
Material
Cells
[0310] The following cell lines were used: HuH-7 cells and HepaRG cells. Both cell lines are immortalized human cells derived from a hepatocellular carcinoma.
Reagents
[0311] Lipofectamine RNAi max transfection reagent, William's E medium and TRIzol were obtained from Invitrogen (Invitrogen, Carlsbad, Calif., USA). FBS and saccharose were obtained from Eurobio (Evry, France). Penicillin/Streptomycin and FBS HyClone III were obtained from Thermo Fisher (Waltham, Mass. USA). WST-1 reagent, geneticin, human insulin, hydrocortisone hemisuccinate, Dimethyl sulfoxide also known as DMSO (D4540), formaldehyde, gelatin, 5-ethylnyl-2′-deoxyuridine, salicylamide, CY5-azide, ethoxyresorufin and methyresorufin, 3-methylcholanthrene and rifampicin were obtained from Sigma-Aldrich (St. Louis, Mo., USA). Methacrylated gelatin (GelMa) was synthetized by the ART Bio-encres (Bordeaux, FR). Lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) was obtained from TCI (Japan) and U0126 was obtained from Promega (Mannheim, GE). Phenobarbital was obtained from the Coopération pharmaceutique française. The Dapi Fluoromount-G was obtained from Southern Biotech (Birmingham, Ala., USA).
[0312] Methods
Cell Culture
[0313] HuH-7 cells (n° 01042712, Health Protection Agency Culture Collections, Salisbury, UK) DNA transfections were carried out using Lipofectamine RNAi max transfection reagent according to the manufacturer's instructions. Briefly, cells were plated in a 35 mm-petri dish. 3 μl of EmGFP-geneticin vector and 5 μl of Lipofectamine RNAi max were mixed in 200 μl Opti-MEM@ and then added to the cells. After overnight incubation, cells were incubated in DMEM medium for 24 h, then selected by treatment with geneticin at 10 mg/mL during 48 h. After selection, HuH-7 GFP+cells were cultured in DMEM comprising 4.5 g/L of both glucose and pyruvate supplemented with 10% (v/v) fetal bovine serum (EuroBio), 100 units/mL penicillin and 100 μg/mL streptomycin, 2 mM L-glutamine and 500 μg/mL geneticin.
[0314] Undifferentiated HepaRG cells (Biopredic, Saint Grégoire, France) were cultured in William's E medium without L-Glutamine supplemented with 10% (v/v) fetal bovine serum Hyclone III, 100 units/mL penicillin, 100 μg/mL streptomycin, 5 μg/mL human insulin, 2 mM L-glutamine and 5×10.sup.−5 M hydrocortisone hemisuccinate (hereinafter referred to as supplemented William's E medium) for 14 days.
[0315] For control experiments in 2D culture (also referred to as 2D-DMSO), hepatic differentiation of HepaRG cells was induced in 2D culture by treatment with 2% (v/v) DMSO (D4540, Sigma-Aldrich, St. Louis, Mo., USA) for an additional 14 days.
Culture According to the Method of the Invention
[0316] Culture in a low attachment plate: immortalized human cells HuH-7 or undifferentiated HepaRG were incubated at 37° C., 5% CO.sub.2, 100% humidity during 15 h-24 h in 6-well low attachment plates (Corning@, Costar®) at a concentration of about 3×10.sup.6 cells per well, in William's E medium without L-Glutamine supplemented with 10% (v/v) fetal bovine serum Hyclone III, 100 units/mL penicillin, 100 μg/mL streptomycin, 5 μg/mL human insulin, 2 mM L-glutamine and 5×10.sup.−5 M hydrocortisone hemisuccinate.
[0317] Preparation of medium comprising GelMa: methacrylated gelatin was prepared as previously described (Kolesky et al., Adv Mater. 2014 May 21; 26(19):3124-30). Briefly, a 10% (w/v) gelatin solution (type A, 300 bloom from porcine skin, Sigma), in PBS, was warmed at 60° C. for 2 h under stirring. Then, the temperature was set to 50° C. and 0.14 mL of methacrylic anhydride (Sigma) per gram of gelatin was added dropwise. The solution was left to react for 4 h, under stirring, then diluted to 5% (w/v) in PBS. The reacted gelatin was precipitated using a 4-fold excess volume of cold acetone. The precipitated gelatin was recovered, vacuum dried for 30 minutes, and then redissolved at 10% (w/v) in PBS at 40° C. It was then placed inside a 12-14 kDa molecular weight cutoff (MWCO) dialysis tubing (Sigma), and dialyzed against deionized water for 3 days, with two daily water changes. The purified GelMa was frozen at −80° C., freeze dried, and stored at −20° C. Small crushed fragments of freeze-dried GelMa (up to 2 mm in length) were dissolved at 37° C. overnight or for a minimum of 6 h in the supplemented William's E medium comprising 10% (v/v) fetal bovine serum Hyclone III, 100 units/mL penicillin, 100 μg/mL streptomycin, 5 μg/mL human insulin, 2 mM L-glutamine and 5×10.sup.−5 M hydrocortisone hemisuccinate as described above. Unless indicated otherwise, a volume of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) at a concentration of 10 mg/mL (in phosphate-buffer saline (PBS)) was then added to obtain a supplemented William's E medium comprising GelMa at a final concentration of 2.5 or 5 g/100 mL, i.e., a final concentration of 2.5 or 5% w/v, and LAP at a final concentration of 100 or 300 mg/100 mL, i.e., a final concentration of 0.1 or 0.3% w/v. Before use, the medium comprising 2.5 or 5% w/v GelMa and 0.1 or 0.3% w/v LAP was kept at 37° C., protected from light. For rheology analyses, photopolymerized GelMa matrices, at 2.5, 5, 7.5 or 10% (w/v) were soaked for 24 h in PBS and then loaded in a Kinexus pro+ rheometer (Malvern Instruments, UK). Frequency sweeps were performed at a constant strain (0.1%) in an angular frequency range of 0.1-100 rad/s, at 37° C.
[0318] Inclusion in a GelMa matrix followed by culture of hepatocytes embedded in the GelMa matrix: HuH-7 or undifferentiated HepaRG were transferred to a suitable container such as an Eppendorf tube (1.5 or 2 mL) or a Falcon tube (15 mL) and centrifuged at 200 g for 2 min. The supernatant was removed and medium comprising 2.5 or 5% w/v GelMa and 0.1 or 0.3% w/v LAP was added to obtain a concentration ranging from about 0.5×10.sup.6 to 2×10.sup.6 cells per mL of medium (for example 0.5×10.sup.6 HuH-7 or 2×10.sup.6 undifferentiated HepaRG cells). For the bioprinting process, 100 μL of the bio-ink, i.e., medium comprising 5% (w/v) GelMa, 0.1% (w/v) LAP and cells, were cooled at 15° C. and extruded through a 23 G or 25 G needle at 1.4-2.4 bar, at 480 mm/min, with the extrusion printer Allevi 2 (Allevi, Philadelphia, Pa., USA) in a 48- or 96-well plate. The 3D construct was designed by the computer aided design software OnShape (Cambridge, Mass., USA).
[0319] Polymerization (i.e., crosslinking) of the GelMa matrix was induced by illumination of the bioprinted 3D construct for 30 seconds to 10 min with a 365, 405 nm or 530 nm LED, preferably by illumination for 60 seconds with a 405 nm ultraviolet light (7 mW/cm.sup.2). After polymerization of the GelMa matrix, a volume of the same supplemented William's E medium (see above) equal to the volume of GelMa matrix was added so that the GelMa matrix was submerged in supplemented William's E medium. The cells embedded in the GelMa matrix were cultured in the supplemented William's E medium as described above for at least 2 days in an incubator at 37° C., 5% CO2, humidity 95%. The medium was changed every 48 h.
[0320] Hereafter, in Example 1, the term “GelMa culture” refers to a culture of immortalized hepatic cells established as described hereinabove, with a first incubation (or culture) of the cells in a low attachment plate followed by the inclusion of the cells in a GelMa matrix, according to the method of the invention. Unless indicated otherwise, any mention of a time of culture, e.g., day 5 of culture, refers to the time of culture in a GelMa (or in a standard multi-well plate for the 2D or 2D-DMSO control condition). Similarly, seeding refers to the inclusion of the cells in a GelMa matrix.
Cell Viability
[0321] Cell viability was assessed by using the WST-1 (water soluble tetrazolium-1) proliferation assay on cell laden 3D constructs at different days or times of culture. Briefly, the cell laden 3D constructs were treated with 100 μL of WST-1 reagent and incubated 30 min at 37° C. Absorbance at 440 nm was read in a microplate reader (SpectrostarNano, BMG labtech, Champigny s/Marne, FR) and the values were converted into percentage of absorbance by comparison with controls assessed at day 0 or time 0. Measurement of lactate dehydrogenase (LDH) was performed on supernatants with the LDH-Glo™ Cytotoxicity Assay (Promega) by following manufacturer's instructions.
Immunostaining
[0322] Cell laden 3D constructs were fixed in 4% formaldehyde, washed 3 times in PBS and stored at 4° C. until use or immediately suspended in a 0.12 M phosphate buffer containing 10% w/v saccharose and incubated overnight. They were embedded in a protective matrix composed of gelatin 7.5% w/v and 10% w/v saccharose, then frozen in isopentane and cryosectionned at a thickness of 7 μm. Slides were stained on the Discovery Automated IHC stainer using the discovery Rhodamin kit (Ventana Mediacl systems, Tucson, Ariz., USA). The concentrations and references of the primary antibodies that were used can be found in Table 1 below. After rinsing, signal enhancement was performed using the Ventana Rhodamin kit and secondary antibody anti-rabbit HRP (760-4311, Roche) or secondary antibody anti-mouse HRP (760-4310, Roche) for 16 min of incubation. After removal from the instrument, slides were manually rinsed, stained when required with albumin antibody then a secondary antibody for albumin detection (Donkey anti goat 488), and mounted with Dapi Fluoromount-G.
TABLE-US-00001 TABLE 1 Primary antibodies Antibody Antibody Antibody Antibody target reference concentration provider Albumin A80229A 1:100 Bethyl Laboratories, TX, USA Cleaved caspase 3 9661S 1:500 Cell Signaling, Leiden, NL E-Cadherin 3195S 1:100 Cell Signaling, Leiden, NL N-Cadherin 610921 1:100 BD Bioscience, San Jose, CA, USA MRP2 Ab3373 1:100 Abcam, Paris, FR Cyclin D1 Ab16663 1:200 Abcam, Paris, FR Ki67 HPA001164 1:400 Atlas Antibodies, Bromma, SE
Proliferation Quantification
[0323] For 5-ethynyl-2′-deoxyuridine (EdU) incorporation, cell laden 3D constructs were treated 48 h with EdU at 1 μM. For negative controls, cells were treated 24 h prior EdU treatment with U126 at 0.05 nM until the fixation of the 3D cell structures. 3D cell structures were fixed and cryosectionned as described above. EdU revelation was carried out by performing permeabilization using 0.1% (v/v) Triton X-100 for 10 min. Then, samples were treated with a mix of CuSO4 (1 μM), Tris pH 8.5 (0.1M), ascorbic acid (0.1M) and CY5-azide (1.5 μM) for 1 h. Cells were mounted with Dapi Fluoromount-G and the nucleus staining was monitored using a fluorescence microscope (Eclipse Ni-E, Nikon, Amsterdam, NL). Image processing was carried out using ImageJ software (http://imagej.nih.gov/ij/).
Metabolic Analysis
[0324] Medium samples were taken for the determination of albumin and urea content every 48 h. Secreted albumin and proCol1a1 (pro-collagen 1 alpha 1) were quantified using the human serum albumin Duoset Enzyme Linked Immunosorbent Assay (ELISA) kit (R&D systems, Minneapolis, Minn., USA) and the human pro-Col1a1 Duoset Enzyme Linked Immunosorbent Assay kits (R&D systems, Minneapolis, Minn., USA), according to manufacturer's instructions. Quantification was performed by measuring absorbance at 450 nm using a microplate reader (SpectrostarNano, BMG labtech, Champigny s/Marne, FR). Urea secretion in the medium was measured using a ChromaDazzle Urea Assay kit (AssayGenie, Dublin, IR) according to the manufacturer's protocol, using medium as standard and reading the absorbance at 420 nm (SpectrostarNano, BMG labtech, Champigny s/Marne, FR)
CYP Activities Measurement by Fluorescence
[0325] After a 24-hour induction with DMSO 0.1% (v/v) or 3-methylcholanthrene (3-MC or 3MC) at 5 μM, ethoxyresorufin-O-deethylation (EROD) was used for measuring CYP1A activity and methyresorufin-O-deethylation (MROD) was used for measuring CYP1A2 activity. EROD and MROD assays were performed as described by Burke and Mayer (Burke & Mayer, Chem Biol Interact. 1983 Jul. 15; 45(2):243-58). Briefly, the cells were washed with PBS at 37° C. and salicylamide (1.5 mM) was added to block phase II conjugation enzymes. 7-ethoxyresorufine or 7-methoxyresorufine was then added 1 min later and the oxidation of the substrates was measured by fluorescence detection every 2 min during 20 min at 37° C. (SpectraMax GeminiXS, Molecular devices, San Jose, Calif., USA). After the detection, a WST-1 proliferation assay was performed as describe above to normalize activity with the quantity of viable cells.
CYP Activities Measurement by Luminescence
[0326] Cytochrome P450 oxidase 2B6 and 3A4 activities were assessed by treating cells with DMSO 0.1% v/v (control) or phenobarbital (0.2 mM, 72 h), and DMSO 0.1% v/v (control) or rifampicin (5 μM, 72 h), respectively. Using the P450 GloAssay (Promega) according to the manufacturer's instructions, a CYP specific luciferin substrate was added on cell laden 3D constructs and incubated at 37° C., 5% CO.sub.2. After 1 hour, CYP-mediated conversion of luciferin substrate to luciferin was determined. The supernatant was incubated with luciferin detection reagent during 20 min at room temperature and the luminescence was measured. After the luminescence measurement, a WST-1 proliferation assay was performed as describe above to normalize activity with the quantity of viable cells.
RNA Extraction and Quantitative Real Time PCR
[0327] After 1, 2 and 4 week(s) of culture, 3D constructs comprising 3D cell structures were harvested, washed twice in PBS and total ribonucleic acid (RNA) was extracted using TRIzol™ reagent. The concentration of total RNA was measured with a NanoDrop ND-1000 (NanoDrop Technologies). Complementary deoxyribonucleic acid (cDNA) was synthetized using the High Capacity cDNA reverse transcriptase kit (Applied Biosystems Foster City, Calif., USA), and real-time polymerase chain reaction (PCR) was performed using the Power SYBR Green PCR master mix (Applied Biosystems Foster City, Calif., USA). Basic local alignment search tool (BLAST) was used to design primer sequences which were purchased from Eurogentec (Searing, BE). All used primers are listed in Table 2 below. The expression levels of the target genes were normalized to those of GAPDH and determined by the ΔΔCt method. Final results were expressed as the n-fold difference in target gene expression in samples when compared with the mean expression values of the target gene for controls: either non-differentiated HepaRG (referred to as 2D) or HepaRG differentiated in standard 2D culture conditions (referred to as 2D DMS0). All results are presented as mean±SD of at least n=3 experiments.
TABLE-US-00002 TABLE 2 Primer sequences Target gene Primer sequence Albumin-forward TGCTTGAATGTGCTGATGACAGG (SEQ ID NO: 1) Albumin-reverse AAGGCAAGTCAGCAGGCATCTCATC (SEQ ID NO: 2) Aldolase B- GTCCATCCAGCCTCGCTATC forward (SEQ ID NO: 3) Aldolase B-reverse ACCAGTCCATTCTGCTGACA (SEQ ID NO: 4) CDH1-forward CGGGAATGCAGTTGAGGAT (SEQ ID NO: 5) CDHl-reverse AGGATGGTGTAAGCGATGG (SEQ ID NO: 6) CDH2-forward GTGCATGAAGGACAGCCTCT (SEQ ID NO: 7) CDH2-reverse ATGCCATCTTCATCCACCTT (SEQ ID NO: 8) CK19-forward GAGCAGGTCCGAGGTTACTG (SEQ ID NO: 9) CK19-reverse GCTCACTATCAGCTCGCACA (SEQ ID NO: 10) CYP 1A1-forward GACATTGGCGTTCTCATCCA (SEQ ID NO: 11) CYP 1A1-reverse AGAAGCTATGGGTCAACCCA (SEQ ID NO: 12) CYP 1A2-forward TGGAGACCTTCCGACACTCCT (SEQ ID NO: 13) CYP 1A2-reverse CGTTGTGTCCCTTGTTGTGC (SEQ ID NO: 14) CYP 2B6-forward TTCCTACTGCTTCCGTCTATC AAA (SEQ ID NO: 15) CYP 2B6-reverse GTGCAGAATCCCACAGCTCA (SEQ ID NO: 16) CYP 2C19-forward GTGAAGGAAGCCCTGATTGA (SEQ ID NO: 17) CYP 2C19-reverse TCCTCTTGAACACGGTCCTC (SEQ ID NO: 18) CYP 2C9-forward GGACAGAGACGACAAGCACA (SEQ ID NO: 19) CYP 2C9-reverse AATGGACATGAACAACCCTCA (SEQ ID NO: 20) CYP 2E1-forward TTGAAGCCTCTCGTTGACCC (SEQ ID NO: 21) CYP 2E1-reverse CGTGGTGGGATACAGCCAA (SEQ ID NO: 22) CYP 3A4-forward CTTCATCCAATGGACTGCATA AAT (SEQ ID NO: 23) CYP 3A4-reverse TCCCAAGTATAACACTCTACA CA GACAA (SEQ ID NO: 24) FXR-forward GGAGGATCAAAGGGGATGA (SEQ ID NO: 25) FXR-reverse CAGTTGCCCCCGTTTTTAC (SEQ ID NO: 26) GAPDH-forward GTGGACCTGACCTGCCGTCT (SEQ ID NO: 27) GAPDH-reverse GGAGGAGTGGGTGTCGCTGT (SEQ ID NO: 28) HNF4a-forward AACCTGTTGCAGGAGATGCT (SEQ ID NO: 29) HNF4a-reverse TTGCCTTGTTCCCATTTTTC (SEQ ID NO: 30) MRP2-forward TGAGCAAGTTTGAAACGCACAT (SEQ ID NO: 31) MRP2-reverse AGCTCTTCTCCTGCCGTCTCT (SEQ ID NO: 32) PXR-forward CCAGGACATACACCCCTTTG (SEQ ID NO: 33) PXR-reverse CTACCTGTGATGCCGAACAA (SEQ ID NO: 34) Serpina1-forward CACCGTGAAGGTGCCTATGATG (SEQ ID NO: 35) Serpina1-reverse GGCATTGCCCAGGTATTTCATC (SEQ ID NO: 36) SOX9-forward GCCTTTTTGTCCATCCCTTT (SEQ ID NO: 37) SOX9-reverse GCTTGCATTGTTTTTGTGTCA (SEQ ID NO: 38) Vimentin-forward CCAAACTTTTCCTCCCTGAACC (SEQ ID NO: 39) Vimentin-reverse GTGATGCTGAGAAGTTTCGTTGA (SEQ ID NO: 40) ACTA2-forward TATTCCTTCGTTACTACTGCT (SEQ ID NO: 41) ACTA2-reverse AAGTCCAGAGCTACATAACA (SEQ ID NO: 42) ADAMTS12- CACGACGTGGCTGTCCTTCT forward (SEQ ID NO: 43) ADAMTS12- CCGAATCTTCATTGATGTTACA reverse ACTG (SEQ ID NO: 44) Col1A1-forward CCTCAAGGGCTCCAACGAG (SEQ ID NO: 45) Col1A1-reverse TCAATCACTGTCTTGCCCCA (SEQ ID NO: 46) Col3A1-forward GGGGAGCTGGCTACTTCTCG (SEQ ID NO: 47) Col3A1-reverse TGAGGTCCTTGACCATTAGG (SEQ ID NO: 48) LPL-forward TCATTCCCGGAGTAGCAGAGT (SEQ ID NO: 49) LPL-reverse GGCCACAAGTTTTGGCACC (SEQ ID NO: 50) MMP2-forward CAAGGACCGGTTTATTTGGC (SEQ ID NO: 51) MMP2-reverse ATTCCCTGCGAAGAACACAGG (SEQ ID NO: 52) TIMP1-forward CACCCACAGACGGCCTTCT (SEQ ID NO: 53) TIMP1-reverse CTTCTGGTGTCCCCACGAACTT (SEQ ID NO: 54) TGFβI-forward CTTCGCCCCTAGCAACGAG (SEQ ID NO: 55) TGFβI-reverse TGAGGGTCATGCCGTGTTTC (SEQ ID NO: 56)
Transcriptomic Analysis
[0328] Total RNAs were purified from freshly isolated human hepatocytes (PHH TO) (n=5), and from differentiated HepaRG after 14 days of culture on 2D (HRG 2D DMSO) (n=5) and at day 14 of culture on GelMa (HepaRG 3D GelMa) (n=4). The samples were concentrated using the RNA Clean & Concentrator-5 (Zymo, Irvine, Calif., USA) and checked for RNA degradation based on the RNA Integrity Number (RIN>6). 3′ sequencing RNA Profiling (3′ SRP-seq) libraries were made at the GenoBIRD facility of Nantes, France, and sequenced using the HiSeq 2500 (Illumina, San Diego, Calif., USA) following the described protocol (Soumillon et al., 2014, BioRxiv 003236). The analysis of the generated data was performed using R packages. The differentially expressed genes (FC>2, p<0.05) between HRG 2D DMSO and HRG GelMa D14 were functionally analyzed by computing enrichments for gene ontology (GO) terms, using the WEB-based database GEne SeT AnaLysis Toolkit (WebGestalT) restricted to protein coding data set. The presented data are selected as the top ten of the enriched categories, sorted by increasing enrichment ratios (FDR<0.05).
SHG/TPEF Microscopy
[0329] TPEF/SHG microscopy imaging was performed on mRIC facility of Biosit, University of Rennes1 (France). The SHG imaging system is composed of a confocal TCS SP5 scanning head (Leica Microsystems, Mannheim, Germany) mounted on a DMIRE2 inverted microscope (Leica Microsystems) and equipped with a MAITAI femtosecond laser (Spectra Physics, Santa Clara, Calif.). 60× water immersion objective (Olympus LUMFL 60W×1.1NA) was used for applying an 820 nm excitation to the sample. The SHG signal was collected in the forward direction using the condenser (S1, NA ¼ 0.9-1.4; Leica Microsystems), and the TPEF was epi-collected in the backward direction. IRSP 715 bandpass and 410 nm infrared (IR) filters (10 nm full width at half-maximum, FWHM) were placed before the photomultiplier tube. The image processing was performed with ImageJ software (National Institutes of Health).
Statistical Analysis
[0330] All variables were expressed as means±standard deviations (SDs) of at least 3 experiments with 3 technical replicates. Evaluation of the difference between the mean values in each group was performed using t-test or one-way analysis of variance (ANOVA) (GraphPad Prism 6, GraphPad Software, Inc.; La Jolla, Calif., USA), in which p values are considered to indicate significance for * or #p≤0.05; ** or ##p≤0.01; *** p≤0.001; **** p≤0.0001.
[0331] Results
Physical Properties of GelMa Matrix
[0332] The physical properties of different methacrylated gelatin matrices (GelMa matrices) were analyzed by assessing the polymerization and bioprinting properties of the matrices as a function of the GelMa concentration, the photoinitiator LAP concentration and the illumination time at 405 nm (
Viability of Immortalized Hepatic Cells in GelMa Cultures
[0333] Next, the short- and long-term viability of HuH-7 cells (an immortalized hepatic cell line derived from transformed hepatocellular carcinoma cells) in 3D GelMa culture (with GelMa 5% (w/v), LAP 0.1% (w/v)) was assessed before and after crosslinking (1 min illumination time) and bioprinting. As shown on
Proliferation of HepaRG Cells in a GelMa Culture
[0334] The method of culturing of the invention was next applied to the gold standard immortalized human hepatic cells, the HepaRG cells. As described above, undifferentiated HepaRG were first cultured in a low-attachment plate for about 15 hours before being embedded in a GelMa matrix. Strikingly, undifferentiated HepaRG cells could not fully differentiate in the GelMa matrix if they were not first cultured in a low-attachment plate. As shown on
[0335] Undifferentiated HepaRG were cultured in a GelMa matrix according to the method of the invention in the absence of DMSO in order to avoid non-specific inductions of the Cyp3A4 cytochrome by DMSO previously observed during the differentiation of HepaRG in 2D standard culture conditions (referred to as 2D-DMSO culture) (Aninat et al., Drug Metab Dispos. 2006 January; 34(1):75-83).
[0336] HepaRG cells in 3D GelMa culture formed small clusters of cells, i.e., spheroids, 7 days after seeding (
[0337] The cells appeared polarized as evidenced by the localization of E- and N-cadherins at the apico and lateral membranes (
Differentiation of HepaRG Cells in a GelMa Culture
[0338] The expression of hepatic genes was compared in freshly isolated primary human hepatocytes, HepaRG cells at day 14 in 2D standard culture conditions (referred to as 2D-DMSO culture) and HepaRG cells at day 14 in 3D GelMa culture (data not shown). Gene expressions was measured by 3′ SRP-RNA-Seq from RNAs HepaRG. The comparison showed that HepaRG cells in 2D-DMSO culture and 3D GelMa culture share a similar hepatic gene expression pattern. When compared to freshly isolated primary hepatocytes, HepaRG cells in both 2D-DMSO culture and 3D GelMa culture showed the same clusters of up- and down-regulated hepatic genes. In particular, 4 specific hepatic markers, ALB, ALDOB, HNF4A and SERPINA1, showed a level of expression similar for freshly isolated primary hepatocytes, HepaRG cells in 2D-DMSO culture and HepaRG cells in 3D GelMa culture.
[0339] More precisely,
[0340] Then, an analysis of liver-specific gene expressions (LiGEP signature) as defined by Kim et al. (Kim et al., Hepatology. 2017 November; 66(5):1662-1674) was carried out. This panel was based on the significantly differential RNA expressions between liver and non-liver samples. Kim et al. developed an algorithm based on RNA-sequencing (RNA-seq) analysis to assess the differentiation or maturation status of 93 liver-specific genes validated by expression profiles in human Protein Atlas database and by quantitative real-time PCR analysis. A modified version of this original list, excluding, inter alia, genes from xenobiotic metabolism, was used for transcriptomic analyzes. RNAs from freshly isolated primary human hepatocyte (PHH TO) were used here as a standard reference. The expression profile of freshly isolated primary human hepatocytes is considered to be closest to the liver in vivo but cannot be reproduced in vitro due to the extremely rapid dedifferentiation of the human hepatocytes in 2D culture. Here, if a decrease in the expression of hepatic genes can be observed during the comparison of HepaRG GelMa and HepaRG 2D DMSO with the freshly isolated PHH, one notes a maintenance of the expression of hepatic genes of the LiGEP signature with a great homology of the expression profiles of HepaRG in GelMa and 2D DMSO, with both HepaRG in 2D and 3D showing a quite similar pattern of many hepatic gene expression (e.g., ALB, ALDOB, SERPINA1, FGA, FGB, FGG).
[0341] As shown on
[0342] Additionally, the decreased mRNA expression level of the biliary lineage markers SOX9 (
[0343] Next, the expression of phase I metabolism genes was compared in freshly isolated primary human hepatocytes, HepaRG cells at day 14 in 2D standard culture conditions (2D-DMSO culture) and HepaRG cells at day 14 in 3D GelMa culture (data not shown). The comparison showed that the expressions of most phase I enzymes are down-regulated in HepaRG cells in 3D GelMa culture, compared to HepaRG cells in 2D-DMSO culture.
[0344] The mRNA expression levels of Phase I enzymes were also quantified using RT-qPCR in HepaRG cells both in 3D GelMa culture and in 2D-DMSO culture, and in undifferentiated HepaRG cells in 2D culture (without DMSO). As shown on
[0345] The mRNA expression levels of Phase II and Phase III enzymes were also assessed in HepaRG cells both in 3D GelMa culture and in 2D-DMSO culture. As seen with the phase I enzymes, the mRNA expression of the Phase II and Phase III enzymes in HepaRG cells in 3D GelMa culture were lower than, equal to or greater than those in HepaRG cells in 2D DMSO culture, depending on the enzymes (data not shown). As mentioned above, the phase II MRP2 transporter was located exclusively at the apico/canicular area showing a good polarization of the HepaRG cells in the 3D GelMa culture throughout the entire culture time. Furthermore, the expression of CDH1 encoding E-cadherin (
[0346] The data on Huh7 and HepaRG cells showed a stable hepatic differentiation, notably close or even higher than that of HepaRG cells cultured in 2D with DMSO according to the gold standard culture method. From the start of the 3D GelMa culture, the differentiation increased until day 15 of the culture. The differentiation was optimal at day 15 and stayed at a high level until day 28, allowing long-term and chronic exposures of drugs and environmental products, in absence of DMSO.
[0347] In conclusion, the data presented above demonstrate that HepaRG cells in 3D bioprinted GelMa display long-term survival and a highly differentiate state with a proliferating capability. The proliferative and differentiated properties of the HepaRG cells may be of great interest to enable genotoxic and mutagen studies of hepatic drugs and environmental products that need to be metabolized by the liver to give genotoxic and/or mutagenic DNA products.
Example 2
[0348] Materials and Methods
[0349] Material
Cells
[0350] The HepaRG cell line was used. Other cells used in the following experiments include human hepatic stellate cells (LX-2 cells derived from activated stellate cells (Xu et al., Gut. 2005 January; 54(1):142-51) and endothelial cells (Human umbilical vein endothelial cells or HUVECs).
Reagents
[0351] DMEM medium L-glutamine was obtained from Gibco (ThermoFisher, Waltham, Mass. USA). EBM™-2 Endothelial Cell Growth basal medium and EGM™-2 BulletKit™ were obtained from LONZA (Aubergenville, France).
[0352] Methods
Cell Culture
[0353] Undifferentiated HepaRG cells (Biopredic, Saint Grégoire, France) were cultured as described above (see Example 1). For control experiments in 2D culture (also referred to as 2D-DMSO), hepatic differentiation of HepaRG cells was induced in 2D culture by treatment with 2% DMSO (D4540, Sigma-Aldrich, St. Louis, Mo., USA) for an additional 14 days.
[0354] LX-2 cells (provided by the Dr N. Theret, Irset, France) were cultured in DMEM comprising 4.5 g/L of both glucose and pyruvate supplemented with 10% (v/v) fetal bovine serum (EuroBio), 100 units/mL penicillin and 100 μg/mL streptomycin and 2 mM L-glutamine.
[0355] Human umbilical vein endothelial cells also referred to as HUVECs (ZHC-2402, CellWorks, Buckingham, UK) were cultured in a mix of EBM™-2 Endothelial Cell Growth basal medium and William's E medium (1:1) supplemented with 10% (v/v) fetal bovine serum Hyclone III, 100 units/mL penicillin, 100 μg/mL streptomycin, 5 μg/mL human insulin, 2 mM L-glutamine and 5×10.sup.−5 M hydrocortisone hemisuccinate. hFGF-B (human fibroblast growth factor-basic), VEGF (vascular endothelial growth factor), ascorbic acid, hEGF (human epidermal growth factor), GA-1000 (gentamicin sulfate-amphotericin) and heparin issued from the EGM™-2 BulletKit™ were added to the medium at concentrations recommended by the supplier.
[0356] HepaRG cells and/or LX-2 cells were included in a GelMa matrix as described hereinabove (see Example 1).
[0357] For coculture constructs, the bioprinted 3D structure comprising HepaRG cells and LX-2 cells was cultured during a week, before being put in a low attachment plate containing 1×10.sup.6 HUVECs per mL of medium.
[0358] Results
Culture of Human Hepatic Stellate Cells in a GelMa Matrix
[0359] Micro-engineered cocultures with HepaRG, LX-2 and HUVECs were developed, with the aim of obtaining an in vitro model for evaluating the fibrotic process in vitro.
[0360] First, the culture of LX-2 cells alone in 3D GelMa culture was assessed, with or without treatment with TGFβ-1, a classical pro-fibrotic cytokine (Czaja et al., J Cell Biol. 1989 June; 108(6):2477-82). From day 2 of culture, cells were treated with vehicle (controls) or TGFβ-1 (5 ng/mL every 48 h) for two weeks (activation) or for one week followed by an additional week without TGFβ-1 (reversion) (
[0361] These results indicate that 3D GelMa provide suitable culture conditions for human hepatic stellate cells.
[0362] SHG microscopy was used to visualize fibrillar collagen, mostly collagen-1 and -3. Briefly, SHG relies on the nonlinear optical interactions with non-centromeric fibrillar structures, such as collagen type I, allowing their detection and quantification in 3D tissues. Very few collagen fibrils or no collagen fibrils at all could be observed after induction of the LX-2 cells by TGFβ-1 (see
Coculture of HepaRG Cells, Human Hepatic Stellate Cells and Human Endothelial Cells
[0363] Then, HepaRG cells, LX-2 cells and HUVECs were cocultured together (
[0364] As controls, HepaRG cells were bioprinted either alone or with HUVECs alone (HepaRG+HUVECs). The green fluorescent protein (GFP) allowed to visualize the homogeneous seeding of fluorescent HUVECs at the surface of the GelMa matrix, including into the channels, 4 days after seeding (
[0365] The mRNA expression of hepatic genes (ALB, ALDOB, PPARA and SERPINA1) and of myofibroblastic transition genes and extracellular matrix remodeling genes (ACTA2, ADAMTS12, COL1A1, COL3A1, MMP2, MMP9 and TIMP1) was assessed at day 21 (i) in HepaRG alone in 3D GelMa culture, (ii) in HepaRG in 3D GelMa culture with LX-2 cells and HUVECs (CoC) and (iii) in HepaRG in 3D GelMa culture with LX-2 cells and HUVECs (CoC) activated with TGFβ-1 treatment for 7 days (
[0366] Basal expression of COL1A1 was significantly higher in HepaRG+LX-2+HUVECs (CoC) than in HepaRG alone or in HepaRG+HUVECs, at a level close to that of LX-2 in GelMa alone (
[0367] In HepaRG+LX-2+HUVECs (CoC), a high basal secretion was measured at day 21 and no significant increase after TGFβ-1 activation could be detected. The level of pro-collagen 1a1 secretion in HepaRG+LX-2+HUVECs was close to that of TGFβ-1 activated HepaRG or HepaRG+HUVECs.
[0368] The deposition of the fibrillar collagen in the HepaRG-LX-2-Huvex cocultures was assessed at day 21 with SHG microscopy (
[0369] In conclusion, the data presented above demonstrate that HepaRG cultured in 3D GelMa in the presence of other types of cells, such as human hepatic stellate cells and human endothelial cells allows the development of complex multicellular hepatic models, that may be used for example as a fibrosis model, for studying collagen synthesis and deposition.