CO-CULTURE OF PRIMARY HEPATIC STELLATE CELLS AND HEPATOCYTES IN
MODELING OF LIVER FIBROSIS IN 2D AND 3D SPHEROIDSRafal P. Witek, Sujoy Lahiri, Bina Khaniya, Mukesh Kumar, Michael P. Connolly, Jennifer C. Hoover, Michael Millet, Debra L. Johnson, Mark Kennedy, Deborah K. Tieberg
Thermo Fisher Scientific, 7300 Governors Way, Frederick, Maryland, 21704

3D Hepatocytes
3Collagen 4A1354Fibronectin

MF-HSC21Fold over Control

Fold over Control

302520151032150003D Co-Cultures of Hepatocytes and Stellate Cells

CYP3A4 - qPCR

0.6
0.4
0.2
0A-HSCa-SMA

Control

Figure 2. Representative flow cytometry analysis of isolated humanquiescent stellate cells. Cells were isolated and labeled with anti-GFAP
antibody (stellate cell marker) and a-SMA (activated stellate cell marker)
followed by detection with alexa fluor 633 labeled secondary antibody.

A.

Hep Alone

Hepatocytes Monocultures
Hep Alone

Co-Culture

Co-culture
Co-Cultures

LX2

A-HSC

LX2Figure 6. Co-culture of primary hepatocytes and MF-HSC resulted in declined CYP3A4activity. As expected, MF-HSC impacted metabolic performance of primary hepatocytes byreducing CYP3A4 activity measured at day 4 by qRT-PCR gene expression analysis and by
LC/MS. This was expected as when MF-HSC are plated on rigid plastic, they produce
cytokines and growth factors that have been implicated in liver regeneration and repair, aprocess that often is characterized by decreased metabolism.

3D Spheroid Co-Cultures of Hepatocytes
and MF-HSC

Figure 7. Workflow of NunclonTM SpheraTM super low attachment U-bottom platesystem that allows assembly of primary hepatocytes and co-cultures into 3Dspheroids.

a-SMAControl

600

Fold over Control

Fold over Control

8003025201540010200

5004Fibronectin

3210Figure 10. Expression of Collagens, and Fibronectin in 3D Co-culture of primary
hepatocytes and MF-HSC and 3D Hepatocytes with and without treatment with
TGFβ and Methotrexate. As expected, there is significant increase in basal expressionof Collagen 1A1 in 3D co-cultures when compared with 3D hepatocytes. This isexpected as activated MF-HSC should display strong expression of this fibrogeniccollagen. Additional increase is observed following induction of fibrogenic response withTGFβ . Note that Methotrexate has not shown any visible change in gene expression
after 7 day treatment suggesting that longer time may be needed to observe any changewith this inducer of fibrosis.

Day 14
Untreated Control

TGFb

Methotrexate

Activated Stellate Cells (MF-HSC)

Quiescent Stellate Cells

B.

1000

Collagen 4A1

35

Hepatocytes

Figure 1. Morphological and Immuno-characterization of quiescent human hepatic
stellate Cells. Cells were isolated from human liver tissue and Immunostained forstellate cell markers. (A) Vitamin A autofluorescence. Inset figure shows magnified viewof stellate cells in phase contrast. (B) GFAP (C) Desmin (D) a-SMA.

0.8

1800
1600
1400
12001000
8006004002000Collagen 1A11200

CYP3A4 – LC/MS

1.211400

Fold over ControlFigure 5. Morphological assessment of monocultures and co-culture of MF-HSCwith primary human hepatocytes. Note that in each case, hepatocyte and MF-HSC
monocultures, and combined hepatocytes and MF-HSC co-cultures formed welldeveloped monolayers at approximately 95-100% confluence. Arrowheads indicate MFHSC mixed between hepatocytes. Note that the addition of MF-HSC did not extend
lifespan of hepatocytes and after 7 days cultures started to decline.

Activation

Co-culture of Hepatic stellate cells with human primary
hepatocytes: For 2D co-cultures, human primary hepatocytes were cocultured along with activated hepatic stellate cells at 1:4 ratio of stellate
cells/hepatocytes in 6 well plates and GibcoTM Collagen Coated 24-wellplates (A1142802). Adv. DMEM media supplemented with 10% FBS for
plating and 0% FBS for maintenance after initial 24hr. Additional
GlutaMAX and P/S were used as needed. There was no overlay used
for this experiment.

Collagen 1A1

Co-Cultures

Cell Isolation:
Hepatic stellate cells were isolated from whole liver human tissue.
Briefly, tissue were digested with a three step EGTA, Pronase andCollagenase solution followed by mechanical digestion and purificationof non-parenchymal cells using density gradient. Primary hepatocytes
were selected from existing inventory and validated for ability to form
3D spheroids. These cells were then used for all 2D and 3D
experiments.

Co-Culture

HepatocytesGFAP

MATERIALS AND METHODS

2D Co-Cultures of Hepatocytes
and MF-HSC

Fold over Control

Isolation and Characterization of Human
Myofibroblastic Stellate Cells (MF-HSC)

CYP3A4 (pmol/min*1E6 Cells)

Human hepatic stellate cells (HSC) are the major contributor to collagendeposition following liver damage. During injury, hepatic stellate cells
activate to a phenotype characterized by increased proliferation,motility, contractility, and synthesis of extracellular matrix componentsthat results in progressive liver fibrosis. To understand the activation
process of q-HSC and to facilitate cell-cell interactions and predict theireffect on hepatocyte function, we have developed co-culture 2D and 3D
spheroid systems to model the progression and reversion of liver
fibrosis in vitro. Following isolation of q-HSC from human cadaveric
tissue, their quiescence was indicated by the presence of vitamin A, thepresence of GFAP and absence of a-SMA. As expected, after platingand culturing for 10 days on rigid plastic, q-HSC activated to themyofibroblastic (MF) phenotype, which is shown by increased a-SMAexpression. Activated MF-HSCs were also positive for Vimentin andCD271 indicating their mesenchymal origin. In order to determine therole of various factors on the activation, q-HSC were maintained inculture until viable culture was establish after which they werecryopreserved at passage 5 and used for all subsequent experiments.
Using 2D and 3D co-culture systems, TGF-b and Methotrexate wereused to induce MF-HSC to produce ECM and to stimulate cytokine,
chemokine, and growth factor production as observed in thephysiological in-vivo setting during fibrogenic liver injury. This response
was evaluated by qRT-PCR that was used to evaluate gene expression.
When co-cultured with hepatocytes, either in 2D plated plates or 3D
spheroid cultures, HSCs stabilized cultures of primary humanhepatocytes and elicited responses observed during liver fibrosis that
included significantly upregulated collagen deposition and a-SMAexpression. In conclusion, we have generated primary human quiescentand activated hepatic stellate cells with high purity and function. Thosecells can be co-cultured in 2D plated system and 3D spheroid system tomodel the liver fibrogenic response. Our data indicates involvement of
HSC produced growth factors, cytokines and chemokines in function ofhepatocytes with implications in modeling fibrosis for drug metabolism
and toxicity testing.

RESULTS

Relative Gene Expression (2-DDCT)ABSTRACT

GFAP

100um

Figure 11. Images displaying morphology of 3D Spheroids of Co-cultures of
Hepatocytes and MF-HSC, and Hepatocytes alone following treatment with TGFβand Methotrexate for 14 days. Note that phenotypic morphology shows differential
density of hepatocyte spheroids that appear more uniform and denser than co-culture
spheroids much more than those at 7 days. Also note appearance of smaller “vesiclelike” structures that are predominantly visible in co-cultures following treatment withMethotrexate.

100um

Gene Expression analysis: Hepatic and co-culture spheroids werelysed using Trizol® followed by mRNA isolation. Following RT, gene
expression was assessed using TaqMan® primers for Collagen 1 Alpha
1 (COL1A1) – HS00164004-M1, Collagen 4 Alpha 1 (COL4A1) –HS00266237-M1, Fibronectin (FN1) – HS00415006-M1; Actin, alpha 2,smooth muscle (ACTA2-aSMA) –HS00909449-M1. GAPDH was used
as internal control.

For Research Use Only. Not for use in diagnostic procedures.

Co-Culture 24hr

Figure 3. Generation of activated HSCs from quiescent cells. (A). Quiescenthepatic stellate cells were grown in uncoated tissue culture plates. After 7-10 days inculture cells activate to Myofibroblastic Hepatic Stellate Cells (MF-HSC) and start
proliferating. (B). After activation and proliferation, immunostaining followed by flowcytometry analysis demonstrated that more than 90% of cells were positive of a-SMA.
Cells were also positive for Vimentin and CD271 (Data not shown).

Co-Culture 72hr

Co-Culture Day 7

Hepatocytes Day 7

Figure 8. Representative images of 3D Spheroids of Co-cultures of Hepatocytes andMF-HSC during their formation using NunclonTM SpheraTM super low attachment Ubottom plate. Approximately 1500 hepatocytes and 500 MF-HSC were used to makeeach spheroid. Note progression of cells going from cell aggregate (24hr) to morecompacted 3D structure (72hr) and finally fully assembled spheroid (7 days). Note themorphological difference of outside surface of co-culture spheroid when compared to moredefined and “smoother” hepatocyte spheroid.

Untreated Control

TGFb

MethotrexateHepatocytes

Day 7Vimentin
MF-HSCaSMA
MF-HSC LX2

LX2
PDGFrbDesminVimentin

Nestin

GAPDH

GAPDH

Figure 4. Characterization pf MF-HSCby IHC and Western blot. Vimentin andaSMA was used to identify MF-HSC andto determine their purity. Additional
Western blot analysis of MF-HSC andLX2 (commercially available humanhepatic stellate cell line) demonstrated
presence of Nestin, PDGFrb, Vimentin
and Desmin in primary MF-HSC but onlyPDGFrb and Vimentin could be detectedin LX2. This supports true identity of ourprimary MF-HSC and their functionality.

Co-Cultures

For 3D spheroids, ratio of 1:3 was used for co-cultures andapproximately 500 stellate cells were used for every 1500 hepatocytes.
Hepatic spheroids were made using 1500 hepatocytes. NunclonTM
SpheraTM super low attachment U-bottom 96-well Microplates (174925)
were applied as an easy to use method for generation of 3D spheroids.
Briefly, after cells were added in 200ul of plating media (Adv. DMEMsupplemented with 10% FBS, GlutaMAX and P/S) to each well, plates
were spun at 200g for 2 min and placed in the incubator for 5 days. Atthat point half of the media was replaced with fresh maintenancemedium (Adv. DMEM media supplemented with GlutaMAX, P/S and 0%FBS) for 2 more days. Experimental treatment started at 7 days after
initiation of co-cultures. At that time, half of the media was removed andreplaced with maintenance medium. This step was repeated 2 times todilute existing medium containing FBS. At that point, half media wasremoved and replaced with equal volume of maintenance medium
containing either TGFβ (100ng/mL) or Methotrexate (200mM). In bothcases this concentration was 2x of the final to which spheroids wereexposed (TGFβ at 50ng/ml and Methotrexate at 100mM at final
concentration). Similarly to this, media change with addition of fresh
TGFβ and Methotrexate was repeated every other day. TGFβ (GibcoTM,PHG9204), Methotrexate (Sigma, A6770-100MG)

CONCLUSIONS AND FUTURE PLANS• Our data indicates that we were able to isolate hepatic stellate cellsfrom human liver tissue as indicated by positive staining for GFAP andDesmin. Those cells activate to Myofibroblastic Hepatic Stellate Cells(MF-HSC) and express Vimentin, Nestin, aSMA and Desmin.
• 2D co-culture of MF-HSC with hepatocytes showed decreased
expression of CYP3A4. Similar decreased activity has been shown in
NAFLD induced liver fibrosis (Woolsey SJ, et al. Drug Metab.Dispos.
2015 Oct;43(10):1484-90).
• 3D co-culture of primary hepatocytes and MF-HSC offer easy toassemble system to generate fibrotic cell model in vitro. The systemeasily allows culture for over 21 days and allows for significant
expression of fibrogenic Collagens. This can be induced by TGFβ .
• Our preliminary data at 14 days treatment suggest that chronic
treatment with Methotrexate may induce fibrogenic changes in vitro.
This will be farther explored in our future studies.
• Future histological and IHC examination will be performed to fully
characterize co-culture fibrotic model generated using NunclonTM
SpheraTM super low attachment U-bottom plate system and primary
hepatocytes mixed with MF-HSC.

Figure 9. Images displaying morphology of 3D Spheroids of Co-cultures ofHepatocytes and MF-HSC, and Hepatocytes alone following treatment with TGFβ andMethotrexate for 7 days. Note that each spheroid was treated individually in each well andthen each treatment group was combined for the imaging. Also note that phenotypicmorphology shows differential density of hepatocyte spheroids that appear more uniform
and denser than co-culture spheroids. Co-culture spheroids appear to be more transparent
containing dense centers.

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