Application Note 8Co-Culturing of Primary Hepatocytes andKupffer Cells in GrowDex® and DAPI/Phalloidin Staining of the Cell Spheroids
Lauri Paasonen, Ph.D.1, Anne-Mari Moilanen, Ph.D.2, Minna Komu, Ph.D.2
1UPM
2
Admescope

INTRODUCTION

It is well known that three-dimensional (3D) culturing of hepatic cells resembles in vivo
better than traditional monolayer cultures. Co-culturing of cells in 3D provides even more
physiologically relevant approach to mimic in vivo tissue morphology and functionality dueto intercellular communication between different cell types. Kupffer cells are specialized
macrophages having an important role in the normal liver function.
Visualization of the cells during culture is an important factor, especially during theestablishment of the model. Some 3D matrices can be problematic due to their inherent autofluorescence. However, GrowDex® is not auto-fluorescent, therefore imaging labelled cells
post staining is straightforward.
In this study we present a fluorescence staining methodology to determine cell viability andmorphology of spheroids co-cultured from primary human hepatocytes and Kupffer cells inGrowDex. Confocal imaging was used to visualise both cell nuclei, stained with DAPI, andfilamentous actin (F-actin), stained with Phalloidin, within the spheroid.

MATERIALS• Human primary hepatocytes and Kupffer cells (Bioreclamation IVT)
• 96 well μ-plate (#89646 Ibidi)• Hepatocyte cell culture medium (CP, Bioreclamation IVT) supplemented with 10 ng/ml hepatocyte growth factor (HGF, H5791, Sigma-Aldrich) and 20 ng/ml epidermal
growth factor (EGF) (E9644, Sigma-Aldrich)• Phosphate buffered saline (PBS)• 4% Paraformaldehyde (PFA) in PBS, 0.1% Triton X-100 in PBS• DAPI (28718-90-3, Sigma-Aldrich), Phalloidin (P1951, Sigma-Aldrich)

METHODSRESULTS

1.

Spheroids were cultured in 0.5% and 0.8% GrowDex for 12 d according to theprocedure presented in Application Note #7 - “Long-term 3D Culture of Human
Primary Hepatocytes in GrowDex®” using Ibidi 96 well µ-plates.
a. The ratio of primary hepatocytes to Kupffer cells was 2:1 (primary hepatoytes:Kupffercells).
b. 10 µl GrowDex containing 5,000 cells was dispensed into each Ibidi μ-well and 20µl medium dispensed on top.
c. The media was renewed every second or third day of culture.
d. From the first medium change onwards, the culture volume was increased to 40 μl
and only half of the medium (20 µl) replaced to avoid disruption of the GrowDex/
cell layer.

2.

Staining of the cells:
q. Culture medium was removed from the wells and cells fixed with 60 µl of 4% PFA in
PBS for 1 h at room temperature.
b. PFA was removed and cells made permeable by the addition of 60 µl of 0.1% Triton
X-100 in PBS and incubated for 30 min at room temperature.
c. Triton X-100 was removed and cells stained with 60 µl DAPI, 10 mg/ml stock
solution diluted 1:500, and Phalloidin-TRITC diluted 1:1000, followed by overnightincubation at +4°C.

3.

Images of the spheroids were captured using a Zeiss LSM780 confocal microscopea. Plan-Apochromat 20x/0.8 objective
b. DAPI: excitation 405 nm, emission 410 nm-533 nm
c. Phalloidin-TRITC: excitation 561 nm, emission 566 nm-670 nm

Primary hepatocytes and Kupffer cells were co-cultured in GrowDex for 12 d and shown toform spheroids. The spheroids were successfully visualised using a confocal microscope afterDAPI/Phalloidin staining of the cell nuclei and filamentous actin (Fig. 1).

Figure 1. Primary hepatocyte/Kupffer co-culture (ratio 2:1). Spheroids stained with DAPI
(blue) and Phalloidin (red) after 12 d culture in A) 0.8% GrowDex, and B) 0.5% GrowDex.

CONCLUSIONS

Relevant in vitro hepatic models are needed in drug development for e.g. hepatotoxicity orefficacy screening. Here we have presented a reliable method for 3D co-culturing of primary
hepatocytes with Kupffer cells to form in vitro liver spheroids. In addition, a reliable andconsistent procedure for staining liver cells with DAPI and Phalloidin, for visualisation of cell
nuclei and actin filaments within spheroids cultured is also presented. Fluorescent dyes diffuse
well and, as GrowDex is not auto-fluorescent, it provides an ideal 3D matrix for fluorescent
staining and imaging of liver cell spheroids.

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