Primary Human Hepatic Kupffer Cell Culture Protocol

• What are Hepatic Kupffer Cells?
• Hepatic Kupffer Cell Culture Materials
• Hepatic Kupffer Cell Culture Methods
• Hepatic Kupffer Cell Culture Results

What are Hepatic Kupffer Cells? 

Hepatic Kupffer Cells, also known as Browicz-Kupffer cells and stellate macrophages, are specialized macrophages in the liver that line the walls of the sinusoids. Kupffer cells can activate macrophages and are constantly exposed to gut-derived bacteria, microbial debris, and bacterial endotoxins.

Hepatic Kupffer cells are intimately involved in the liver's response to infection, toxins, ischemia, resection, and other stresses. Upon activation, Kupffer cells release various products, including cytokines, prostanoides, nitric oxide, and reactive oxygen species. These released molecules regulate other Kupffer cells and neighboring cells, such as Hepatocytes, Stellate cells, Endothelial cells, and other immune cells that traffic through the liver. Human hepatic Kupffer cells are often characterized using flow cytometry for population distributions, and are positive for CD11b, CD14, and CD68¹. In vitro, these cells do not proliferate and cannot be passaged.

Our cryopreserved hepatic Kupffer cells are derived from the human liver, with each donor providing documented consent for research use of non-transplantable organs or tissues. Following the primary culture, these cells are cryopreserved. Each lot undergoes testing for specific cell markers, fold activation of cytokine production following LPS stimulation and comes with a guarantee of ≥70% post-thaw viability. In this protocol, we demonstrate how to thaw, plate, and culture primary human hepatic Kupffer cells.

Hepatic Kupffer Cell Culture Materials

• Normal Human Hepatic Kupffer Cells
  Note: Upon receipt, immediately store cryovial(s) in vapor phase liquid nitrogen.
• Collagen Type I, Rat Tail
• Tissue culture treated multiwell plates
• Kupffer Maintenance Medium²:

Components	Catalog Number	Working Stock	Final Dilution	Final Conc.	Final Volume (mL)
Williams’ Medium E	W1878-500ML			1x	470
FBS	ES-009-M			5%	25
Insulin	I9278-5ML	10mg/mL (1.7mM)	1700x	1µM	0.29
Pen/Strep	P7539	100x	100x	1x	5
				Total volume	500

HEPATIC KUPFFER CELL CULTURE METHODS

These protocols were performed using a Class II laminar flow biohood unless it is otherwise specified directly. The incubators used were humidified and set to 37°C and 5% CO₂. Researchers should wear PPE including safety glasses, gloves, and a lab coat.

 Preparing a Collagen-Coated Culture Plate

1. Create a collagen solution with a final concentration of 56µg/mL in sterile 70% ethanol. Mix gently until the collagen is fully solubilized.
2. Completely cover the bottom of the well or flask with the collagen/ethanol solution.
3. Gently swirl the cell culture plate so the collagen/ethanol solution evenly coats the wells or flask bottom.
4. Air dry the plates or flasks in a laminar flow hood and leave it over night with the cover or the cap ajar to prevent condensation.

Thawing and Plating Kupffer Cells

Before starting, please prepare the collagen I coated cultureware (see above protocol). DO NOT pre-warm the medium to thaw cells. Kupffer cells easily attach to the walls of the conical tube at 37°C. Therefore, use of pre-warmed media is not recommended.

1. Place Kupffer cell vial in a 37°C water bath and gently rotate the vial until the cells are completely thawed. Immediately remove the vial from the water bath, wipe dry and rinse with 70% ethanol, and transfer to a sterile field. Remove the cap and be careful not to touch the interior threads.
2. Transfer the thawed cells to a 15 mL conical tube containing 9 mL of COLD (4°C) Kupffer Cell Medium and place the tube on ice.
3. Centrifuge tube at 500xg for 5 minutes. After centrifugation, aspirate the medium and resuspend cell pellet in 1mL COLD Kupffer Cell Medium.

Note: the cell pellet will be very small. Resuspend using a P1000 micropitetter, as resuspension with a serological pipette may lead to clumping of the cells.

1. Count the cells using the trypan blue exclusion assay.
2. Dilute the cells in WARM Kupffer Cell Medium to 400,000 cells/mL.
3. Plate 100,000 cells/cm² on culture ware coated with collagen type I (Refer to the table below)

Format	cm2 per well	Cells per cm2	Number of cells per well	Total number of cells per plate
384 Well Plate	0.056	100,000	5,600	2.15 x 106
96 Well Plate	0.32	100,000	32,000	3.07 x 106
48 Well Plate	1.02	100,000	102,000	4.89 x 106
24 Well Plate	1.94	100,000	194,000	4.66 x 106
12 Well Plate	3.87	100,000	387,000	4.64 x 106
6 Well Plate	9.62	100,000	962,000	5.77 x 106
10cm Dish	56	100,000	N/A	5.60 x 106
60mm Dish	21	100,000	N/A	2.10 x 106
35mm Dish	8	100,000	N/A	800,000
T-25 Flask	25	100,000	N/A	2.50 x 106
T-75 Flask	75	100,000	N/A	7.5 x 106

7. Place the cells in a humidified 37°C/5% CO₂ incubator and allow them to attach for 4–6 hours to overnight.

Note: Human Kupffer cells can be removed easily using vacuum aspiration. Use caution.

8. After attachment, replace the medium with fresh WARM Kupffer Cell Medium.
9. After 24 hours, replace the medium with warmed Kupffer Cell Medium and proceed with your experiment. 

Note: These cells DO NOT proliferate and cannot be passaged.

Hepatic Kupffer Cell Culture Results

Figure 1 Example microscopy images of Kupffer cell culture after a.) 6 hours, b.) 24 hours, and c.) 48 hours.

References

1. Seki S, Ikarashi M, Nakashima M, Nakashima H. 2014. New Findings about Liver Kupffer Cells/Macrophages, B Cells and their Functions. J Hepat Res. . 1(1):1003. https://austinpublishinggroup.com/hepatitis/fulltext/hepatitis-v1-id1003.php

2. Su GL, Goyert SM, Fan M, Aminlari A, Gong KQ, Klein RD, Myc A, Alarcon WH, Steinstraesser L, Remick DG, et al. 2002. Activation of human and mouse Kupffer cells by lipopolysaccharide is mediated by CD14. American Journal of Physiology-Gastrointestinal and Liver Physiology. 283(3):G640-G645. https://doi.org/10.1152/ajpgi.00253.2001