Harvesting Hepatitis E Virus Particles from Human Liver Cancer Cells

Abstract

Source: Meister, T. L., et al. A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks. J. Vis. Exp. (2020).

This video demonstrates the process of isolating hepatitis E virus (HEV) particles from transfected human hepatocellular carcinoma cells. Extracellular viral particles are collected from filtered supernatants, while intracellular particles are released by subjecting the cells to repeated freeze-thaw cycles. The lysate is centrifuged, and the supernatant containing HEV particles is aliquoted and stored at an ultra-low temperature for future use

Protocol

1. Preparation of HepG2 cells for cell culture-derived HEV (HEVcc) production
NOTE: To avoid contamination, preparation of cells, electroporation, infection, harvesting and cell fixation were carried out under sterile conditions in a biosafety level 2 facility. Incubation steps at 37 °C that involve cells were accomplished in a 5% CO₂ incubator.

1. To prepare HepG2 cells for HEVcc production (Figure 1), seed cells in complete Dulbecco's Modified Eagle Medium (DMEM) (see Table 1) onto a 15 cm collagen (see Table 1, sterile filter phosphate-buffered saline (PBS)- acetic acid mix through 0.2 µm mesh before adding collagen) coated culture dish. Incubate at 37 °C until cells are 90% confluent.
   NOTE: Each 15 cm dish containing 90% confluent cells will generate enough cells for up to 4 electroporations.
2. Gently remove the medium from the plate and wash cells once with 10 mL of 1x PBS. Trypsinize cells by adding 3 mL of 0.05% trypsin-EDTA onto the cells and incubate at 37 °C until cells are detached completely. Resuspend cells in 10 mL DMEM complete medium and transfer the cell suspension into a 50 mL tube. Determine the total number of cells.
3. Since each electroporation requires 5 x 10⁶ cells, transfer the appropriate volume into a new 50 mL tube and fill up to at least 35 mL with 1x PBS. Centrifuge cells at 200 x g for 5 min and carefully discard the supernatant without disturbing the cell pellet.
4. Wash cells once again with 35 mL of 1x PBS at 200 x g for 5 min. Place cells on ice and do not remove 1x PBS yet, to keep the cells in suspension.

2. Electroporation of HepG2 cells

1. For electroporation of HepG2 cells (Figure 2) prepare 400 µL of Cytomix complete per electroporation by supplementing 384 µL of Cytomix (see Table 1) with 2 mM ATP and 5 mM Glutathione. Prepare fresh right before use and place directly on ice.
   NOTE: A correct yet quick execution of the following steps is crucial. Therefore, make sure that everything is prepared properly.
2. Carefully remove 1x PBS without disturbing the cell pellet from step 4.4. Resuspend 5 x 10⁶ cells in 400 µL of Cytomix complete and add 5 µg RNA. Transfer the solution into a 4 mm cuvette and pulse once with 975 µF, 270 V for 20 ms with an electroporation system.
   1. After electroporation, transfer cells as quickly as possible with a Pasteur pipette into 11 mL complete DMEM per electroporation.
      NOTE: To make sure that RNA is successfully transfected into HepG2 cells, a transfection control (TC) will be performed. Expected transfection rates vary between 40-60% of ORF2-positive cells.
3. Transfer 10 mL of electroporated cells into a 10 cm culture dish coated with collagen. Add 1.3 x 10⁵ electroporated cells (300 µL) into one well of a collagen-coated 24-microtitre plate carrying a cover slip (later used as a transfection control). Distribute cells evenly and incubate at 37 °C.
   1. After 24 h, change the medium in the 10 cm dish and replace with 10 mL fresh complete DMEM. Do not change the medium of the transfection control. Incubate for another 6 days at 37 °C.

3. Harvesting of intra- and extracellular cell culture-derived HEV (HEVcc)

1. To harvest extracellular HEVcc (Figure 3), filter the supernatant obtained from the 10 cm dish after 6 days through a 0.45 µm mesh to remove any cell debris. Store harvested extracellular HEVcc at 4 °C for the same-day infection; otherwise store at -80 °C.
2. To harvest intracellular HEVcc (Figure 3), wash cells with 1x PBS and trypsinize by adding 1.5 mL of 0.05% trypsin-ethylenediamine tetraacetic acid (trypsin-EDTA). Incubate at 37 °C until cells are detached completely. Add 10 mL of completeDMEM, flush the plate to detach cells and transfer the cell suspension into a 50 mL tube. Wash cells twice with PBS. Centrifuge at 200 x g for 5 min.
   1. Discard the supernatant and resuspend the cell pellet in 1.6 mL of complete DMEM per electroporation. Transfer the cell suspension into a 2 mL reaction tube.
      NOTE: Do not use larger volumes, as it would dramatically decrease viral loads.
   2. Freeze (in liquid nitrogen) and thaw cells. Repeat this sequence 3 times.
      NOTE: Do not pool more than one electroporation (1.6 mL) for the 3 freeze-and-thaw cycles, as the lysis efficiency would be impaired. Do not vortex the cell suspension in between the cycles. Make sure to thaw the cell suspension slowly (e.g., room temperature or on ice) to maximise viral loads.
   3. High-speed centrifuge the lysed cells for 10 min at 10,000 x g to separate cell debris. Transfer the supernatant to a new tube. Take the supernatant for infection; otherwise, store at -80 °C.
   4. Optionally, concentrate the extra- and intracellular HEVcc using a concentrator to increase viral loads.

Table 1: Table of Buffer Composition.

Working solution	Concentration	Volume
Collagen	40 mL	PBS
	40 µl	Acetic acid
	1 mL	Collagen R solution 0.4% sterile
Cytomix	120 mM	KCl
	0.15 mM	CaCl2
	10 mM	K2HPO4 (pH 7.4)
	25 mM	HEPES
	2 mM	EGTA
DMEM complete	500 mL	DMEM
	5 mL	Pen/Strep
	5 mL	MEM NEAA (100X)
	5 mL	L-Glutamin
	50 mL	Fetal bovine serum
MEM complete	500 mL	MEM
	5 mL	Sodium pyruvate
	5 mL	Gentamycin
	5 mL	MEM NEAA (100X)
	5 mL	L-Glutamine
	50 mL	ultra-low IgG

Results

Figure 1: Schematic experimental setup for the preparation of HepG2 cells for cell culture-derived HEV (HEVcc) production.

Figure 2: Schematic experimental setup for the electroporation of HepG2 cells.

Figure 3: Schematic experimental setup for harvesting intra- and extracellular HEVcc.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.45 µm mesh	Sarstedt	83.1826	Harvest extracellular Virus
MEM	gibco	31095-029	Cell culture
MEM NEAA (100×)	gibco	11140-035	Cell culture
Trypsin-EDTA (0.5 %)	gibco	15400054	Cell culture
ultra-low IgG	gibco	1921005PJ	Cell culture
Acetic acid	CarlRoth	6755.1	Collagen working solution
ATP	Roche	11140965001	In vitro transcription and electroporation
BioRender	BioRender		Figure Generation
Calcium chloride	Roth	5239.2	Cytomix
Collagen R solution 0.4 % sterile	Serva	47256.01	Collagen working solutio
DMEM	gibco	41965-039	Cell culture
EGTA	Roth	3054.3	Cytomix
GenePulser Xcell Electroporation System	BioRad	1652660	Electroporation
HEPES	Invitrogen	15630-03	Cytomix
Di-potassium hydrogen phosphate	Roth	P749.1	Cytomix
KCL	Roth	6781.3	Cytomix
Potassium di-hydrogen phosphate	Roth	3904.2	Cytomix
L-Glutamin	gibco	25030081	Cell culture
L-Glutathione reduced	Sigma-Aldrich	G4251-5G	Cytomix
MEM	gibco	31095-029	Cell culture
MEM NEAA (100×)	gibco	11140-035	Cell culture
Magnesium chloride	Roth	2189.2	Cytomix
Microvolume UV-Vis spectrophotometer NanoDrop One	Thermo Fisher	ND-ONE-W	DNA/RNA concentration
PBS	gibco	70011051	Cell culture
Pen/Strep	Thermo Fisher	15140122	Cell culture