CELL LINE DESCRIPTION

BioVector's A549-hACE2 cells were generated from the A549 lung carcinoma

cell line, a commonly used cellular model for the study of respiratory

infections. BioVector's A549-hACE2 cells were stably transfected to express the

human ACE2 (hACE2) gene. Thus, unlike their parental cell line, they

are permissive to infection with pseudotyped lentiviruses expressing

the SARS-CoV-2 Spike protein.

.BioVector's A549-hACE2 cells are resistant to

Puromycin.

The additional expression of the human TMPRSS2 gene in the A549-

hACE2-TMPRSS2 cells significantly increases their permissivity to

infection by SARS-CoV-2 Spike-pseudotyped lentiviruses.

BACKGROUND

ACE2 (angiotensin I-converting enzyme-2) is a type I membrane

protein that belongs to the angiotensin-converting enzyme family1

. It

is expressed in arteries, heart, kidneys, and epithelia of the lung and

small intestine2

. Human ACE2 is the established host receptor for

the Spike (S) protein of SARS-CoV-2, the causative agent of COVID19, enabling its entry into target cells3-5. In particular, SARS-CoV-2

gains entry to host cells through the binding of the Spike receptorbinding domain (RBD) to ACE2 at the cell surface4,5. Following

this, host proteases, such as TMPRSS2 and Cathepsin L, allow the

cleavage of the S protein into two subunits (S1 and S2), at the cell

surface or in the endosomes, respectively. S2 mediates the fusion

between the viral and host membranes, thereby releasing the viral

contents into the cell.

APPLICATIONS

A549-hACE2 cells are permissive to infection by SARS-CoV-2 and/or

spike-pseudotyped lentiviral particles. Thus, they are ideal for studying

viral entry into host cells, as well as for screening small molecule

inhibitors and neutralizing antibodies. These cells can be used for

comparative studies with BioVector's A549-hACE2-TMPRSS2 cells which express

both ACE2 and TMPRSS2 and are more permissive to SARS-CoV-2

infection than BioVector's A549-hACE2.

HANDLING PROCEDURES

Required Cell Culture Medium

? Growth Medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine,

10% heat-inactivated fetal bovine serum (FBS; 30 min at 56 °C),

100 μg/ml Normocin?, Pen-Strep (100 U/ml-100 μg/ml)

? Freezing Medium: DMEM, 4.5 g/l glucose, 10% FBS, 10% DMSO

? Required Selection Antibiotic: Puromycin

Initial Culture Procedure

The first propagation of cells should be for generating stocks for

future use. This ensures the stability and performance of the cells for

subsequent experiments.

1. Thaw the vial by gentle agitation in a 37 °C water bath. To reduce

the possibility of contamination, keep the O-ring and cap out of the

water. Thawing should be rapid (approximately 2 minutes).

2. Remove the vial from the water bath as soon as the contents are

thawed, and decontaminate by dipping in or spraying with 70% ethanol.

Note: All of the steps from this point should be carried out under strict

aseptic conditions.

3. Transfer cells to a larger tube containing 15 ml of pre-warmed

growth medium. Do not add selection antibiotics until the cells

have been passaged twice.

4. Centrifuge tube at 200-300 x g for 5 minutes.

5. Remove supernatant containing the cryoprotective agent and

resuspend cells with 1 ml of growth medium without selective

antibiotics.

6. Transfer the contents to a T-25 tissue culture flask containing 5 ml

of growth medium without selective antibiotics.

7. Place the culture at 37°C in 5% CO2

.

Frozen Stock Preparation

1. Resuspend cells at a density of 5-7x 106 cells/ml in freshly

prepared freezing medium.

Note: A T-75 culture flask typically yields enough cells for preparing 1-2

frozen vials.

2. Dispense 1 ml of cell suspension into cryogenic vials.

3. Place vials in a freezing container and store at -80°C overnight.

4. Transfer vials to liquid nitrogen for long-term storage.

Note: If properly stored, cells should remain stable for years.

Cell maintenance

1. BioVector's A549-hACE2 cells grow as adherent cells. To detach cells, rinse

the cell layer with PBS, then incubate with 0.25% trypsin-EDTA for

2-5 minutes.

2. After cells have recovered and are growing well (following at least

2 passages), maintain and subculture the cells in growth medium

supplemented with 0.5 μg/ml of Puromycin.

3. Renew growth medium twice a week.

4. Cells should be passaged when a 70-80% confluency is reached. Do

not let the cells grow to 100% confluency.

Note: The average doubling time for the BioVector's A549-hACE2 cells is ~25 hours

using the conditions described above.

Cell Handling Recommendations

To ensure the best results, use BioVector's A549-hACE2 cells with less than 20

passages.

Supplier: BioVector NTCC質(zhì)粒載體菌株細(xì)胞蛋白抗體基因保藏中心

www.biovector.net