Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

Summary

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

Abstract

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Introduction

The development of replication-defective, pseudotyped lentiviruses (LV) has provided researchers with a safe and efficient method to deliver transgenes in vitro ¹. Due to their long-term expression, these LV could also be used for the delivery of therapeutic agents in vivo ^2,3. The production of LV requires co-transfection of human embryonic kidney (HEK) cells with several plasmids: transfer vector, packaging gag/pol, packaging rev, and envelope vesicular stomatitis virus glycoprotein (VSV-G) plasmids. Third-generation packaging systems are considered safer than second-generation systems because the rev gene is encoded on a separate packaging plasmid, thereby reducing the possibility of recombination to produce a replication competent lentivirus. Although second generation packaging systems yield five fold higher total transduction units, the split of the original viral genome to create the third generation system has decreased the level of transduction only minimally ^3,4.

While cell lines can be transduced more easily and efficiently, researchers have shifted their focus to primary cells, because these are more representative of in vivo tissues ^5,6. However, primary cells are refractory to transduction. While transduction of fully differentiated airway epithelial cells have been reported, the overall efficiency has not exceeded 15% ⁷.

Described here is a protocol that allows production of high-titer LVs. A step-by-step approach is outlined to achieve almost 100% transduction efficiency into primary HBE cells. More specifically, the protocol describes the optimal culture conditions instrumental to succeed ³. Briefly, HEK 293T cells are transfected using the lentiviral expression vector pCDH with the EF-1 promoter driving expression of enhanced green fluorescent protein (eGFP) or mCherry, a red fluorescent protein, and a third-generation packaging system. LVs released into the media are collected 24 to 72 hr later. The virus particles are concentrated with polyethylene glycol (PEG), a convenient and easy method of viral concentration, before titer estimation using a p24 antigen enzyme-linked immunosorbent assay (ELISA) kit. Subsequently, undifferentiated primary HBE cells are transduced in proliferation media (bronchial epithelial growth medium or BEGM) ⁸, while attaching (after being trypsinized), at a multiplicity of infection (MOI) factor of 4, adding hexadimethrine bromide and incubating for 16 hr (overnight). The cells are then allowed to differentiate. Since the viral transgene is expressed long-term (using the appropriate promoter, see discussion), expression will be maintained throughout differentiation into a pseudostratified airway epithelium or can be induced (using an inducible promoter) after differentiation.

This protocol is of broad interest to researchers who want to use primary HBE cells instead of cell lines, and might be adaptable to other difficult to transduce cell types.

Protocol

1. Stage 1: Culture of HEK 293T

1. Prepare HEK cells media (referred as HEK media) by adding 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin to high glucose Dulbecco's Modified Eagle's Medium (DMEM).
2. Coat one 10 cm tissue culture dish with collagen I. Mix 30 µl of collagen I in 2 ml of distilled water. Spread the mix onto the dish and incubate for 2 hr at 37 °C and 5% CO₂.
3. Remove the mix and let the dish dry in a laminar flow cabinet for 15 min.
4. Plate HEK cells onto the coated dish at a density of 1 x 10⁶ cells in 10 ml HEK media and incubate at 37 °C and 5% CO₂.
5. When HEK cells are 50-60% confluent (Figure 1a, 2-3 days after plating), proceed to stage 2. Visual assessment of confluency is sufficient. No quantification is needed.
   Note: HEK cells are cultured in HEK media (see above). When they reach confluency, they can be split (e.g. 1/5) or frozen down for later use.

2. Stage 2: Production and Concentration of Lentiviruses (LVs) in HEK Cells

Note: The protocols should be executed in line with institutional and governmental regulations under BSL-2+ (enhanced BSL-2) conditions in the USA.

1. Transfect using a calcium precipitation procedure (lipofectamine and other transfection agents were not as efficient as the calcium precipitation procedure). Use of a commercial transfection kit is recommended to assure reproducibility, though the procedure is modified as follows. Bring all reagents to room temperature. This is day 0.
2. For each 10 cm dish of HEK293 cells, mix 4.5 µg of envelope DNA pMD2-VSVG, 7.5 µg packaging DNA pMDLg/pRRE, 3.75 µg DNA pRSV-rev, 10 µg DNA of lentiviral expression vector, 86 µl of a 2 M calcium solution, and sterile water in a 15 ml centrifuge tube to a final volume of 700 µl (the latter two solutions are provided in the commercial transfection kit).
3. Drop-wise, add 700 µl of 2x HEPES-buffered saline (HBS, provided in the transfection kit) while vortexing (in the laminar flow cabinet) and incubate at room temperature for 30 min (perform step 4 while waiting).
4. During the early incubation time, deposit 5 µl of the mix on a slide. Check the droplet under a microscope with a 10X objective, focusing slowly up and down. A fine precipitate should be visible, confirming the calcium phosphate precipitation process (Figure 1b).
5. Stop the precipitation after 30 min by adding 10 ml of HEK cells media and pipette up and down to mix.
6. Take the dish of HEK cells from step 2.1-2.5, and remove the medium (day 0). Add the precipitate solution from step 2.5 carefully and slowly along the edge of the dish.
7. On the next day (day 1), remove and discard the medium containing the precipitate and replace it with 10 ml of HEK medium. Check for the fine precipitate under the microscope: it should be visible in the dish in empty spaces between the cells (Figures 1c and d).
8. On day 2, collect media with a syringe, filter through a 0.45 µm syringe filter into a 15 ml centrifuge tube containing 3.8 ml of a 40% polyethylene glycol (PEG) solution. Invert 5x to mix and store at 4 °C for at least 24 hr (for the PEG precipitate to form, but no longer than 5 days) until all of the virus collections are complete.
9. Repeat step 2.8 on days 3 and 4. On day 4 do not replace with HEK medium but decontaminate with 10% bleach and discard the dish.
10. Centrifuge all the collection tubes (usually all collections together on day 5) at 1,650 x g for 20 min at 4 °C to pellet the virus. Remove and discard the supernatant, and spin again at 1,650 x g for 5 min at 4 °C to collect and remove any remaining of PEG supernatant.
11. Resuspend the pellet of each tube in 200 µl of bronchial epithelial growth medium (BEGM) without amphotericin B ⁸. Pool them together and aliquot at 200 µl. Store virus at -80 °C. Aliquot an additional 10 µl to perform the HIV-1 Gag (p24) antigen ELISA assay.
12. Using a commercial ELISA kit, perform a p24 antigen assay according to manufacturer's protocol. The assay gives an estimate of viral p24 in pg/ml.

3. Stage 3: Culture Primary HBE Cells

1. Plate HBE cells in 10 ml BEGM media (with 100 µl amphotericin B if passage 0 cells are used to eliminate possible fungal contamination) at a density of 1 x 10⁶ cells in a collagen I coated 10 cm tissue culture dish. Incubate at 37 °C and 5% CO₂.
2. On the next day, remove the medium and replace with 10 ml BEGM (with 100 µl amphotericin B for passage 0 cells) for a total of 4 days. Return to incubator.
3. After 4 days, use only BEGM without amphotericin B. Change media every other day.
4. When cells are 80-90% confluent (Figure 2a; ~7 days after plating), proceed with transduction (pay attention to 3.5 for preparation before transduction). Again, visual assessment of confluency is sufficient. No quantification is needed.
5. Prior to transduction, coat the permeable support inserts with a collagen IV solution diluted 1/10 with sterile distilled water. Add 200 µl to each insert and let dry overnight in the laminar flow cabinet.

4. Stage 4: Transduction of HBE Cells

1. Remove media, add 3 ml of 0.1% trypsin in 1 mM ethylenediaminetetraacetic acid in phosphate buffered saline (EDTA/PBS) and incubate 5 min at 37 °C and 5% CO₂. Check under the microscope (10X objective) to see if all the cells are detached. If not, return to the incubator for an additional 5 min.
2. When all cells are detached, add 3 ml of soybean trypsin inhibitor (SBTI 1 mg/ml), mix and transfer to a 15 ml centrifuge tube. Pellet the cells by spinning at 460 x g for 5 min at room temperature.
3. Remove supernatant and resuspend pellet into 3 ml of BEGM and proceed with counting.
4. Resuspend HBE cells (now passage 1 or P1) at a ratio of 2 x 10⁵ cells per one 12 mm permeable support insert in 400 µl of BEGM (see Table 2). Extrapolate these numbers based on the amount of permeable support inserts that will be transduced.

Surface	Surface area	Cell count	Media	Volume (media)
10 cm dish	52.7 cm2	1 x 106	BEGM	10 ml
12 mm filter	1.13 cm2	2 x 105	BEGM	400 μl
24 mm filter	4.52 cm2	8 x 105	BEGM	800 μl

Table 2: Media extrapolation per cell count.

5. Using a multiplicity of infection (MOI) factor of 4, add the appropriate volume of virus to the cell suspension.
6. Add hexadimethrine bromide to a 2 µg/ml final concentration.
7. Dispense 400 µl of the mix (BEGM, HBE cells, virus and hexadimethrine bromide) per permeable support insert into the apical compartment, and add 1 ml of BEGM alone to the basolateral compartment. Incubate overnight at 37 °C and 5% CO_2. Always plate HBE cells without virus as control and test puromycin selection if applicable.
8. On the next day, remove apical and basolateral media, wash with PBS, and replace both compartments with air-liquid interface (ALI) media ⁸.
9. When cells are confluent, remove apical fluid (known as establishing an air-liquid interface). Continue changing the medium (ALI) in the bottom compartment every other day until they reach full maturity; usually 3 to 4 weeks later.
   Note: If the lentiviral vector contains a selection gene such as puromycin, cells can be selected by adding puromycin according to a selection concentration curve. For HBE cells, a concentration of 1 µg/ml has been determined to be ideal for consistent selection of transduced cells. However, this concentration might differ for other cells or conditions and should be determined by killing curves. Addition of puromycin can start as early as one day after the media has been replaced by ALI media or later (2-3 days) to allow the full expression of the selection gene. Be sure to add puromycin to one insert that has not been transduced. Puromycin can be added until the cells are fully differentiated.

Results

Figure 1 depicts different key steps of assessing cells and solutions during the transfection process. Figure 1a shows the optimal confluency of HEK293T cells prior to transfection for virus production. It is important that the cells are distributed evenly throughout the dish. Figure 1b reveals the precipitate in a drop of the transfection mixture using bright field optics and a 10X objective. The more the precipitates look like sand grai...

Discussion

The protocol outlined here assures close to 100% transduction efficiencies. However, there are steps during the process that are important to achieve this goal. For instance, during the transfection process, the presence of precipitates in the transduction mix (drop) or in the dish the day after transduction (Figures 1b and d) are both relevant for a good transfection. The absence of a precipitate or the presence of agglomerates can be due to an omission of one of the transfection reagen...

Materials

Name	Company	Catalog Number	Comments
HEK293T/17 cells	ATCC	CRL-11268
Fetal bovine serum (FBS)	Sigma	12306C	use at 10%
DMEM	Thermo Scientific	11995-040
Penicillin/Streptomycin (100x)	Thermo Scientific	15140-122	use at 1x
Collagen I	BD Biosciences	354231	dilute 1:75 in distilled water
Calphos	Clontech	631312	Transfection kit containing 2M Calcium solution, 2X HEPES-Buffered solution (HBS) and sterile H2O
Polythylene glycol 8000	VWR scientific	101108-210	stock of 40%
Amphotericin B	Sigma	A9528	use at 1x
Trypsin	Sigma	T4799
Soybean trypsin inhibitor	Sigma	T9128
Transwell 12mm	Corning	3460
Collagen IV	Sigma	C7521	dilute 1:10 in distilled water
Hexadimethrine bromide	Sigma	H9268	stock of 2mg/ml
Puromycin (10.000x)	Thermo Scientific	A11138-03	use at 1x
Alliance HIV-1 p24 ELISA	PerkinElmer	NEK050001KT
F12	Thermo Scientific	11765-054
pCDH-EF1-MCS-IRES-Puro	System Biosciences	CD532A-2	lentiviral expression vector
pMD2-VSVG	Addgene	12259	packaging DNA
pMDLg/pRRE	Addgene	12251	packaging DNA
pRSV-rev	Addgene	12253	packaging DNA