Harvesting and Concentration of Lentiviral Vectors

Harvesting and Concentration of Lentiviral Vectors May 8, 2007

TRP Doc. D423

By: Olga Liu and Max Richardson

Modified by Richard Carroll

Page 1 of 4

Harvesting and Concentration of Lentiviral Vectors

Introduction:

This protocol describes procedures for harvesting and concentrating lentiviral vectors from the supernatant of transfected 293T cells. Production of lentiviral vectors is described in TRP Document D420. It is not always necessary to concentrate vector-containing supernatants, but it is frequently desirable to add as little vector supernatant to cultures as possible. This protocol describes the use of high speed centrifugation to concentrate vectors. Experiments have shown this procedure to be more reliable than ultracentrifugation.

Materials:

A.Flask of vector-producing 293T cells.

B.50 ml Conical Centrifuge Tubes (Fisher Cat. #14-959-49A)

C.0.45 um Syringe Filters, 25 mm (Fisher Cat. #09-719D)

D.60 ml Luer-Lock Syringes (Becton Dickinson Cat. #309653)

E.Oak Ridge 50 ml PSF Centrifuge Tubes (Nalgene Cat. #3115-0050)

1.Cleaning and maintenance of tubes is discussed in Procedures, Section III.

F.Wet Ice

G.Dry Ice

H.Cryotubes, 1.8ml (Fisher Cat. #12-565-163N)

I.Pipettes, mixed

J.Aspirating Pipette, 2 ml, (BD/Falcon Cat. # 357558)

K.R10 Culture medium (see SOP TRP210 for preparation procedures)

L.Virkon Solution (Gallard Schlesinger Industries Cat. #222015406)

M.Water: both distilled and Milli-Q

N.Autoclave Bags (provided by Research Facilities)

Equipment:

A.Sorvall Legend Tabletop Centrifuge

B.Sorvall RC5C High Speed Centrifuge

C.Biosafety Cabinet

D.SA-600 Rotor

E.Balance AND Model FX-3200

F.Autoclave (research facilities)

Procedures:

I.Harvesting of Virus

A.Collect supernatant from T150 flasks and place in 50 ml conical centrifuge tubes.

B.Centrifuge in Sorvall Legend for 10 minutes at 2000 rpm, 4oC, to clarify the supernatant.

C.After spinning, place tubes on ice.

1.They should be kept cold for the remainder of the procedure.

D.Remove the plunger from a 60 ml syringe and equip the syringe with a 0.45 m luer-lock syringe filter.

E.Place the syringe and filter inside a new 50 ml conical tube.

F.Transfer the cleared viral supernatant into the syringe.

1.It is probably best not to pour it, but rather to pipette into a spare conical tube.

G.Re-insert the plunger and filter the supernatant.

H.If you wish to concentrate your virus, proceed to Part II. If you only want un- concentrated virus, continue with Step I below.

I.Aliquot the filtered supernatant into labeled cryovials and freeze at -80oC.

II.Concentration of Virus

A.The starting material for this step is the cleared vector supernatant (Step C from Part I above).

B.0.45 uM filter the cleared supernatant as described in Part I, Steps D-G, and keep the filtered supernatants on ice.

C.Prepare the SA-600 rotor and the RC5C centrifuge for use:

1.Clean out the SA-600 rotor with Virkon to remove any bacterial contaminants left over from maxi/midi preps.

2.Clean out the inside of the centrifuge itself as well, and wipe down the top of the centrifuge.

3.Note: Make sure to use only the centrifuge on the right (as you face them); the centrifuge on the left frequently shuts down on extended runs.

D.Transfer the supernatant into a sterile Nalgene Oakridge 50 ml PSF centrifuge tube.

1.35-40 ml is the maximum volume that can be loaded.

3.It is recommended that you not run much less than 35-40 ml.

E.Fasten the cap tightly, and mark both the top of the cap and the corresponding side of the tube.

1.This will serve to help locate the pellet. See Step L below.

F.Place the tube on ice after securing and marking the cap and tube.

G.Tubes should be weighed prior to placing in the rotor. Tubes should weigh within 0.5 g of each other.

1.If tube weights differ by more than 0.5 g, the appropriate volume of R10 culture medium can be added (in the Biosafety cabinet) to the lighter tube.

H.Insert the centrifuge tubes in the SA-600 rotor, oriented such that the mark on the cap faces the outside edge of the rotor. The vector pellet will thus correspond with the mark.

I. Spin down the vector at 8400 rpm (10,000 x g) for 18 hours at 4oC.

J.After 18 hours, stop the rotor (it is okay to stop it with the brake on).

K.Remove the tubes and place on ice gently (make holes in ice ahead of time with a 50 ml conical tube).

1.When transferring tubes into the hood, placing an ice bucket in the hood is not recommended. Instead, fill a small plastic tray (for example, an empty P1000 tip box) with ice and place the tube in that.

2.Alternatively, one tube at a time can be brought into the hood and resuspended rapidly (not on ice) as described below.

L.Using a 10 ml pipette or a 2 ml aspirating pipette equipped with a yellow tip, remove supernatant down to where the bottom of the tube starts to curve in.

1.This will leave a residual volume of 2-3 ml.

2.Be certain to pipette or aspirate on the opposite side of the tube from where your mark or pellet is located (see Figure 1).

Figure 1. Proper Alignment of Tube When Removing Supernatant.

M.Using a 2 ml pipette, pipette the residual 2-3 ml up and down vigorously, concentrating on the (marked) area where your pellet is located.

1.Under the right contrast, a white pellet can be seen on the lower side of the tube.

2.The pellet should be pipetted up and down a maximum of ~ 10-20 times total (this should take ~ 30 seconds to perform).

N.Aliquot the resuspended vector into anywhere from 2 to 8 appropriate cryovials. O. Place the virus aliquots on dry ice immediately, and store subsequently at –80oC.

P.Proceed to titer the virus (see SOP TRP350)

III.Cleaning and Maintenance Oakridge Centrifuge Tubes

A.Fill tubes partway with Virkon solution and re-cap.

B.Shake the tubes energetically.

C.Discard the Virkon.

D.Repeat Virkon washing step.

E.Rinse tubes and caps extensively in distilled water.

F.Rinse tubes and caps one time with Milli-Q water.

G.Replace the caps on the tubes, but leave the caps loose (do not tighten them).

H.Insert tubes in a blue autoclave bag (do not overfill the bag).

I.Autoclave using Gravity Cycle, 60 minute exposure time.

1.This should be performed using the small autoclave outside room 583.

J. Retrieve bags and store in drawer in HIV Room.

References:

A.TRP Document D150: Basic Sterile Technique

B.TRP Document D420: Lentiviral Vector Preparation by Fugene-Mediated Transient Transfection of 293T Cells

C.SOP TRP210:Preparation of R10 Medium

D.SOP TRP350: Titering Lentiviral Vectors on Primary Human T Cells

E.SOP TRP355: Transduction of Human T Cells by Lentiviral Vectors