Optimization of Lentiviral Spinfection

Last!Mo dified:!April!2018!

Last!Review:!October!2018!

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Lentiviral!transduction!is!an!effective!method!for!creating!a!stable!cell!l ine!with!a!DNA!

cassette!of!interest!integrated!into!its!genomic!DNA,!e.g.!an!s gRNA!or!gene!expression!

cassette.!Transducing!cells!at!a!concentrated!seeding!density!in!12-well!plates!while!

centrifuging!(‘spinfection’),!is!a!method!to!achieve!efficient!transduction!of!a!large!number!of!

cells.!Cells!are!typically!infected!in!the!pres ence!of!polybrene,!a!polycation!that!neutraliz es!the!

charge!repulsion!between!the!virus!and!cell!target!surface!and!helps!viral!integration !into!the!

cell.!!

The!ideal!sp infection!conditions!can!be!highly!variable!across!cell!types!and!should!be!

optimized,!as!cell!l ines!vary!in,!e.g!infectability,!po lybrene!sensitivity!and!response!to!dense!

culture.!A!balance!between!seeding!density,!polybrene!concentration!and!other!spinfection!

variables!will!be!determined!to!ensure!successful!transduction!and!minimal!cost!to !cell!

viability.!This!protocol!uses!the!pRosetta!GFP-vector!as!a!lentiviral!con trol!and!as!an!optional,!

but!highly!recommended,!simple!assessment!of!the!infectability!of!the!cell!li ne.!Transduction!

efficiency!is!determin ed!as!t he!percentage!of!GFP-positive!cells,!via!flow !cytometry.!!

F?(*5<8AB57=(5;G("85H8;<=(

The!following!materials!are!required:!

• 30!million!cells!

• Growth!media,!PBS!and!trypsin!

• Cell!counter!

• Trypan!Blue!(for!suspension!lines)!

• 12-well!plates!

• Tissue!culture!flasks!

• Polybrene!(Sigma-Aldrich,!Cat!#H9268),!make!a!stock!of!8!mg/mL!in!water)!

• Pipettes!

• 15!mL,!50!mL!tubes!

• 2!mL!of!pRosetta!virus!(aka!pLKO_TRC060)!lentivirus!(PuroR,!BlastR,!GFP),!available!

from!GPP!(gpp-reagents@broadinstitute.org)!!

The!following!materials!are!optional,!but!highly!recommended:!

• Flo w!cytometer!

• 96-well!V-bottomed!plates!

• Flo w!buffer!(PBS,!2%!FBS,!5!uM!EDTA)!

I?(# D<BJBK5<B9;(9:(1DB;:8C<B9;(%9;GB<B9;=(

1$/!(>'(%877(28;=B<L(5;G(!97L6A8;8(*5<ABM(

Perform!a!spinfectio n!using!a!range!of!cell!densities!and!polybrene!concentrations.!!

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A:!Prepare!12-well!plates!and!master!mixes!

1. Label!two!12-well!plates!as!follows:!

!!!!!!!!!!!!!!!!

!!!!! !

0"PB"

NIC"

0"PB"

4"PB"

2"PB"

8"PB"

1.5E6"

1.##

2.##

3.##

4.##

5.## 6.## 7.## 8.##

9.## 10.## 11.## 12.##

0"PB"

NIC"

0"PB"

4"PB"

2"PB"

8"PB"

3E6"

1.##

2.##

3.##

4.##

5.## 6.## 7.## 8.##

9.## 10.## 11.## 12.##

2. Col lect!the!cells:!

• For!adherent!cells:!

i. Aspirate!media!and!wash!flask!with!warm!PBS.!!

ii. Add!trypsin!and!allow!cells!to!detach!from!fl ask.!

iii. Add!warm!media!(2-3x!the!amount!of!trypsin)!to!the!flask!to!quench!the!

trypsin.!Transfer!the!cell!suspension!into !a!conical.!

iv. Wash!the!flask!with!media!and!add!the!wash!to!the!same!conical.!

v. Mix!the!cell !suspension!thoroughly!and!count.!!

• For!suspension!cells:!

i. Collect!the!cell!suspension!from!the!flask!into!a!conical.!

ii. Spin!down!the!cells!at!335-524!g!for!5!min!and!aspirate!

media!sup ernatant.!!

iii. Re-suspend!the!cells!thoroughly!in!fresh,!warm!media!and!count.!

3. Dilute!the!cells!in!media!to!the!following!concentrations:!

5? 1.5E6!cells/mL!in!6!mL!total!(low-density)!

6? 3E6!cells/mL!in !6!mL!total!(high-density)!

4. Make!the!following!media,!8!mg/mL!polybrene!stock!and!pRosetta!virus!solutions:!

C? 3!mL!of!media!

G? 2.7!mL!media!+!0.3!mL!virus!

8? 2.7!mL!media!+!0.3!mL!virus!+!1.5!uL!polybrene!(final!=![ 2!ug/mL])!

:? 2.7!mL!media!+!0.3!mL!virus!+!3!uL!polybrene!(final!=![4!ug/mL])!

H? 2.7!mL!m edia!+!0.3!mL!virus!+!6!uL!polybrene!(final!=![8!ug/mL])!

B:!Seed!cells!in !12-well!plates!

1. Add!1!mL!of!C877(=N=D8;=B9;!and!1!mL!of!J8GB5W!@WXBAN=(=97N<B9;!to!the!

corresponding!labeled!wells,!as!follows:!!

Note:&the&wells&labeled&‘0&PB,&NIC’&are&the&no&polybrene,&no-infection&controls.&&

!!!!!!!Each!well!will!have!a!total!of!2!mL.!!

!!!!!!!!!!!!!! !!!!! !

0"PB"

NIC"

"a"+"c"

0"PB"

a"+"d"

2"PB"

a"+"e"

4"PB"

a"+"f"

8"PB"

a"+"g"

1.5E6"

1.##

2.##

3.##

4.##

5.## 6.## 7.## 8.##

9.## 10.## 11.## 12.##

0"PB"

NIC"

"b"+"c"

0"PB"

"b"+"d"

2"PB"

"b"+"e"

4"PB"

b"+"f"

8"PB"

"b"+"g"

3E6"

13.$$

14.$$

15.$$

16.$$

17.$$ 18.$$ 19.$$ 20.$$

21.$$ 22.$$ 23.$$ 24.$$

C:!Centrifuge!the!plates!

1. Spin!the!plates!at!931!g!for!2!hours!at!30°C.!!

D:!Add!media!to!plates!and!incubate!

1. After!2!hours!of!spinning,!add!2!mL!of!media!(without!polybrene)!dropwise!on!top!o f!

each!well.!!

2. Place!in!37°C!incubator !overnight.!!

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E:!Inspect!cells!visually!

1. Th e!next!day!(16-24!hrs!later),!remove!the!plate!from!the!incubator!and!inspect!the!

cells!under!a!microscope.!!

2. Take!note!of!the!fol lowing!things:!

• If!the!cells!clumped!together!and!lifted!off!the!plate&

• The!confluency!of!the!wells!

• How!adhered!the!cells!are!to!the!plate!(suspension!cells!will!be!loosely!adherent)!

• The!amount!of!dead!cells!(typically!shriveled!and!floating)!

General'Assessment:''

• If!in!all!conditions:!

o The!cells!clumped!together!and!lifted!off!the!plate,!they!may!be!sensitive!to!a!

high!confluency!and/or!centrifugation.!Be!sure!to!gently!break!up!the!clumps!

before!counting!i n!1$/!(>Y(25L(FY(!5A<(4.!!

o There!appear!to!be!a!lot!of!loosely!adherent!cells,!they!may!be!sensitive!to!

density.!!

o There!appear!to!be!a!lot!of!dead!cells,!they!may!be!sensitive!to!centrifugation.!!

F:!Collect!the!cells!from!the!plates!

1. Col lect!the!cells!from!the!plate:!

• For!adherent!cells:!

i. Label!10!conicals!with!the!condition!names!from!the!plate.!

ii. Collect!the!media!from!each!well!into!the!appropriate!conical.!

iii. Add!1!m L!of!PBS!to!each!well!and!collect!it!into!the!appropriate!conical.!!

iv. Add!250!uL!of!trypsin!to!each!well!and!let!the!cells!detach!from!the!plate.!!

v. Add!750!uL!of!media!to!each!well!to!quench!the!trypsin!and!transfer!the!

cell!susp ension!to!the!appropriate!conical.!

vi. Add!1!mL!of!media!to!wash!each!well!and!transfer!the!wash!to!the!

appropriate!conical.!!

vii. Spin!the!conicals!at!335-524!g!for!5!min.!

viii. Aspirate!the!media!and!re-suspend!the!‘low-density’!cells!in!1!mL !of!fresh!

media!and!the!‘high-density’!cells!in!2!mL!of!media.!!

• For!suspension!cells:!

i. Label!10!conicals!with!the!condition!names!from!the!plate.!

ii. Gently!pipette!the!media!in!each!well!up!and!down!to!dislodge!the!cells!

and!then!collect!the!cell!suspension!into!the!appropriate!conical.!

iii. Add!1!m L!of!media!to!wash!each!well!and!transfer!the!wash!to!the!

appropriate!conical.!!

iv. Spin!the!conicals!at!335-524!g!for!5!min.!

v. Aspirate!the!media!and!re-suspend!the!low-density!cells!in!1!mL!of!fresh!

media!and!the!high-dens ity!cells!in!2!mL!of!media.!!

G:!Coun t!the!cells!and!assess!the!recovery!from!the!12-well!plates!

1. Count!the!cells,!using!Trypan!blue!for!suspension!cells.!!

2. Record!yields!–!note&more&than&25%&loss&or&cell&death&in&any&condition.!!

General'Assessment:'

• Compare!the!recoveries!between!the!(-)polybrene,!(-)virus!wells!and!the!(-)polybrene,!

(+)virus!wells!(#1!vs .!#5!and!#13!vs.!#17):!if!the!numbers!are!lo wer!with!virus,!

lentivirus!may!be!toxic!to!the!cells.!'

• Compare!the!recoveries!between!the!(-)polybrene!wells!and!the!(+)polybrene!wells!

(#5!vs.!#s!6,!7,!8!and!#17!vs.!#s!18,!19,!20):!if!the!numbers!are!lower!with!polybrene,!

polybrene!may!be!toxic!to!the!cells.!'

• Compare!the!recoveries!between!the!‘low’!vs.!‘high-density’!wells!(ex.!#5!vs.!#17):!if!

more!cells!are!lost!from!th e!‘high-density’!wells,!the!cells!may!be!sensitive!to!density.!'

H:!Seed!the!cells!into!fl asks!!

1. Choose!appropriately!sized!flasks!based!on!cell!size!and!doubling!time!to!ensure!the!

cells!will!not!become!confluent!within!2-4!days.!

2. Label!the!10!flasks!with!each!well!condition!name.!!

3. Seed!the!cells!from!each!conical!into!the!corresponding!labeled!flask!and!add!the!

appropriate!volume!of!media.!!

4. Incubate!the!cells.!!

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I:!Monito r!the!cells!post-spinfection!and!seeding!

1. Check!the!cells!under!the!microscope!over!the!next!2-4!days!and!note!any!significant!

cell!death!and/or!debris!in!any!of!the!conditions.!!

2. Before!th e!cell s!get!confluent,!stop!the!ass ay!and!conti nue!with!1$/!(F.!!

1$/!(F:!);:8C<B9;(/::BCB8;CL(5;G(%877(0B56B7B<L("85G(#N<!

Determine!the!infection!efficiency!and!cell!viability!via!several!methods.!!

A:!Visually!assess!the!confluency!and!health!of!the!cells!

1. Check!each!flask!under!the!microscope.!

2. Take!note!of!the!fol lowing!things:!

• Cell!confluency!

• Morphological!changes!

• Amount!of!cell!death!and/or!debris!!

B:!Collect!and!count!the!cells!from!the!flasks!to !obtain!yields!

Note:&there&must&be&cells&left&over&from&each&flask&to&perform&flow&cytometry.& &

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1. Col lect!the!cells!from!each!flask,!ensuring!that!every!condition!is!kept!separate.!

2. Count!the!cells!and!record!yields.!!

3. Save!the!cells!in!suspension!to!perform!flow!cytometry.!!!

C:!Optional,!but!highly!recommended:!assay!the!percentage!of!GFP-positive!cells!via!flow!

cytometry!

1. Take!200!uL!of!each!cell!suspension!and!seed!into!a!V-bottomed!96-well !plate!for!flow!

cytometry.!!

2. Add!100!uL!of!flow!buffer!(PBS,!2%!FBS,!5!uM!EDTA)!to!each!well!and!mix!by!pipetting!

up!and!down.!!

3. Assay!the!10!conditions!via!flow!cytometry,!using!the!no -polybrene,!no-infection!

control!well!to!draw!the!appropriate!gates!for!GFP-negative!cells.!!

4. Record!the!%!GFP-positive!cells!for!each!condition,!indicating!the!percentage!of!

infected!cells.!!

General'Assessment:'

• The!density!and!polybrene!combination!with!the!highes t!yield!from!manual!counts!

(and!the!highest!%!viable!cells!from!Trypan!counts!for!suspension!cells)!and!the!

highest!percentage!of!GFP-positive!cells!will!l ikely!be!the!best!to!use!for!future!

spinfections.!!!

Troubleshooting'[also'see'the'28CB=B9;($A88\''

• If!in!1$/!(>Y(25L(FY(!5A<(/!the!cells!clumped!and!lifted!off!the!plate!and/or!there!was!

more!than!25%!loss!AND!in!1$/!(FY(!5A<(@(5;G(%!th ere!were!poor!yields!and/or!low!

GFP!percentages,!the!cells!are!likely!s ensitive!to!a!high!confluency!and/or!

centrifugation .!Repeat!the!protocol!trying!one!or!more!of!the!following!options:!!

o Continue!with!1$/!(>(25L(F(!5A<(.,!4-6!hours!post-spin!on!Day!1.!!

o A!no-spin!lentiviral!transduction!in!flasks!(see!!A9<9C97'(-9Z=DB;(B;:8C<B9;(:9A(

5GQ8A8;<(C877(7B;8=).!

o A!low er!seeding!density!of!8E5!–!1E6!cells/12-well.!

• Notes!o n!viral!toxicity:!

o If!there!is!a!significant!loss!of!cell!yield!when!comparing!the!(-)polybrene,!!!!!!!!!!!

(-)virus!flasks!to!the!(-)polybrene,!(+)virus!flasks,!lentivirus!is!likely!toxic:!

§ Use!a!low!range!of!virus!volumes!and!a!low!seeding!density!when!

titrating!with!another!virus.!

§ If!lentivirus!remains!toxic,!seek!an!alternative!method!of!delivery.!

• Notes!o n!comparing!polybrene!concentrations:!

o The!lowest!concentration!that!does!not!affect!cell!yield!should!be!used.!!

o If!there!is!a!significant!loss!in!cell!yield!at!all!concentrations,!polybrene!is!likely!

toxic.!Repeat!the!protocol!trying!one!or!more!of!the!following!options:!

§ Instead!of!adding!add itional!media!on!top!of!the!wells!post-spin,!g ently!

aspirate!the!media!from!the!wells.!Then,!dropwise!and!down !the!side!of!

the!well,!add!2 !mL!of!fresh!media!on!top!of!the!cells!–!Note:!do&not&

aspirate&media&from&suspension&cells!&

§ Instead!of!incubating!the!cells!overnight,!continue!with!1$/!(>(25L(F(

!5A<(.,!4-6!hours!post-spin!on!Day!1.!!

§ A!lower!seeding!density!of!8E5!–!1E6/12-well!and!no!polybrene!duri ng!

the!spinfectio n.!

• Notes!o n!comparing!low!vs.!high!d ensity:!

o If!there!are!poor!infection!efficiencies!at!the!‘low!density’!(<25%),!the!cells!have!

very!low-infectability.!Repeat!usi ng!an!even!lower!seeding!dens ity!of!8E5!–!

1E6/12-well.!!

o If!there!are!poor!infection!efficiencies!at!the!‘high!density’!(<25%),!the!cells!

have!low-infectability.!Use!the!‘low!density’!when!infecting!with !another!virus.!!

o If!there!is!not!a!significant!loss!in!cell!yield/infection!efficiency!with!the!‘high!

density,’!it!can!be!used!with!high-titer!viruses!–!Note:&this&is&especially&useful&f or&

some&genome-wide&libraries.!!

T?(!8A:9AJ(<Q8(1DB;:8C<B9;(RB<Q(,;9<Q8A(0BAN=(

The!chosen!sp infection!conditions!can!now!be!applied!to!futur e!l entiviral!infections!with!other!

viruses!besides!the!pRosetta!control.!For!a!list !of!available!vectors!p lease!visit!

https://intranet.broadinstitute.org/gpp/db/!(Broad!login!required),!or!email!gpp-

reagents@broadinstitute.org!regarding!pooled!libraries.!!

• Most!of!these!vectors!have!an!antibiotic!sel ection!marker!to!confirm!successful!

integration!of!the!vector!into !the!cell’s!genome.!The!puromycin!or!blasticidin!dose!for!

selection!of!the!cells !must!be!optimized!prior!to!infection:!

!Puromycin!or!blasticidin!dosing!(see!!A9<9C9 7'(!NA9JLCB;(5;G(675 =<BCBGB;(<B<A5<B9;)(

• A!commonl y!used!vector!is!pXPR_111!(pLEX_311Cas9v2)!for!introducing!Cas9!into!

cells.!T his!vector!produces!very!low!titer!virus,!and!it!can!therefore!be!challenging!to!

obtain!a!high!infectio n!efficiency.!Project!Achilles!has!a!high!success!rate!with!the!

followi ng!conditions:!

o 1.5E6!cells!per!12-well!

o Polybrene!concentration!cell!line!dependent!!

o 750!uL!of!pLEX_311Cas9v2!virus!per!12-well!

o Infecting!several!wells!at!the!same!time!to!obtain!more!screenable!cells!faster!

(with!one!non-in fectio n!control!well!to!assess!infection!efficiency!and!compl ete!

blasticidin!selection)!

• Before!beginning!a!screen,!a!6-well!seeding!density!test!and!viral!titration!with!the!

library!virus!should!be!performed!first:!!

!Cell!Density!and!Doubling!Rate!assay!(see!!A9<9C97'(!9978G(1CA88;(0BA5 7($B<A5<B9;)!

(

!Viral!titration!with!library!virus!(see!!A9<9C97'(!9978G(1CA88;(0BA57($B<A5<B9;)(

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O?(/M5JD78(1DB;:8C<B9;(%9;GB<B9;=(

Here!are!some!example!spi nfections!conditions!for!reference:!!

],>/!–!very!fast!doubling!time,!with!high-titer!virus!

• 1.5E6!cells!p er!12-well!!

• 4!ug/mL !polybrene!

• Collected!cells!out!of!12-wells!4-6!hours!post-spinfection!to!avoid!clumping!and!lifting!

off!of!plate!!

$%IF!–!sensitive!to!pol ybrene,!moderate!infectability,!with!high-titer!virus!

• Optimized!2E6 !cells!per!12-well!!

• 2!ug/mL !polybrene!

• Aspirated!and!replaced!media!after!2!hour!spin!

@/[F\%!–!low!infectability,!not!polybrene!sensitive,!with!low-titer!virus!

• 1.5E6!cells!p er!12-well!

• 8!ug/mL !polybrene!

,TPV!–!high-titer!virus!!

• 3E6!cells!per!12-well!

• 4!ug/mL !polybrene!

S?(1DB;:8C<B9;(28CB=B9;($A88(

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Did$the$cells$clump$or$die$

post-spinfection?$

Yes$ No$

Try$these$tips:$

Were$the$cells$infectable$at$

the$low$density?$

No$

•  Collect'and'seed'

cells'4-6'hrs'post-

spinfection'

•  No-spin'infection'

•  Seeding'density'of'

8E5'–'1E6'

Were$the$cells$infectable$at$

the$high$density?$

Yes$No$

Use'the'

'low$density$

Was$lentivirus$toxic$to$

the$cells?$

Yes$ No$

•  Use'a'low'range'of'

virus'volumes,'and'

the'low$density$for'

future'titrations'

•  Seek'alternative'

delivery'method'

Was$polybrene$toxic$to$

the$cells?$$

Yes$ No$

Try$these$tips:$

•  Aspirate'and'replace'

media'post-spin'

•  Collect'and'seed'cells'4-6'

hrs'post-spinfection'

•  Seeding'density'of'8E5'–'

1E6,'no'polybrene'

Pick'lowest'polybrene'

dose'that'does'not'

affect'cell'viability'

Yes$

Try$this:$

Seeding'density'

of'8E5'–'1E6'

Piece'of'

cake!'$