Optimization of Lentiviral Spinfection

Last!Modified:!September!2020!

Last!Reviewed:!February!2019!

Version:!1.0.1!

Protocol:!Optimization!of!lentiviral!transduction!using!

spinfection!!

Table&of&Contents&

1. Brief&Description………………………………………………………………………………………………………1&

2. Materials&and&Reagents…………………………………………………………………………………………….1&

3. Optimization&of&Spinfection&Conditions…………………………………………………………………….2&

a. STEP &1:&Cell&Density&and&Poly brene&Matrix…………………………………………………….2&

b. STEP &2:&Infec tion&Efficiency&and&Cell&Viability&Read&Out…………………………………5&

4. Polybrene&Sensitivity&Curve………………………………………………………………………………………7&

5. Perform&the&Spinfection&with&Another&Virus……………………………………………………………..9&

6. Example&Spinfection& C ondit ions……………………………………………………………………………..10&

7. Spinfection&Decision&Tree……………………………………………………………………………………….11&

1.&Brief&Description&

Lentiviral!transd u ction!is!an!effective!method!for!creating!a!stable!cell!line!with!a!DNA!cassette!of!

interest!integrated!into!its!genomic!DNA,!e.g.!an!sgRNA!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!presence!of!polybrene,!a!polycation!that!neutralizes!the!charge!repulsion!between!the!virus!

and!cell!target!surface!and!helps!viral!integration!into!the!cell.!!

The!ideal!spinfection!conditions!can! be!highly!variable!across!cell!types!and!sh ould!be! optimized,!

as!cell!lines!vary!in,!e.g ! infectability,!polybrene!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!control!and!as!an!optional,!but !highly!recommended,!

simple!assessment!of!the!infectability!of!the!cell!line.!Transduction!efficiency!is!determined!as!the!

percentage!of!GFP-positive!cells,!via!f low!cyto metry.!!

2.&Materials&and&Reagents&

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,!store!at!-20C!

for!long-te rm!use,!keep!at!4C!for!2-3!weeks,!do!not!freeze/thaw!multiple!times)!

• Pipettes!

• 15!mL,!50!mL!tubes!

• 2!mL!of!pRosetta!(aka!pLKO_TRC060)!(PuroR,!BlastR,!GFP)!or!pRosettav2!(PuroR,!BlastR,!

HygroR,!GFP)!lentivirus!available!from!GPP!(gpp-reagents @broadinstitute.org)!!

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

• Flow!cyto meter!

• 96-well!V-bottomed!plates!

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

3.&O ptimization&of&Spinfection&Conditions&

STEP &1:&Cell&Density&and&Polybrene&Matrix&

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

Day&1:& &

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

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

!!!!!!!!

!!!!! !

2. Collect!the!cells:!

• For!ad h erent!cells:!

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

ii. Add!trypsin!and! allow!cells!to!detach !from!flask.!

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.##

iii. Ad d!warm! media!(2-3x!the!amount!of!trypsin)!to!the!flask!t o!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!sus pension!cells:!

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

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

media!su pernatant.!!

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

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

a. 1.5E6!cells/mL!in!6!mL!to tal!(low-density)!

b. 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!

d. 2.7!mL!media!+!0.3!mL!virus!

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

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

g. 2.7!mL!media!+!0.3!mL!virus!+!6!uL!polybrene!(final!=![8!ug/ mL])!

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

1. Add!1!mL!of!cell&suspension!and!1!mL!of!media/PB/virus&solution!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.!!

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

C:!Centrifuge!th e!plates!

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

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.$ $

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

1. After!2!hours!of!spin n ing,!add!2!mL!of! media!(without!polybrene)!dropwise!on!top!of!each!

well.!!

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

Day&2:& !

E:!Inspect!cells!visually!

1. The!next!day!(16-24!hrs!later),!remove!the!plate!fro m! the!incubato r!and!ins pect!the!cells!

under!a!microscope.!!

2. Take!note!of!the!following!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!in!STEP&1,&Day&2,&Part&G.!!

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. Collect!the!cells!from!the!plate:!

• For!ad h erent!cells:!

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

ii. Collect!the!media!f rom!each!well!into!the!appropriate!conical.!

iii. Add!1!mL!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!

suspension!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!medi a.!!

• For!sus pension!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!int o!the!appropriate!conical.!

iii. Add!1!mL!of!media!to!wash!each!well!and!transf er!the!wash!to!the!

appropriate!conical.!!

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

v. Aspirate! th e!media!and!re-suspend!the!lo w-density!cells!in!1!mL!of!fresh!

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

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

1. Cou nt!the!cells,!using! Trypan!blue!for!suspension!cells.!!

2. Record!yields!–!note&more&than&25%&loss&or&cell&deat h&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!lower! 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!the!‘high-density’!wells,!the!cells!may!be!sensitive!to!density.!'

H:!Seed!the!cells!into!flasks!!

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!12-well!condition!name.!!

3. Seed ! the!cells!from!each ! conical!into!the!corresponding!labeled! f lask!and! add!th e!

appropriate!volume!of!media.!!

4. Incubate!the!cells.!!

Day&4-6:&&

I:!Monitor!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!the!cells!get!confluent,!stop!the!assay!and!continue!with!STEP&2.!!

STEP &2:!Infection&Efficiency&and&Cell&Viability&Read&Out!

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!following!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.&&

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

2. Cou nt!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! co nditions!via!f low!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' A ssessment:'

• The!density!and!polybrene!combination!with!the!highest!yield!from!manual!counts!(and!

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

percentage!of!GFP-positive!cells!will!likely!be!the!best! to!use!for!fu ture!spinfections.!!!

Troubleshooting'(also'see'the'Decision&Tree):'

• If!in!STEP&1,&Day&2,&Part&E!the!cells!clumped!and!lifted!off!the!plate!and/or!there!was!more!

than!25%!loss!AND!in!STEP&2,&Part&B&and&C!there!were!poor!yields!and/o r!low!GFP!

percentages,!the!cells!are!likely!sensitive!to!a!high!confluency!and/or!centrifugation.!

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

o Continue! with!STEP&1&Day&2&Part&F ,!4-6!hours!post-spin!on!Day!1.!!

o A!no-spin!lentiviral!transdu ction!in!flasks!(see!Protocol:&No-spin&infection&for&

adhere nt&c ell&lines).!

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

• Notes!on!viral!toxicity:!

o If!there!is!a!significant!loss!of!cell!yield!when!com paring!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!on!comparing!po lybrene!concentratio ns:!

o The!highest!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!additional!media!on!top!of!the!cells!post-spin,!gently!

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!th e!cells! overnight,!continue!with!STEP&1&Day&2&Part&

F,!4-6!hours!post-spin! on!Day!1.!!

§ A!lower!seed ing!density!of!8E5!–!1E6/12-well!and!no!polybrene!during!the!

spinfectio n.!

• Notes!on!comparing!low!vs.! high!density:!

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

very!low-infectability.!Repeat! u s ing!an!even! lo wer!seedin g!density!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&for&

some&genome-wide&libraries.!!

4.&Polybrene&Sensitivity&Curve&

If!the!ideal!spinfection!conditions!for!the!cells!are!already!known,!a!polybrene!sensitivity!assay!

may!be!performed!instead!of!the!above!cell!density!and!polybrene!matrix.!!

Day&1:& &

A:!Prepare!a!12-well!plate!and!cell!suspensions!

1. Label!one!12-well!plate!as!follows:!

2. Collect!the!cells! at!the!desired!concentration!in!10!mL!of!media!total.!

3. Label!4!conicals !with!the!po lybrene!concentrations:!0,!2 ,!4,!8.!

4. Add!2!mL!of!cell!suspension!to!each! of! the!conicals.!

0"PB 2 PB 4"PB 8 PB

5. Add!the!following!volumes!of!8!mg/mL !polybrene!s tock!to!the!corresponding!conicals!and!

mix!thoroughly:!

a. 0!

b. 0.5!uL!!(final!=![2!ug/mL])!

c. 1!uL!!(final!=![4!ug/mL])!

d. 2!uL!!(final!=![8!ug/mL])!

B:!Seed!cells!in!12-well!plate!and!centrifuge!

1. Seed ! each!cell+polybrene!sus p ension!into!the!co rresponding! 12-well.!!

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

3. After!the!spin,!add!2 ! mL!of!media! (without!polybrene)!dropwise!on!top!of ! each!well.!

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

Day&2:& &

C:!Inspect!and!collect!the!cells!

1. Visually!inspect!each!well!and!note!any!significant!amount!of!cell!debris.!!

2. Collect!the!cells!from!each!well!into!a!labeled!conical,!count!(using!Trypan!blue!with!

suspension!cells),!and!reco rd!yields.!!

D:!Seed!the!cells!

1. Choose! an!appropriately!sized!well-plate!or!flask!based!on!cell!size!and!doubling!time!to!

ensure!the!cells!will!not!become!confluent!within!3-4!days.!!

2. Label!4!wells/flasks!with!the!p o lybrene!concentrations.!

3. Seed ! the!cells!from!each ! conical!into!the!corresponding!wells/flasks!and!add!the!

appropriate!volume!of!media.!!

4. Incubate!the!cells.!!

Day&5-6:&!

E:!Collect!and!count!the!viable!cells!from!each!well/flask!(using!Trypan!blue!for!suspension!cells).!!

General' A ssessment:''

• The!highest!concentration!of!polybrene!that!results!in!less!than!10%!cell!toxicity!compared!

to!no!polybrene!should!be!used.!!

• If!po lybrene!is!toxic!at!all!concentrations,!repeat!the!protocol!trying!one!or!more!of!the!

following!options:!

o Instead!of!adding!additional!media!on!top!of!the!cells!post-spin,! gently!aspirate!the!

media!from!the!wells .!Then,!dropwise!and!down!the!side!o f!the!well,!add!2!mL!of!

fresh!media!–!Note:!do&not&aspirate&media&from&suspension&cells!!

o Instead!of!incubating!the!cells!overnight,!continue!with!Day&2&P a rt&C,!4-6!hours!

post-spin!on!Day!1.!!

5.&Perform&the&Spinfection&with&Another&Virus!

The!chosen!spinfection!conditions!can!now!be!applied!to!future!lentiviral!infections!with!other!

viruses!besides!the!pRosetta!control.!For!a!list!of!available!control!viruses!please!visit!the!GPP!

Web!Portal!or!email!gpp-reag ents@broadinstitute.org!regarding!pooled!libraries.!!

• Most!of!these!vectors!have!an!antibiotic!selection!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:!

!Selection!drug!dosing! (see!Protocol:&Puromycin,&blasticidin&an d&hygro my cin&titra ti on)!

• A!commonly!used!vector!is!pXPR_111!(pLEX_311Cas9v2)!for!introducing!Cas9!into!cells.!

This!vector!produces!very!low!titer!virus,!and!it!can!therefore!be!challenging!to!obtain!a!

high!infection!efficiency.!Project!Achilles!has!a!high!success!rate!with!the!following!

spinfectio n!conditions:!

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

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

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

o Infect!several!wells!at!the!same!time!to!obtain!more!screenable!cells!faster!(with!

one!non-in fection!control!well!to!assess!infection!efficiency!and!complete!

blasticidin!selection)!

o Add!blasticidin!48!hours!pos t-spinfection!!

• 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!Protocol:&Pooled&Screen&Viral&Titration)!

!Viral!titration!with!library!virus!(see!Protocol:&Pooled&Screen&Viral&Titration)&

10!

6.&Example&Spinfection&Conditions&

Here!are!some!example!sp infections!conditions!for!reference:!!

HA1E!–!very!fast!doubling!time,!with!high-titer!virus!

• 1.5E6!cells!per!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!!

TC32!–!sensitive!to!polybrene,!moderate!infectabi lity,!with !high-titer!virus!

• Optimized!2E6!cells!per!12-well!!

• 2!ug/mL!polybrene!

• Aspirated!and!replaced!media!after !2!hour!sp in!

BE(2)C!–!low!infectability,!not!polybrene!sensitive,!with!low-titer!virus!

• 1.5E6!cells!per!12-well!

• 8!ug/mL!polybrene!

A549!–!high-titer!virus!!

• 3E6!cells!per!12-well!

• 4!ug/mL!polybrene!

11!

7.&Spinfection&Decision&Tree&

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

post-spinfection ?

Yes No

Try$these$tips:

Were$the$cells$infectable at$

the$low $d ensity?

• 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?

YesNo

Use'the

low $d ensity

Was$lent i virus toxic to$

the$cells?

Yes No

• Use'a'low'range'of'

virus'volumes,'and'

the'low$dens i t y$for'

future'titratio ns

• 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'highest polybrene'

dose'that'does'not'

affect'cell'viability

Yes

Try$this:$

Seeding'density'

of'8E5'– 1E6

Piece'of'

cake!