TITLE: Whole-genome CRISPR Screening of stably expressing Cas9 Cancer Organoid Lines
AUTHORS: Tessa Fowler, Jade Smith, Adam Jackson, Agnieszka Andres, Emily Souster, Hazel Rogers, Alexandra Beck, Charlotte Beaver, Mathew Garnett

[DESCRIPTION]
This protocol is for whole-genome CRISPR screening of stably expressing Cas9 cancer organoid lines in triplicate using the commercially available minimal genome-wide human CRISPR Cas9 library. The protocol uses lentiviral transduction as a method for gRNA delivery. This method can be adapted for other gRNA libraries. 

The protocol can be followed assuming the following is known:
- The number of days required for screening
- The size of the gRNA library
- The required coverage of the library

To allow for efficient scale up of the organoid culture for screen, a 5% suspension culture method can be used. This is not essential to be able to perform whole-genome CRISPR screening of stably expressing Cas9 cancer organoid lines, but provides a more scalable, ergonomic and cost efficient culturing method. 

Prior to commencing the screen a puromycin antibiotic titration is used to identify the most suitable puromycin concentration for the selection of Cas9 positive cancer organoid lines transduced with gRNA library virus.

A gRNA library titration is then performed to determine the volume of library virus required to transduce Cas9 cancer cells at 30% transduction efficiency which is calculated using FACS analysis. This is to avoid host cells taking up more than one gRNA copy per cell, therefore an MOI of 30% should be aimed for.

Process Diagram
 

 

This protocol uses a 21-23 day screen process.


[BEFORE_START]
Prior to each process, pre-warm culture media to room-temperature.
Where required, prepare an aliquot of 1mg/ml puromycin (working concentration) by diluting a 10mg/ml stock 1:10 with sterile water.
On Day 4 of the puromycin titration process, bring Cell Titer-Glo 2.0 reagent to room temperature. (It is light-sensitive and so it is advisable to keep the reagent covered at all times to avoid exposure to light when using).
Prior to gRNA library transduction, thaw an aliquot of (10mg/ml) polybrene 
Where required, thaw the required volume of lentivirus for each process stage.
When fixing organoids, dilute 37% formaldehyde solution 1:10 with DPBS and store at 4 °C 


[GUIDELINES]
It is important that all stages are carried out using the stably expressing Cas9 version of each cancer organoid line.

Ensure the cell suspension is mixed thoroughly at all stages to create an even single cell suspension before plating.

Avoid maintenance of organoids in a single-cell state for prolonged periods to retain viability.

Avoid freeze/thaw cycles of lentivirus stocks. Prioritising freshly prepared virus stocks will ensure viral integrity is maintained. 

Puromycin titration;

It is advised to use black 96-well plates in this protocol, as luminescence can carry over into surrounding wells in clear plates.

All steps involved in the plate set up, including seeding cells, media, antibiotics and CellTiter-Glo should be carried out using reservoirs and multi-channel pipettes where possible to reduce ergonomic strain and to maintain homogeneous solutions throughout. 

gRNA library titration;

The volumes of library titrated may differ depending on the batch of lentivirus or library used. To achieve a 30% MOI there may therefore be a requirement to dilute the virus prior to titrating. For this reason, an increased number of titration points may also be required. This may require some R&D to determine optimal volumes to titrate.

gRNA library transduction and screen;

The gRNA library transduction is carried out in triplicate with 12.5 x10^6 cells seeded per replicate. Expansion of the line to a minimum of 40 x10^6 cells is required for this process. 

Following selection at Day 6 the organoid suspension should remain in puromycin selection media for the remainder of the screen.


Unless otherwise stated, all steps should be performed under sterile conditions in a microbiological safety cabinet.


[STEPS]
SECTION: Puromycin titration
1. Day 1: Titration plate set up

 

SECTION: Puromycin titration
1.1. Pre-warm organoid specific culture media to room temperature.
SECTION: Puromycin titration
1.2. Aspirate media from each well of the organoid culture plate plate and add 2 mL TrypLE to each well.
SECTION: Puromycin titration
1.3. Using a cell-scraper detach BME2 drops containing the cancer organoids from the plate and transfer the organoid suspension to an appropriately sized tube.
SECTION: Puromycin titration
1.4. Pipette the suspension up and down multiple times to dissociate organoids from the BME2.
SECTION: Puromycin titration
1.5. Incubate at 37 °C 5% CO2.
SECTION: Puromycin titration
1.6. Check the organoid suspension under the microscope every 15 minutes to assess and 
monitor the dissociation of the organoids. Mix the suspension thoroughly prior to each check to help dissociate the organoids.
 

SECTION: Puromycin titration
1.7. Centrifuge at 800 x g for 2 min.
SECTION: Puromycin titration
1.8. Aspirate the supernatant and resuspend the pellet in an 10 ml of organoid specific culture media.
SECTION: Puromycin titration
1.9. Perform a cell count to calculate the total number of cells.

 

SECTION: Puromycin titration
1.10. Resuspend 2.4x106 cells in 2.7 ml of organoid specific culture media + 300 µL BME2 (this will give a final seeding density of 8x104 cells per well once plated, Rows B-G of Fig 1).

SECTION: Puromycin titration
1.11. Prepare a control stock solution containing organoid specific culture media with 5% BME2 (Row A of Fig 1).
SECTION: Puromycin titration
1.12. Set up the titration plate as detailed in Fig 1 below;


 

 

SECTION: Puromycin titration
1.13. Incubate the plate at 37 °C 5% CO2 for 10 min to allow the BME2 to polymerise.
SECTION: Puromycin titration
1.14. Using a 1 mg/ml stock, prepare puromycin antibiotic solutions at 2x concentrations in organoid specific culture media in 5 ml tubes as outlined below. (Table 1).

 


 

 

SECTION: Puromycin titration
1.15. Remove the plate from the incubator and pipette 100 µL of the relevant 2x puromycin stock into each well (Rows B-G) to achieve the final require puromycin concentration according to the plate layout in Fig 1. 

SECTION: Puromycin titration
1.16. Incubate the plate for 4320 min at 37 °C 5% CO2. 
SECTION: Puromycin titration
1.17. Plate any remaining cells from step 1.10 into a 5% BME2 suspension culture by seeding cells into a solution of 19 mL organoid specific culture media + 1 mLBME2 using an ultra-low attachment T75 flask.

 

SECTION: Puromycin titration
2. Day 4: Assessing cell viability using CellTiter-Glo
SECTION: Puromycin titration
2.1. Thaw CellTiter-Glo 2.0 reagent and equilibrate to room-temperature prior to use.

 


 

SECTION: Puromycin titration
2.2. Run a CellTiter-Glo 2.0 viability assay following the manufacturer’s instructions. 

  

 

SECTION: Puromycin titration
2.3. Using the luminescence data plot a kill curve to ascertain the lowest concentration of puromycin which results in approximately 100% cell death after 72 hours. (Fig 2).

 


 


SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3. Day 1: gRNA library titration set up
SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.1. Pre-warm organoid specific culture media to room temperature and thaw a 10 mg/ml aliquot of polybrene.
SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.2. Prepare transduction media, by adding 12.5 µL of 10 millimolar (mM) Y-27632 (Rock inhibitor) to 50 mL organoid specific culture media (2.5 micromolar (µM) final concentration). 

 

SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.3. Collect the organoid suspension culture (from step 1.18) in a 50 ml tube and centrifuge at 800 x g for 2 min.

SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.4. Aspirate the supernatant and resuspend the pellet in 15 ml to 20 ml of TrypLE.
SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.5. Pipette the suspension up and down multiple times to dissociate organoids from the BME2.
SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.6. Incubate at 37 °C 5% CO2.
SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.7. Check the organoid suspension under the microscope every 15 minutes to assess and 
monitor the dissociation of the organoids. Mix the suspension thoroughly prior to each check to help dissociate the organoids.
 

SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.8. Centrifuge at 800 x g for 2 min.
SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.9. Aspirate the supernatant and resuspend the pellet in an appropriate volume of transduction media.
 

SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.10. Perform a cell count to calculate the total number of cells.
SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.11. Prepare a preparation mix using the cell suspension and transduction media to achieve a final concentration of 5.6x106 cells in a total volume of 11.2 mLplus 14 µL 10 mg/ml polybrene.

 

SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.12. Transfer 1.6 ml of the preparation mix to 6x bioreactor tubes (8x105 cells per tube) and add relevant transduction media volumes. (see media volume in Table 2).

 

SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.13. Thaw an aliquot of the gRNA library to room temperature. If the gRNA library is not being used 
neat, dilute the required volume of the gRNA library in organoid complete culture media.
 

 

SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.14. Add the appropriate volume of gRNA library virus (Table 2) to each bioreactor tube.
SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.15. Mix well by pipetting and transfer the bioreactor tubes to the incubator at 37 °C 5% CO2 for 
overnight incubation.

SECTION: Guide RNA library titration of Cas9 expressing organoid lines
3.16. Passage all remaining organoids in 5% BME2 organoid specific suspension culture media. 
 

SECTION: Guide RNA library titration of Cas9 expressing organoid lines
4. Day 2: Plating 
SECTION: Guide RNA library titration of Cas9 expressing organoid lines
4.1. Transfer the 6x bioreactor tubes to the centrifuge ensuring the centrifuge bucket lid is 
secure.
 

SECTION: Guide RNA library titration of Cas9 expressing organoid lines
4.2. Centrifuge at 800 x g for 2 min.
SECTION: Guide RNA library titration of Cas9 expressing organoid lines
4.3. Aspirate the supernatant from the tubes and resuspend each cell pellet in1.9 mL of transduction media (prepared on Day 1) and add 100 µL of 100% BME2 to make a 5% BME2 solution. Mix well and transfer all 2 ml into each well of a low attachment 6wp. (Fig 3).


 


SECTION: Guide RNA library titration of Cas9 expressing organoid lines
4.4. Incubate at 37 °C 5% CO2 until Day 6.


SECTION: Formaldehyde fixation of organoids
5. Day 6: Fixing and staining organoids for flow cytometry analysis.

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6. Day 1: gRNA library transduction
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6.1. Pre-warm organoid specific culture media to room temperature. Thaw a 10 mg/ml aliquot of polybrene and a  10 millimolar (mM) aliquot of Y-27632 (Rock inhibitor).
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6.2. Collect the organoid suspension culture from step 3.16    in a 50 ml tube and centrifuge at 800 x g for 2 min.

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6.3. Aspirate the supernatant and resuspend the pellet in 30 ml of TrypLE.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6.4. Pipette the suspension up and down multiple times to dissociate organoids from the BME2.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6.5. Incubate at 37 °C 5% CO2.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6.6. Check the organoid suspension under the microscope every 15 minutes to assess and 
monitor the dissociation of the organoids. Mix the suspension thoroughly prior to each check to help dissociate the organoids.
 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6.9. Perform a cell count to calculate the total number of cells.
 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6.11. Carry out each library transduction in triplicate. Prepare each replicate in a 250 ml erlenmeyer flask as per Table 3. Mix gently by pipetting.


 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6.12. Prepare an un-transduced control using the appropriate volumes of reagents listed in Table 4. Approximately 1.6 x106 cells should be seeded into a 50 ml bioreactor tube.  


 


SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6.13. Incubate the control bioreactor and replicate flasks prepared in step 6.11 and 6.12 overnight at 37 °C5% CO2.

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6.10. Thaw an aliquot of the gRNA library to room temperature. If the gRNA library is not being used 
neat, dilute the required volume of the gRNA library in organoid complete culture media.
 

 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
7. Day 2: Plating
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
7.1.       
Collect and transfer the replicate solutions into labelled 50 ml  tubes and centrifuge alongside the control bioreactor tube at 800 x g for 2 min.
 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
7.2. Aspirate the supernatant and resuspend each replicate pellet in 19 mL organoid specific culture media with 5 µL of   10 millimolar (mM) Y-27632 (Rock inhibitor) and 1 mL of BME2  to make a 5% BME2 solution.   

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
7.3. Mix the suspension well by gentle pipetting and transfer each replicate into x1 ultra low-attachment T75 flask and incubate at 37 °C 5% CO2.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
7.4. For the control sample, aspirate the supernatant from the tube and resuspend the cell pellet in 1.9 ml of organoid specific culture media containing 0.5 µL  10 millimolar (mM) Y-27632 (Rock inhibitor) and add 100 µL of BME2 to make a 5% BME2 solution. 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
7.5. Mix well by pipetting and transfer into 1 well of an ultra-low attachment 6wp and incubate at 37 °C 5% CO2.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
8. Day 6: Puromycin selection and flow cytometry analysis.
SECTION: Formaldehyde fixation of organoids
5.1. Prepare Live/Dead stain solution or antibodies.
 

SECTION: Formaldehyde fixation of organoids
5.2. Collect the suspension culture from each well of the ultra-low attachment 6wp into individual 2 ml tubes.
SECTION: Formaldehyde fixation of organoids
5.3. Centrifuge at 800 x g for 2 min.
SECTION: Formaldehyde fixation of organoids
5.4. Aspirate the supernatant and resuspend in 1 mL of Trypsin-EDTA (0.25%). 


 

SECTION: Formaldehyde fixation of organoids
5.16. Resuspend the pellet in 500 µL (dependent on pellet size) PBS or alternative FACs buffer, and store at 4 °C until ready for analysis by flow cytometry. 

SECTION: Formaldehyde fixation of organoids
5.19. Using the %BFP expression for each titration point, plot a titration curve to calculate the volume of virus required to obtain 30% BFP expression. (Fig 5).
 

 

SECTION: Formaldehyde fixation of organoids
5.5. Incubate for 15 min, until organoids have broken down to single cells. 
 

SECTION: Formaldehyde fixation of organoids
5.6. Once organoids have broken down to single cells stop the reaction by adding 1 mL (diluting 1:1) of media containing serum.
SECTION: Formaldehyde fixation of organoids
5.7. Centrifuge at 800 x g for 2 min.
SECTION: Formaldehyde fixation of organoids
5.8. Aspirate supernatant and resuspend pellets in 200 µL Live/Dead dye solution (or specific antibody of choice).
SECTION: Formaldehyde fixation of organoids
5.9. For the Live/Dead solution, incubate at room temperature for 5 min. (Follow specific guidelines for your antibodies).
SECTION: Formaldehyde fixation of organoids
5.10. Add 1 mL PBS to each tube

SECTION: Formaldehyde fixation of organoids
5.11. Centrifuge at 800 x g for 2 min.
SECTION: Formaldehyde fixation of organoids
5.12. Aspirate supernatant and resuspend in 500 µL of 3.7% formaldehyde. Mix well by pipetting to ensure cells are fixed as single cells.
 

SECTION: Formaldehyde fixation of organoids
5.13. Incubate at 4 °C for 10 min.
SECTION: Formaldehyde fixation of organoids
5.14. Centrifuge at 800 x g for 2 min.
SECTION: Formaldehyde fixation of organoids
5.15. Carefully aspirate supernatant (in chemical fume hood). 
 

SECTION: Formaldehyde fixation of organoids
5.17. Using collected flow cytometry data create a plot showing side scatter area vs BFP+ expression, gating the positively expressed BFP+ cells. 
SECTION: Formaldehyde fixation of organoids
5.18. For each titration point create an overlay plot (Fig 4) using your un-transduced control (Well 1 of Fig 3).
 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
8.1. Using a pipette, collect 0.5-1 ml from each replicate and transfer to individual 2 ml tubes. Collect all of the control and transfer to another 2 ml tube, this is done to test infection efficiency using flow cytometry.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
8.2. Fix each organoid sample and analyse by flow cytometry following steps 5.1 to 5.17.  
 


SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
8.3. The pass rate for transduction efficiency is 30%, calculated from step 5.19  .  However a value between 15-50% is acceptable. This is to try and ensure only 1 copy of the library has been transduced per cell. If a value outside of this range is obtained at this stage the screen should be failed. (Fig 6). If the pass rate has been reached, continue to step 8.4. 
 


SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
8.4. For each replicate; collect the suspension culture in a 50 ml tube using a stripette or by pouring the suspension.
 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
8.5. Mix the suspension well by pipetting to break down any larger clumps of BME2/aggregates 
before centrifugation.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
8.6. Centrifuge at 800 x g for 2 min.
 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
8.7. Aspirate the supernatant and resuspend the pellet in 10 mL organoid specific culture media.

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
8.8. Prepare the ultra-low attachment T75 flask with the appropriate remaining volume of media, BME2 and puromycin (Table 5 and 6) (Puromycin should be added at a concentration previously calculated in  Fig 2).

 

 


 


 


SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
8.9. Mix the cell suspension well by pipetting, then transfer cell suspension to the individual ultra-low attachment T75 flasks.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
8.10. Incubate at 37 °C 5% CO2.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
9. Day 9-20: Maintenance of screens. (The following steps outline a 1:2 passage)
 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
9.1. Collect suspension for each replicate in 50 ml tubes.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
9.2. Centrifuge at 800 x g for 2 min.
 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
9.3. Aspirate the supernatant and resuspend the pellet in an appropriate volume of TrypLE.
 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
9.4. Mix well by pipetting and incubate at 37 °C 5% CO2 for 10 min.

 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
9.5. Centrifuge at 800 x g for 2 min.
 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
9.6. Aspirate the supernatant and resuspend each pellet in 20 mL organoid specific culture media.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
9.7. Prepare 2x ultra-low attachment T75 flasks with the appropriate remaining volume of media, BME2 and puromycin per replicate (Table 5 and 6).   

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
9.8. Mix the cell suspension well by pipetting, then transfer half the cell suspension to each new ultra-low attachment T75 flask.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
9.9. Incubate at 37 °C 5% CO2.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
10. Day 21-23: Assessment of final selection efficiency 
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
10.1. Using a pipette, collect 0.5-1 ml from each replicate and transfer to separate 2 ml tubes.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
10.2. Fix each organoid sample and analyse by flow cytometry following steps 5.1 to 5.17.  
 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
10.3. The pass rate for post-puromycin selection is between 60-100%. This ensures efficient selection of organoids that have been transduced with the viral library. If a value below this range is obtained at this stage the screen should be failed. (Fig 7). If the pass rate has been reached continue to step 11.1.
 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
11. Day 21-23: Pelleting using Cell Recovery Solution
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
11.1. Collect suspension for each replicate in 50 ml tubes.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
11.2. Centrifuge at 800 x g for 2 min.
 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
11.3. Aspirate the supernatant and resuspend each pellet in an appropriate amount of Cell Recovery Solution (up to 30 mLper replicate) to remove BME. Mix well by pipetting. 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
11.4. Incubate on ice for60 min.

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
11.5. Once the sample tubes have been on ice for 30 min; mix the suspensions well by pipetting and take aliquots from each replicate to perform a cell count. 

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
11.6. Following 60 min on ice, based on the required number of cells per pellet, transfer the required volume of cells per pellet into 15 ml tubes.
 


SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
11.7. Centrifuge at 800 x g for 2 min.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
11.8. Aspirate the supernatant and resuspend in 10 ml of 4 °C  PBS to wash and remove the Cell Recovery Solution from the pellets.
 


SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
11.9. Centrifuge at 800 x g for 2 min.
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
11.10. Aspirate the PBS and store the pellets at -80 °C ready for downstream processing.

SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6.7. Centrifuge at 800 x g for 2 min
SECTION: Guide RNA Library transduction and screen of Cas9 expressing organoid lines
6.8. Aspirate the supernatant and resuspend the pellet in an appropriate volume of organoid specific culture media.
 

SECTION: Puromycin titration
1.18. Incubate the ultra-low attachment T75 flask at 37 °C 5% CO2.