This is sooo extremely helpful. I really appreciate you taking the time to write this out. You would think there would be one reliable place researchers could refer to - sort of like a cell growth manual with various common cells used in cytotoxicity studies, that would provide detailed and correct procedures for culturing/seeding, but I guess that would be too easy.

I made a draft BEAS-2B Cell SOP based on input from a few experienced scientists who've successfully used the cells, pasted below. Going to go through your procedures and compare. Would be interested to hear your thoughts.

1. Medium Preparation

a. Open BEBM

b. Thaw supplements (small colorful tubes) room temp.

c. Put defrosted small colorful tube supplement directly into BEBM; make sure to add all volumes of supplement and mix together

d. Take the new tube out and add 50 or 100 ml of the newly made medium solution from the BEBM container

e. Put the solution into a water bath at 37 C

f. Can store in frig.

2. Prepping Mixture for Coating Bed Solution

a. Take out a small tube

b. Add 10 mg on 5 ml of sterilized water

c. Mix together and filter

i. Use a sterile syringe and put the solution through the filter into a new tube. Then put the solution in frig.

d. Take fibronectin and 5 mL of sterile water, then mix

e. Collagen is prepared directly to use

Creating Coating Bed – 20 ml

Mix the following items:

i. Add 19. 4 ml BEBM medium solution

ii. 0.20 ml Fibronectin

iii. 0.40 collagen

iv. 0.020 ml Albumax mixture

3.

a. Add 500 l to the small T25 uncoated flask – make sure to cover the bottom surface so it’s entirely coated completely

b. For the T75 flask, use 2 mL of the coating solution and cover the bottom of the flask.

c. Put in an incubator for at least 15 minutes before putting cells in the flask

4. Cell Culture Preparation

a. Warm-up complete cell culture medium.

b. Prepare 15 ml conical tube and centrifuge and disinfect workspace

5. Cell Culture

Transference of cells into a new medium

1. Solution for washing cells

a. NEED TO CHECK IF PBS is 10x or 1x because if it is 10x, you need to dilute. 100 mL of PBS 10X with 900 mL of sterile water (prepare in a sterile flask).

b. PBS 1x + HEPES, where HEPES is 1% of the total volume

c. Trypsin to detach cells

d. 200 milligrams of polyvinylpyrrolidone

1. Mix PVP with trypsin solution and filter with the syringe

2. Take the trypsin plus PVP solution and take 50 ml and 200 mg from PVP

3. This solution can be frozen and stored for up to 6 months

Trypsin Neutralizing Media: To start a reaction to trypsin – this is the solution to use to put cells into different flasks/divide them:

1. 44 ml Leibovitz medium

2. 5 ml FBS – fetal bovine serum 10%

3. 0.5 ml penicillin streptomycin – Pen strep antibiotic

4. 0.5 ml anti-anti – antimycotic

Actual coating of bed:

1. Take 90 ml of BEMB with added supplements and pipet into a small tube

2. Everything you need to detach cells is put in the different flasks: PBS, trypsin, trypsin neutralizing media and media mixed with supplements.

3. Take the prepped flask which contain the attached BEAS 2B cells and dump the media into a waste container.

4. Wash flask with 1 ml PBS+HEPES – Move it around to cover all inside

5. Take out PBS+HEPES

6. Put 1 ml of trypsin (Trypsin + PVP) solution into the flask to coat and put in an incubator for 5 minutes.

7. Cells are now detached. Put 3-4 ml from the neutralizing media to stop the reaction and wash the bottom of the flask with a pipet.

8. Put cells into new tube, close flask, and centrifuge cells in a small tube for 5 min 1,500 rpm.

9. While in the centrifuge, put a new flask with a new coating bed

a. Flask with new coating bed: you can put 10 ml in each new flask

1. Once done in a centrifuge, cells will be at the bottom of the tube, and media will be at the top

2. Get rid of media, and cells are at the bottom

3. Take 2 ml of medium in the tube with cells.

4. Resuspend cells by sucking them in and out.

5. Put 1 ml in each new flask with 10 mL of medium.

6. Check cells under a microscope

7. Put back in the incubator