User:Hendry Cahaya/Notebook/Culturing Human Lung Fibroblast IMR 90/Entry Base

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 Culturing Human Lung Fibroblast IMR-90
Materials: (Note: materials may be different from lab to lab, but the basic media or buffer is similar)

Culture Holding: T-75 flask or 55.6 cm^2 dish (from Nunc)

Complete Media: DMEM Hi Glucose (Invitrogen 10566, supplemented with GlutaMax). 10% FBS (non heat activated) 1% Penicillin/Streptomycin

Reagents: D-PBS without calcium and magnesium. Absence of Ca and Mg helps in detaching the cells. 1X Trypsin (0.05) with EDTA

 Protocol (from frozen vial/stock ~ 1 million frozen cells): 

1. Warm up complete media and D-PBS.

2. In a T-25 flask, add 10 ml of full media.

3. Add the entire thawed cell stock into the T-25 containing media.

4. Incubate at 37C/5% CO2 for 24 hours.

5. Check morphology of the culture the next day. If most of the cells have attached, aspirate the old media and add 10 ml of warm full media. Otherwise, wait for another 24 hours for media exchange.

Note: Old media contains a small trace of DMSO from the frozen vial.

6. It usually takes 4 days to reach 90% confluency. When this stage is reached, cells must be detached and are ready for expansion. (It can be challenging to judge when cells are 90% confluence. My rule of thumb is that when when viewed under microscope, cells are nicely spread with some gaps. They are not packed one-another. IMR-90 fibroblast is sensitive to contact inhibition)

 Expansion from T-25 to T-75: 

1. Aspirate old media.

2. Wash attached cells with 10 ml 1X D-PBS (No Ca/Mg) twice.

3. Add 0.5 ml of 1X Trypsin/EDTA. Incubate ~ 3 min/37C.

4. Transfer the entire content to T-75 containing 20 ml full media.

6. Cells should be 90% confluent within 4 days. At this stage, IMR-90 are ready for split or life-span study.

 How to split IMR-90? 

From a ~ 90% confluent IMR-90 cells in T-75:

1. Aspirate old media. Wash cells once with 10 ml warm 1X D-PBS (No Ca/Mg)

2. Add 1 ml of warm 1X Trypsin/EDTA. Incubate ~ 3-5 min at 37C incubator.

3. Cells should be detached by 5 min. If not, a gentle shake can help. Quench 1X Trypsin/EDTA with 1 ml of warm full media.

4. Counting cells can be done with Coulter counter or Trypan Blue. Follow counting protocol according to your system. I count using Trypan Blue at 1:5 dilution.

5. Calculate the volume of cell suspension needed for propagation. I usually culture IMR-90 at a density of 0.5 million cells per T-75 flask (20 ml warm full media). At this density, cells achieve 90% confluency within 5-7 days depending on their PDL.