Culturing MRC-5 cell lines

Hello, i am Stanley and i will be doing my very first blog posting. I am sorry for the delay as i have only started my experiment this week. I have managed to perform some simple experiment today and i will like to share my experience with you guys. These experiments were done in a bio safety level 2 laboratory and are related to Mammalian cell technology (MCT).



The aim of major project is to evaluate the effect of green tea on large lung cancer cells. I am given two different cell lines which are namely the MRC-5 cell line ( healthy lung cell) and the NCI-H460 ( large cell lung cancer). Both cell lines are previously purchased from ATCC and are both in passage number 2 when they are given to me. The objective for today's experiment is to culture both cells in their respective media therefore allowing healthy cell growth and expansion.



First of all, i will have to prepare the respective media for each cell lines. I will be stating the steps involved in preparing the modified Dulbecco's modified eagle media (DMEM), which will be use to culture the MRC-5 cells.


The steps to prepare 1 litre of DMEM are:

1) Thawing of the Fetal bovine serum(FBS), L glutamin, penicillin streptomycin, sodium pyruvate
in a 37 Degree Celsius water bathe.

2) Dissolve 1 packet of DMEM powder and 37g of sodium bicarbonate into of 1 litre ultra pure
water.

3) Add 10ml of penicillin streptomycin

4) Add 10ml of L- glutamin

5) Add 10ml of sodium pyruvate

6) Add 50ml of FBS

7) Filter sterilize



Although the above steps might seem simple and rather straight forward. I made a mistake of introducing too much bubbles which resulted in overflowing of media into the electric vacuum. Although, i had learnt how to filter sterilize DMEM media in Mammalian cell technology. The modified DMEM media is completely different as it contains 10% FBS and many other constituents. I later learnt from my supervisor that the presence of FBS is the main reason behind the large production of bubbles.

Following the preparation of DMEM media, i am ready to culture my MRC-5 cells. The steps are as followed:

1) Pipette a small amount of DMEM media into a centrifuge tube

2) Pipette 2 tubes of MRC-5 cell line into the centrifuge tube

3) Centrifuge it at 1000 rpm for 5mins

4) Pipette out the supernatant

5) Add 20ml of DMEM media to resuspend the cells

6) Pipette the resupended cells into a T75 flask
There are a few key points to note while culturing cell
7) Incubate the culture under 5% Co2 level at 37 Degrees Celsius



While culturing fragile cells for example the MRC-5. We will have to ensure that the media is properly warmed to around 37 Degrees Celsius which is near to our body temperature before it can use to culture the cells. Similarly, no bubbles should be present in the culture as this might lead to cell death.


Strict aseptic techniques have to be observed throughout the whole experiment as any contamination will results in the discarding of media or materials. It is rather scary as my supervisor had previously ordered the materials in week 2 but the it only arrive on week 6 of my major project.

Here are how both cells look like under an inverted microscope.

Mrc-5 cells--------------------->

H460 cells ----------------->

Thank you