Tissue Culture

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Cell culture guidelines

The following is a general guideline for culturing of cell lines. All cell culture must be undertaken in microbiological

safety cabinet using aseptic technique to ensure sterility.

1. Preparation of cell growth medium

Before starting work check the information given with the cell line to identify what media type, additives and

recommendations should be used.

Most cell lines can be grown using DMEM culture media or RPMI culture media with 10% Foetal Bovine Serum

(FBS), 2 mM glutamine and antibiotics can be added if required (see table below).

Check which culture media and culture supplements the cell line you are using requires before starting cultures.

General example using DMEM media:

DMEM - Remove 50 ml from 500 ml bottle then add the other constituents. 450 ml

10% FBS 50 ml

2 mM glutamine 5 ml

100 U penicillin / 0.1 mg/ml streptomycin 5 ml

2. Checking cells

1. Cells should be checked microscopically daily to ensure they are healthy and growing as expected.

* Attached cells should be mainly attached to the bottom of the flask, round and plump or elongated

in shape and refracting light around their membrane.

* Suspension cells should look round and plump and refracting light around their membrane. Some

suspension cells may clump.

* Media should be pinky orange in colour.

2. Discard cells if

* They are detaching in large numbers (attached lines) and/or look shrivelled and grainy/dark in

colour.

* They are in quiesence (do not appear to be growing at all).

3. Sub-culturing

1. Split ratios can be used to ensure cells should be ready for an experiment on a particular day, or just to keep the

cell culture running for future use or as a backup. Suspension cell lines often have a recommended subculture

seeding density. Always check the guidelines for the cell line in use. Some slow growing cells may not grow if a

high split ratio is used. Some fast growing cells may require a high split ratio to make sure they do not overgrow.

Note that most cells must not be split more than 1:10 as the seeding density will be too low for the cells to survive.

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As a general guide, from a confluent flask of cells:

1:2 split should be 70-80% confluent and ready for an experiment in 1 to 2 days

1:5 split should be 70-80% confluent and ready for an experiment in 2 to 4 days

1:10 split should be 70-80% confluent and ready for sub-culturing or plating in 4 to 6 days.

2. If cells are less then 70-80% confluent but you wish to subculture them on (eg Friday before the weekend) then

they should be split at a lower split ratio in order to seed the cells at a high enough density to survive e.g. use 1:2

or 1:5 split.

4. Splitting

1. When the cells are approximately 80% confluent (80% of surface of flask covered by cell monolayer) they should

still be in the log phase of growth and will require sub-culturing. (Do not let cells become over confluent as they will

start to die off and may not be recoverable).

2. To sub-culture, first warm the fresh culture medium at 37oC water bath or incubator for at least 30 minutes. Then

carry out one of the appropriate following procedures:

1 X 252cm flask

Split 1:3

3 X 252 cm flasks

Or

1 X 752 cm flask

Attached cell line split ratios are done on volume of flask surface area:

100 ml cell suspension

Split 1:4

25 ml cell suspension + 75 ml fresh

media in 5 separate new flasks

Or

50ml cell suspension + 150 ml

Fresh media in 2 larger flasks

Suspension cell line split ratios are done on volume of culture cell

suspension:

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3. Make sure flasks are labelled with the cell line, passage number, split ratio, date, operator initials and the vial

number of the cells. Place flask(s) straight into 37oC CO2 incubator. Write down the details of the sub-culturing in

the culture record log sheet. There should be a separate log sheet for each vial of cells resuscitated and in use.

(A) Sub-culturing loosely attached cell lines requiring cell scraping for sub-culture

1. When ready, carefully pour off media from flask of the required cells into waste pot (containing approximately

100ml