SOURCES OF CELLS

1). FRESH (isolated by investigator usually)

a). Cells in blood

- RBC
- Leucocytes
- Platelets

Single cells in suspension (i.e. blood) can be isolated by:

i). layering on density gradient medium (see below)
ii). then centrifugation

• cells separate on basis of size/density
  
• may need to repeat process several times but can isolate virtually all cells in blood using these techniques
  

 

b). Isolation of Cells From tissues.

Difficulty depends on cell type: e.g. fibroblasts v. easy

Procedures vary, but an e.g. might be:

1. Chop-up tissue with Scissors or scalpel to ­ surface area.
2. Add one/more enzymes, e.g.: collagenase, pronase, etc.

-break down extracellular matrix (proteins) joining cells together
- to give single cells suspension

3. Separate cells from debris by slow-speed centrifugation

4. Layer cells on density gradient medium & centrifuge

5. Select cells of correct density

6. Culture

NOTE: Fresh isolated cells - eventually die (4 - 8 passages is good!!)

2). FROM CELL BANKS (Commercial)

i. do not die
ii. because they are immortal
iii. have been made from cancers/ tumours (tumour cell lines)

TUMOUR CELL LINES:

• unregulated growth = immortal
• isolated from human/animal tumours
• made by:

  (i). Fuse: tumour cell + normal cell

Tumour line
(ii). Add genes from
· tumour cells
· tumour viruses

ADVANTAGES
· grow continuously (not contact-inhibited)
· homogenous - grow from one cell Þ clones
\ reproducible
· can be stored frozen in liquid (-190° C)
· commercially available (tissue culture banks)
(ETC C / ATTC)

DISADVANTAGES

· can lose differentiated properties
· "foreign" genes generate further genetic changes,
· cells "switch off" desirable genes
· all energy à á growth,

Tumour/immortal cells used for:
· large scale/batch cultures
· Incorporate "genes" for desirable products
- better than yeast or bacteria (E coli.) as carry out unique "post - translational" modifications of proteins:
i.e. glycosylation,
esterification


SOURCES OF CELLS:
a). Public access:
· ECACC: European Collection Of Animal Cell Culture (Portondown, Salisbury)
· ATCC; American Type Culture Collection (Rockville, USA)

b). Commercial sources i.e. Clonetechs
c). Other banks
· Drug/research companies
· Cancer research institutes
d). Other research groups, i.e. cancer research institutions.