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Contents

Making Primary Cells from Surgery Tissue

Materials

To help you make some decisions about what might be suitable this is what we did to make primary cells from chunks of a human ADPKD kidney that I estimated as weighing ~0.5g each:


Grow Cells on Collagen Coated Surfaces

Refer to (Collagen Coating of Cell Culture Vessels) for protocol.

Cell Culture Media Tissue Collected in (no additives)

DMEM/F-12 (1:1)

Cell Culture Media Cells Grown in (with additives)

Reagent 50mL 500mL Final Concentration
DMEM/F-12 (1:1) 46.85mL
insulin-transferrin-selenium (100x) 375uL3.75mL0.75x
dexamethasone (20ug/mL) 250uL2.5mL100ng/mL
triiodotyronine (20ug/mL) 15uL150uL6ng/uL
murine EGF (0.1mg/mL) 5uL50uL10ng/mL
pen/strep 0.5mL5mL
FCS 2.5mL25mL5%


Insulin-Transferrin-Selenium (100x)

Insulin-transferrin-selenium, 10mL, Invitrogen #41400-045
Store at 4 degrees C
Ready to use


Dexamethasone (20ug/mL)

Dexamethasone, 1mg, Sigma #D8893
Lyophilised reagent stored at 4 degrees C

Triiodotyronine (20ug/mL)

Triiodotyronine, 1mg, Sigma #T5516

Murine Epidermal Growth Factor

mEGF, 0.1mg, Sigma #E4127


Collagenase Type II Solution

Work out volume of collagenase type II solution required (depends on how many tissue samples and sizes you'll be harvesting)
Here's some examples below of tissue that primary cells have been successfully made from.
Any excess collagenase solution can be aliquoted and stored at -20 (avoid freeze/thawing more than 2x)

Tissue Sample Collagenase Type II Solution Average Weight of Tissue Sample Volume Collagenase/Sample Amount Collagenase/Sample
HsADPKD kidney chunks 18.7.05 1mg/mLestimate 0.5g15mL30mg/g tissue
Mouse Embryonic Kidneys example 1 1mg/mL~5mg each0.2mL40mg/g tissue
Mouse Embryonic Kidneys example 2 2mg/mL~5mg each0.1mL40mg/g tissue



Method