Isolation and Differentiation of Primary Adipose SVF cells

Gupta Lab version: 05/07/13

Reagents

1. SVF medium:

   10% FBS in high glucose DMEM/F12 media (1:1 Invitrogen) + Glutamax + Pen/Strep + Gentamicin
   To 500 mL bottle of DMEM/F12 add:
   55 mL of filtered FBS (Lot tested to support differentiation)
   5 mL of Pen/Strep
   0.5 mL of Gentamicin (optional)

2. 100 um and 40 um cell strainer

3. Digestion buffer:

   Stock Buffer:

   Stock concentration	For 500 mL	Final Concentration
   1 M HEPES	50 mL	100 mM HEPES pH 7.4
   5 M NaCl	12 mL	120 mM NaCl
   3 M KCL	8.3 mL	50 mM KCL
   100 mM Glucose	25 mL	5 mM Glucose
   2.5 M CaCl2	0.2 mL	1mM CaCl2
   ddH20	404.5 mL

For 1x Working Digestion Buffer

For 10 mL:
10 mL stock buffer
10 mg Collagenase D (Roche) (1 mg/mL= final concentration)
0.15 g of BSA (1.5%=final concentration)

SVF isolation

Procedure

1. Sacrifice mouse and carefully dissect inguinal WAT. Immediately place into sterile 1x PBS.
2. Combine 4 fat pads total (i.e. from 2 mice) into 10 mL beaker. Mince tissue with spring scissors until tissue is homogeneous.
3. Transfer tissue (pour from beaker) to 50 mL falcon tube containing 10 mL of “1x digestion buffer.” Place tube into 37 degrees shaking water bath. Shake for 2 hrs until tissue is digested. Vortex tube gently every 30 minutes.
4. Pipette digest up and down with 10 mL pipette and pass through 100 um cell strainer into fresh 50 mL falcon tube. Add 30 mL SVF media to new tube to dilute digestion buffer. Spin 600 x g for 5 minutes.
5. Remove floating adipocyte fraction. (Discard or save for analysis)
6. Resuspend SVF cells in 10 mL SVF media and pass through 40 um cell strainer into new 50 mL falcon tube. Add 30 mL SVF media to new tube to wash cells. Spin 600 x g for 5 minutes.
7. Aspirate media. Resuspend pellet in 5 mL SVF media and plate cells onto 6-cm (60 mm) collagen-coated plate. Next day, gently change media. Split cells into 1 x 10-cm plate when cells reach 80-90% confluence. For 1st passage you will need 0.25% trypsin to remove cells from 6-cm plate. Maintain subsequent passages as described below.

Growth and Maintenance

Grow cells at 10% CO2 in high glucose DMEM/F12 media (1:1 Invitrogen) + Glutamax containing 10% FBS + Pen/Strep. Split 1:2 or 1:3 every 2-3 days. These cells like to remain in contact with one another (i.e. ~70-80% confluent at all times). To do not passage cells for more than ~7-8 times or cells may begin losing differentiation capacity. Maintain many frozen vials.

Freezing cells

Freeze cells in 90% serum and 10% DMSO.

Differentiation

When cells are confluent (really packed, growth arrested) add the following to induce differentiation:

Day0:
10% FBS in high glucose DMEM/F12 media (1:1 Invitrogen) + Glutamax + Pen/Strep
5 ug/ml insulin
1uM Dexamethasone
0.5 mM isobutylmethylxanthine (IBMX)

Note: Can add 1 uM Rosiglitazone to enhance differentiation

Day2 (48hrs after adding induction cocktail):
Change media to 10% FBS + 5ug/ml insulin
Do not rinse cells before adding maintenance media.
Pipette carefully, as cells may come off.

Days 4,6,8,10: Change the media to fresh 10% FBA+ 5ug/ml insulin
SV cells will be mostly adipocytes by Day6.

Stock solutions

Insulin: 5 mg/ml = 1000X

1. Make 1M HCl: 2.5ml 12M HCl + 27.5ml ddH2O
2. Make 50mM HCl: 2ml 1M HCl+ 38ml ddH2O
3. Dissolve 100mg of insulin (Sigma I6634) in 20ml of 50mM HCl.

IBMX: 250mM = 500X

Dissolve 832.5 mg of IBMX (Sigma I7018)
in 15ml of DMSO.

Dexamethasone: 1mM = 1000X

Dissolve 5.88mg of dexamethasone (Sigma D4902) in 15ml EtOH.