TITLE: Human Adipose Dissociation and Cell Culture -- University of Minnesota TMCs
AUTHORS: Elizabeth Thompson, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Purpose: To dissociate human adipose to obtain adipose progenitors and endothelial cells for cell culture experiments. 
  

Adapted from Miltenyi Biotec - Adipose Tissue Dissociation Kit (attached). 
  


[STEPS]
SECTION: I. Materials
1. Adipose Tissue Dissociation Kit mouse and rat (Miltenyi Biotec; 130-105-808)
Kit Components: 5 vials containing:
2 vials of Enzyme D (lyophilized powder)
1 vial of Enzyme R (lyophilized powder)
1 vial of Enzyme A (lyophilized powder)
1 mL of Buffer A
SECTION: I. Materials
1.1. Fibroblast Media (made ahead of time)
500mL MEMα (Gibco; 32561037)
10µL EGF 500 µL/mL (R&D Systems; 263-EG-200 = recon in 400 µL PBS, aliquot and store at -80C )
2.5 µL FGFbasic 50 µg/mL (R&D Systems; 233-FB-025 = recon in 500 µL PBS, aliquot and store at -80C)
50 mL FBS
5 mL MEM Non-essential amino acids (NEAA) (Gibco;11140050)
5 mL Pen/Strep (Gibco;15140122)
SECTION: II. Reagent Preparation
2. Prepare Enzyme D by reconstitution of the lyophilized powder in each vial with 3 mL of DMEM and sterile filter. Prepare 0.5 mL aliquots to avoid repeated freeze-thaw-cycles. Store aliquots at –20 °C. This solution is stable for 6 months after reconstitution.
SECTION: II. Reagent Preparation
3. Prepare Enzyme R by reconstitution of the lyophilized powder in the vial with 2.7 mL of DMEM. Prepare 250 µL aliquots to avoid repeated freeze-thaw-cycles. Store aliquots at –20 °C. This solution is stable for 6 months after reconstitution.
SECTION: II. Reagent Preparation
4. Prepare Enzyme A by reconstitution of the lyophilized powder in the vial with 1 mL of Buffer A supplied with the kit. Do not vortex. Prepare 50 µL aliquots to avoid repeated freeze-thaw-cycles. Store aliquots at –20 °C. This solution is stable for 6 months after reconstitution.
SECTION: III. Adipose Tissue Dissociation Protocol
5. Prepare Enzyme Mix with prepared enzymes from step 1. 2-4 samples are typically processed at a
time and enzyme mix should be prepared as master mix for all the samples.
Enzyme Mix:
 


SECTION: III. Adipose Tissue Dissociation Protocol
6. Aliquot 2.5 mL of Enzyme mix into gentleMACS C Tube for each sample and label tube.
SECTION: III. Adipose Tissue Dissociation Protocol
7. Transfer 0.5g of adipose tissue into the gentleMACS C Tube containing the enzyme mix. Use a small sterile scissors to cut tissue into small pieces while in the C Tube. Tightly close tube beyond the first resistance.
SECTION: III. Adipose Tissue Dissociation Protocol
8. Use the heating function of the gentleMACS Octo Dissociator with Heaters run program 37C_mr_ATDK_1.
SECTION: III. Adipose Tissue Dissociation Protocol
9. After termination of the program, detach C Tube from the gentleMACS Dissociator.
SECTION: III. Adipose Tissue Dissociation Protocol
10. (Optional) Perform a short centrifugation step up to 300×g to collect the sample material at the tube bottom and resuspend cells.
SECTION: III. Adipose Tissue Dissociation Protocol
11. Label a 15 mL tube for each sample. Place a MACS SmartStrainer (100 µm) on each 15 mL tube.
SECTION: III. Adipose Tissue Dissociation Protocol
12. Pour the cell suspension through the MACS SmartStrainer (100 µm) into the labeled 15 mL tube.
SECTION: III. Adipose Tissue Dissociation Protocol
13. Rinse the C tube and wash the MACS SmartStrainer with 5–10 mL of DMEM for each sample.
SECTION: III. Adipose Tissue Dissociation Protocol
14. Discard the MACS SmartStrainer and centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
SECTION: III. Adipose Tissue Dissociation Protocol
15. Gently resuspend cell pellet with 1 mL ACK Lysis Solution (Gibco A10492-01) to lyse red blood cells. Incubate samples at room temperature for 3 minutes and add 3 mL DMEM to each sample.
SECTION: III. Adipose Tissue Dissociation Protocol
16. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant leaving ~0.5mL.
SECTION: III. Adipose Tissue Dissociation Protocol
17. Resuspend cell pellet and transfer to labeled 10cm plate with 10 mL Fibroblast media for each sample.
SECTION: IV. Cell Culture
18. Place 10 cm plates in 37C/5% CO2 incubator.
SECTION: IV. Cell Culture
19. Change media on the cells after 18-24 hours and change the media every 3-4 days until confluent using Fibroblast media.
SECTION: IV. Cell Culture
20. Harvest cells by removing media and adding 2 mL TrypLE to 10 cm plate. Incubate at room temperature until cells begin to detach.
SECTION: IV. Cell Culture
21. Resuspend cells and transfer to 15 mL tube with 2 mL Fibroblast media.
SECTION: IV. Cell Culture
22. Centrifuge cells at 1500 rpm for 3 minutes. Remove supernate and resuspend cells in residual volume.
SECTION: IV. Cell Culture
23. Transfer 1/10 of cells to new 10 cm plate with 10 mL Fibroblast media to continue to growing.
SECTION: IV. Cell Culture
24. Freeze down the remainder of the cells in fibroblast freezing media (50% FBS/40% Fibroblast media/10% DMSO) in 1 mL aliquots (2-3 aliquots/10cm plate)