Rat Tail Collagen Extraction (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Jeffrey Pippin; Stuart Shankland; Racheal Jappert; Ying Zheng

Keywords

[‘Collagen isolation’]

Subjects

[‘Cell culture’, ‘Tissue culture’]

Release Date

2017-05-26

Abstract

Describes an inexpensive method for obtaining rat tail collagen used for casting three-dimensional microvascular culture systems.

Reagents

Rat Tails (Rockland RT-T297, 1 Pack of 50 = $179.00)

Equipment

Tools:

(All tools should be steam sterilized)

Equipment:

• Biosafety cabinet
• Magnetic stirrer
• -80°C Freezer
• Lyophilizer

Procedure

Procedure

1) Perform subsequent procedures in biosafety cabinet. 2) Thaw frozen rat tails (10) in 70% ethanol. 3) Cut off thick end of tail. 4) Slowly cut through the skin longitudinally from thick tip to thin tip. Take extreme care not to cut yourself!!! 5) Cut off the thin tip of tail. 6) Peel off skin using hemostatic forceps and submerge in 70% ethanol. Do Not Allow Tail to Air Dry 7) Starting at the thin tip, grab above the first joint using hemostat and break joint. 8) Pull out and string of collagen will be attached. Using scissors, cut collagen from joint into beaker of 70% ethanol. 9) Repeat at each joint going down the length of the tail until all of the collagen strands have been obtained. 10) Wash collagen in new beaker of 70% ethanol at least 3x. 11) Place collagen in pre-weighed 50mL conical tube, drain off as much liquid as possible, and obtain total weight of collagen. 12) Transfer collagen strands into sterile glass bottle. 13) Add 0.1% acetic acid (1mL acetic acid/gram collagen). 14) Dissolve collagen by placing on magnetic stir plate and stirring continuously for 3-4 days at 4°C. 15) Place dissolved collagen into sterile 50ml conical tubes (JA-20 rotor holds 8 tubes). 16) Centrifuge for 90 minutes at 4 degrees at 9800G (9000 rpm in JA-20 rotor). 17) In culture hood, pour off supernatant into sterile bottle. 18) Add about 30ml 0.1% acetic acid (made in sterile water) to the pellet. 19) Re-suspend pellet and combine with supernatant in step 14. 20) Leave for couple days at 4 degrees for additional extraction. 21) Repeat steps 12-14. 22) Place 30ml of extracted collagen into separate 30ml conical tubes and freeze at -80°C overnight. 23) Loosen caps and place on lyophilizer for 3-4 days (until dry). 24) Store at -80°C until needed for 3D MPS assembly.

Consortium

(Re)Building a Kidney (RBK) Consortium