Massachusetts
Human Stem
Cell Bank
SOP-RP-005
Preparation of Collagenase Solution
PREPARATION OF COLLAGENASE SOLUTION
OBJECTIVE: Collagenase is one of the enzymes used to remove human Embryonic Stem
Cells (hESCs) grown on Mouse Embryonic Fibroblast (MEF) feeder layers during
passaging. This Standard Operating Procedure (SOP) details how to prepare collagenase
solution at a concentration of 1 mg/ml.
SCOPE: This procedure applies to all Massachusetts Human Stem Cell Bank laboratory
personnel responsible for preparation of collagenase solution.
RESPONSIBILITY: It is the responsibility of the Laboratory Operations Manager and
Quality Assurance Officer to ensure all laboratory personnel are properly trained in and
follow this SOP.
SAFETY: All laboratory personnel should be in compliance with UMASS Employee
Health and Safety regulations when working in the laboratory. Protective equipment,
such as a lab coat and disposable gloves must be worn when working in the lab.
ABBREVIATIONS AND DEFINITIONS
hESCs: Human Embryonic Stem Cells
MEF: Mouse Embryonic Fibroblast
DMEM: Dulbecco’s Modified Eagle Medium
SOP: Standard Operating Procedure
UMASS: University of Massachusetts Medical School
REFERENCES
Not applicable
1. MATERIALS REQUIRED
1.1. EQUIPMENT
• Sterile biosafety cabinet (tissue culture hood)
• Pipette-aid
• Balance
1.2. SUPPLIES
• Weigh boat
• 25 ml sterile serological pipettes
• 150 ml Stericup
• Absorbent paper towels or Kimwipes
• Black extra-fine alcohol proof pen
• 70% ethanol spray
•
Disposable nitrile gloves
(Fisher brand 08-732-113)
(Fisher brand 13-678-36D)
(Millipore SCGPU01RE)
(Fisher 06-666A-C)
(VWR 52877-150)
(Fisher NC 9608803 diluted
from 95% ethanol)
(World Wide Medical
Supplies 71011000-3)
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Human Stem
Cell Bank
1.3. REAGENTS
• DMEM-F12 media
• Collagenase Type IV
SOP-RP-005
Preparation of Collagenase Solution
(Invitrogen 11330-057)
(Invitrogen 17104-019)
2. PREPARATION
2.1. DOCUMENTATION
1. From the binder containing the current Solution/Media Preparation Log
Sheets, take out the Collagenase Solution Preparation Log Sheet.
2. Assign a batch number to the solution to be prepared as: Collag-yy-mm-dd.
For example, for Collagenase solution prepared on 10/01/2008, the batch#
will be: Collag-08-10-01.
3. Estimate how much Collagenase solution will be needed for the next
two weeks.
4. Record the batch number, date, initials, and the volume to be prepared on the
Log Sheet.
5. Take the Log Sheet to the lab for recording of the manufacturer’s lot number
of the collagenase powder.
2.2. PREPARE THE BIOSAFETY CABINET (HOOD)
1. Make sure there are sufficient materials listed below near the hood:
• 25 ml sterile serological pipettes
• Absorbent paper towels (or Kimwipes)
• 70% ethanol spray
• Appropriate-size disposable gloves
• An appropriate-size filter unit and bottle
3. PROCEDURE
3.1. STERILIZATION PREPARATION BEFORE WORKING IN THE HOOD
1. Wash hands and arms thoroughly (about one minute) with soap.
2. Rinse completely with warm tap water.
3. Dry hands and arms with paper towel.
4. Put on appropriate-size gloves.
5. Spray 70% ethanol on the gloves and a paper towel until fully saturated.
6. Thoroughly clean the surface in the hood with the ethanol-sprayed
paper towel.
7. Make sure the following items are in the hood.
• 50 ml sterile centrifuge tubes or other sterile containers
• Black extra-fine alcohol proof pen
• DMEM-F12 medium sufficient for preparation of collagenase solution.
• 150 ml filter and bottle unit.
3.2. PREPARATION OF COLLAGENASE SOLUTION AT 1 MG/ML
Note: The following procedure describes how to prepare 100 ml collagenase
solution at a concentration of 1 mg/ml. If preparing another quantity,
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02/24/2009
Massachusetts
Human Stem
Cell Bank
SOP-RP-005
Preparation of Collagenase Solution
change the amount of collagenase Type IV powder and DMEM-F12
medium proportionately.
1. Remove the container of collagenase Type IV powder from the refrigerator.
2. Record all necessary information (e.g., lot#, expiration date) on the
Collagenase Solution Preparation Log Sheet.
3. On a balance, weigh 100 mg of Collagenase Type IV powder in a weigh boat.
4. Place the weigh boat containing collagenase in the hood.
5. To the 150 ml filter cup, pour 100 ml of room temperature
DMEM-F12 medium.
6. Using a 25 ml pipette, transfer 20 ml DMEM-F12 medium to the weigh boat
containing the collagenase.
7. Pipette DMEM-F12 medium up and down in the boat to dissolve the
collagenase Type IV powder. The collagenase should dissolve
almost instantly.
8. When the collagenase is completely dissolved, transfer the solution to the
150 ml Stericup.
9. Rinse the boat to collect residual collagenase solution and transfer the solution
to the Stericup.
10. Filter the solution and tighten the cap.
11. Record the following on both the cap and the bottle:
• The collagenase batch number as Collag-yy-mm-dd
• Initials
• Expiration date mm/dd/yyyy (14 days after preparation)
12. Place the bottle of collagenase solution in a 4°C refrigerator for storage.
13. Complete the Collagenase Solution Preparation Log Sheet.
14. Return the Log Sheet to the appropriate binder.
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Human Stem
Cell Bank
SOP-RP-005
Preparation of Collagenase Solution
COLLAGENASE SOLUTION PREPARATION LOG SHEET
Note 1: Collagenase Solution Batch# is assigned as: Collag-yy-mm-dd.
Note 2: Expiration date is 14 days from the date of preparation.
Manufacturer
Collagenase Type IV DMEM-F12 Medium
Date
Initials
Lot#
Expiration Date
Lot#
Expiration Date
Collagenase Solution
Vol
Batch#
Expiration Date
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