GM501 Collagenase

• NaCl, KCl, KH₂PO₄, MgSO₄^.7H₂O, NaHCO₃, CaCl₂^.2H₂O, D(+)-Glucose anhydrous, Na-Lactate (50 % solution), Na-Pyruvate, EDTA, Alanyl-Glutamine, water, non-essential and essential Amino Acids, HEPES, Phenolred
• It has an activity of 1000 CDU/ml (Collagen Digestion Units). The Collagenase is obtained from culture filtrates of Clostridium histolyticum.

Product specifications and quality control:

• All raw materials are of highest available purity (European Pharmacopoeia and/or USP standard) if applicable.
• A certificate of analysis is available for each batch upon request from our website with respective lot number.
• The MSDS for GM501 Collagenase is available upon request and can also be downloaded from our website.
• GM501 Collagenase is manufactured and tested according to the following specifications:

pH (at 37°C)                                 7.20-7.70

Osmolality (mOsm/kg)              270-310

Sterility                                         sterile – SAL^10-3 (Sterility Assurance Level)

Sperm Survival test (%)             ≥ 80 survival after exposure of density selected spermatozoa to the test medium

NOTE:
Performing LAL-endotoxin- and MEA-tests is not possible with these media as the activity of the Collagenase inactivates the enzymes (LAL) and damages the mouse embryos (MEA), which are used for these assays, respectively.

Instructions for use:

1. Extract 1.5 ml GM501 Collagenase out of original bottle and fill into a 5 ml round-bottom tube.
2. Warm the GM501 Collagenase at 37°C. GM501 Collagenase is HEPES-buffered. Incubation in a CO2-Incubator will lower the pH.
3. For digestion of testicular tissue carefully pick up the chosen tissue pieces with a fine syringe cannula. For easier handling, if necessary, fill the tissue suspension into a 60 mm petri dish. Let adhesive transport or cryo medium drop off as much as possible and transfer into Collagenase tubes.
4. Close the tube completely. Now put the tube for 60 minutes in the incubator or ideally in a heat cabinet for digestion of the tissue. Slight agitation every 20-30 min will facilitate the formation of a single cell suspension.
5. Suspend the digested tissue by carefully pipetting up and down. Under ideal conditions a suspension of single testicular tissue cells and free semen cells has been formed. If coarse tissue pieces are still visible, repeat step 4 for a further 20 to 30 minutes.
6. Now centrifuge the tissue cell suspension and wash twice with 1–2 ml HEPES-buffered sperm processing medium (e. g. GM501 SpermAir). Discard the obtained supernatant. Alternatevily, the cell suspension can be processed using a density gradient system (e. g. GM501 Gradient).
7. After the last centrifugation re-suspend the pellet in a small volume of 30–80 µl HEPES-buffered sperm processing medium. Add a few µl of this suspersion into a dish.
8. Control by microscope whether viable semen cells are present.
9. Continue IVF treatment according to internal standard procedures.

Precautions and warnings

• GM501 Collagenase does not contain any antibiotics.
• The long term safety of ICSI including the use of Collagenase, on children following this procedure is still under investigation.
• Always wear protective clothing when handling specimens.
• Always work under strict hygienic conditions (ISO 5 environment, e.g. LAF-bench) to avoid possible contamination.
• Only for the intended use.
• The user facility of this device is responsible for maintaining traceability of the product and must comply with national regulations regarding traceability, where applicable.

Storage instructions and stability

• The shelf life is 9 month from time of manufacture.
• Store between 2-8°C.
• Do not freeze before use.
• Keep away from (sun) light.
• The product can be used safely up to 7 days after opening, when sterile conditions are maintained and the products are stored at 2-8°C.
• Stable after transport (max. 5 days) at elevated temperature (≤ 37°C).
• Content cannot be re-sterilized after opening.
• Do not use after expiry date.