GM501 OvarStore Cryo^TM

• Ethylenglykol, DMSO, DSS, Gentamicin

GM501 OvarStore Thawing^TM

• Sucrose, HSA, Gentamicin

GM501 OvarStore Dissection^TM

• Leibowitz L-15, HSA, Gentamicin

GM501 OvarStore Transport^TM

• Hypo Thermosol

GM501 Cult with Gentamicin

• NaCl, KCl, KH₂PO₄, MgSO₄^.7H₂O, NaHCO₃, CaCl₂^.2H₂O, D(+)-Glucose anhydrous, Na-Lactate (50 % solution), Na-Pyruvate, EDTA, Na-Alanyl-Glutamine, water, non-essential and essential Amino Acids, 1 % human serum albumin, Gentamicin (10.00 mg/liter).

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.
• GM501 OvarStore is manufactured and tested according to the following specifications:

GM501 OvarStore Cryo^TM

GM501 OvarStore Thawing^TM

GM501 OvarStore Transport^TM & GM501 OvarStore Dissection^TM

GM501 Cult with Gentamicin

Principle of the method:

• It is to be applied for patients who want to make sure that in spite of an upcoming chemotherapy/radiotherapy measure, they will be able to fulfill their desire to have children as well as for therapy independently from baby-planes. For these two reasons, they have ovarian tissue extracted prior to their therapy. The tissue is treated using the media and can be returned into the patient´s body at a later date without overloading this tissue by the therapy.

Instructions for use:

• The intended users are IVF professionals (lab technicians, embryologists or medical doctors).

In general: The distance between the OP and the biological laboratory decides between the application of GM501 OvarStore Transport or GM501 OvarStore Dissection.

• Option A: If the transport time lies between 25 minutes and maximum 26 hours, the tissue should be handled in GM501 OvarStore Transport. The ideal transport temperature for GM501 OvarStore Transport is 2°C to 8°C.
• Option B: If the transport time lies below 25 minutes, it is recommended to handle the tissue in GM501 OvarStore Dissection. The ideal transport temperature for GM501 OvarStore Dissection is between room temperature and 37°C.

Preparation for extraction of ovarian tissue:
The extraction of ovarian tissue is possible in any phase of the cycle. The attending doctor decides on the preparations to be done for the extraction.

1 to 2 days before extraction of ovarian tissue:

• Procure and store 25-50 ml GM501 OvarStore Transport at 2-8°C in the operation area.

1 day before extraction of ovarian tissue:

• Transport time “Option A” (see In general) - procure 25-50 ml     GM501 OvarStore Transport in a fridge at 2-8°C.
• Transport time “Option B” (see In general) - procure 25-50 ml    GM501 OvarStore Dissec­­tion in a fridge at 2-8°C.

Day of ovarian tissue extraction:

• Take procured GM501 OvarStore Dissection or Transport out of the refrigerator and let them warm to room temperature (maximum 37°C). Prepare under a sterile LAF-bench with 3 sterile petri-dishes (100 mm), 1 sterile scalpel, 1 sterile urine sample container, 2 sterile forceps and sterile gloves. Open Cryo-packing and mark the single components (tissue straws, high security straws, cryo tubes) with the details of the patient and procure on the LAF-bench.

Ovarian tissue extraction:
The extraction of ovarian tissue is to be solely effected by a qualified specialist.

• Put extracted tissue in a prepared 50 ml tube with medium (“Option A”: 25-50 ml GM501 OvarStore Transport; “Option B”: 25-50 ml GM501 OvarStore Dissection).
• Transport of tissue tubes to biological laboratory.

Preparation of strips and freezing:

• Dissection of the ovary medulla (stroma) from the cortical part of the ovarian tissue using scalpel and forceps.
• Preparation of strips with supposition “one strip for one tube”.
• Induce saturation of the ovarian tissue with cryoprotectants over 30 minutes until 1 hour at room temperature in the sterile urine sample container (with 30 ml of OvarStore Cryo).
• Fill the prepared cryo-packing (tissue straws, high security straws or cryo tubes) with GM501 OvarStore Cryo using a syringe with long needle.
• After equilibration, transfer the tissue strips into the cryo-packing using sterile tweezers, close these cryo-packing and place it 10 min into icewater, then position cryo packing in the rack of the freezer and freeze as described above:

Stage 1: From room temperature to 0°C, at a rate of -2°C/min.

Stage 2: From 0°C to -7°C, at a rate of -1°C/min.

Seeding (manual or automatic): At -7°C with subsequent 10 min holding at 7°C.

Stage 3: From -7°C to -35°C, at a rate of -0.3°C/min.

Stage 4: After achieving of 35°C, the cryo-packing with ovarian tissue immerse into fluid nitrogen and locate it for long-term storage in cryobank.

Thawing

Step 1: there are 2 possibilities:

• Method 1: Leave the Cryo-Straw for 10 seconds at room temperature and give them afterwards into boiling water (100°C) for another 20 seconds.
• Method 2: Leave 1.8 ml Cryo-Vials for 30 seconds at room temperature and afterwards add them into boiling water (100°C) for another 60 seconds.

The thawing procedure has to finish quickly, that´s the reason we suggest to have the temperature definitely at 100°C and keep it with a magnetic stirrer in the flow.

Step 2:

• The thawing of the tissue in the water is visually inspected for the presence of ice as soon as the existing ice is only about 1 mm thick, take the tissue out of the water. The final temperature of the frozen liquid is after the removal of the Cryo-packing round about at 4°C and 10°C.

Step 3:

• After thawing up place the ovarian tissues quickly (5-7 sec) into a 100 ml sample-container with 10- 12 ml OvarStore Thaw. The sample-container has to be in a rotation shaker (200 oscillations/min for 15 min at room temperature).

Step 4:

• The rehydration of the ovarian tissues takes place if you steadily add with the pipette 50 ml OvarStore Transport Medium during a time of 30 min at room temperature. It is helpful to use a perfusion unit or an infusion set for a steadily release of the OvarStore Transport Medium.
• The final saccharose concentration should have 0,08–0,09 M like an isotonic solution.

Step 5:

• Wash the ovarian tissues three times in OvarStore Transport medium for 10 min.

Step 6:

• Put the ovarian tissue for the in-vitro culture in culture medium or in OvarStore-Transport Medium for the following transplantation.

Precautions and warnings:

• GM501 OvarStore contains the antibiotic Genta­micin ­Sulfate. Appropriate precautions should be taken to ensure that the patient is not sensitized to this antibiotic.
• Standard measures to prevent infections resulting from the use of medicinal products prepared from human blood or plasma include selection of donors, screening of individual donations and plasma pools for specific markers of infection and the inclusion of effective manufacturing steps for the inactivation/removal of viruses. Despite this, when medicinal products prepared from human blood or plasma are administered, the possibility of transmitting infective agents cannot totally be excluded. This also applies to unknown or emerging viruses and other pathogens. There are no reports of proven virus transmissions with albumin, manufactured to European Pharmacopoeia specifications by established processes.
• Therefore, handle all specimens as if capable of transmitting HIV or hepatitis. Always wear protective clothing when handling specimens.
• Always work under strict hygienic conditions (ISO 5 enviroment, e.g. LAF-bench) to avoid possible contamination, even when GM501 OvarStore medium contains Gentamicin.
• Only for the intended use.

Pre-use checks:

• Do not use the product if bottle, seal of the container or package is opened or defect when the product is delivered.
• Do not use the product if it becomes discoloured, cloudy or shows any evidence of microbial contamination.

Storage instructions and stability:

• Shelf life from GM501 Cult with Gentamicin is 6 months from time of manufacture.
• Shelf life from GM501 OvarStore CryoTM, GM501 OvarStore ThawTM, GM501 OvarStore DissectionTM and GM501 OvarStore TransportTM is 9 month from time of manufacture.
• Store between 2-8°C.
• Do not freeze before use.
• Keep away from (sun) light.
• After opening the container do not use the product longer than 7 days.
• Do not use after expiration date.

Rev01_00 Stand: 05.02.2013