Methods and procedures:

• Embryos (reproductive entities), stem (primitive, undifferentiated) cells and somatic cells (body cells like liver, pancreas, lung, kidney, etc) will be collected from super-ovulated (hormone injected) sheep ewes and laboratory mice by sterile surgical procedures.  The ewes will be under full general anesthesia during the embryo and cell collection procedures, and the mice will be sacrificed before collection.  All embryos and cells will be thoroughly washed through five steps in culture medium (see appendix 1).  

• The stem and somatic cells will be collected about one month before the commencement of the space flight, to ensure that they are healthy and able to grow and multiply under in vitro (incubator) conditions.  Just before transfer to the portable incubator (one to two days before the launch), the cells will be harvested with a trypsin enzyme, which loosens them from the bottom of the dish.  After a series of washes they will be transferred to the culture tubes in the incubator.  After transfer half of the volume of the culture medium (see appendix 1) has to be changed every second day, as the developing cells and embryos utilize the nutrients in the culture medium, which has to be replaced.  A second reason for replacing the medium is to dilute out the metabolic waste products produced by the growing cells and embryos.

• The 1-cell to 2-cell stage of the embryos will be collected as shortly as possible before take-off, but not more than 36 hours.  The developmental rate of the embryos will be slowed down until launching occurs.  Attempts will be made to do the embryo collections in Russia (if at all possible), to have the earliest possible developmental stage of the embryos before launching.  The reason for this is that ovine (sheep) embryos will hatch in vitro from its surrounding capsule (the zona pellucida) within seven to eight days after ovulation (shedding of the female reproductive eggs), which corresponds to six to seven days after collection.  Collection is between 16 to 24 hours after ovulation.  

• Mouse embryos on the other hand hatch even faster, between four to five days after ovulation (i.e. 3,5 to 4.3 days after collection). Not much further development occurs in vitro (in the incubator) after hatching.  We will therefore try to take development of the embryos through all of its stages during the flight, with hatching not occurring too long before the return of the flight.  If the embryos die off during the flight due to weightlessness, they will arrest at that stage and the timing can be correlated with the time after ovulation.

• Fifty embryos from mouse and sheep origin will be transferred after washing to two incubator tubes each.  One million stem cells and one million somatic cells will also be transferred after trypzinising and washing to two incubator tubes each.

• The incubator (see appendix 1) with dimensions 270x210x260 mm, and weighing not more than 3 kg, will therefore contain twelve plastic culture tubes of 5 ml culture medium each, as well as three tubes of 50 ml reservoir culture medium each.  An empty 150 ml tube will serve as waist receptacle.

• Inside the incubator will also be a rechargeable battery pack that will keep the incubator at 38.5 degrees Centigrade (body temperature) for a period of up to 48 hours.  The incubator can run on 220V AC or 12V DC.

• The incubator with its contents of 12 tubes, with two tubes each with different types of embryos or cells in a growth medium and tightly sealed, will be delivered to the launch site the day before launching.  The battery pack will keep the incubator and its contents at body temperature for the launching period and up to docking to the ISS.

• On arrival at the ISS the incubator must be plugged into a 12V DC power supply for the duration of stay at the ISS.

• On day 1, 3 5 and 7 after launching, half the culture medium of each sealed tube must be replaced with new culture medium.  An injection needle, fitted to a sterile syringe, is pushed through each of the rubber stoppers of the 12 tubes inside the incubator (each containing 5 ml of medium and cells/embryos), and 2.5 ml of the contents of each tube drawn off.  A special filter (of 1.5 micron pore size) halfway inside the tube will prevent any cells or embryos from entering the syringe when half of the medium is drawn off.  This used medium is then injected through the rubber stopper of another empty 150 ml test tube used for storage of the waste.  Another needle and syringe of 30 ml is then used to draw off 30 ml of the 150 ml reservoir tube of replacement culture medium, and 2.5 ml is injected into each of the 12 tubes inside the incubator.  During this whole procedure no medium or cells/embryos will be leaked into the atmosphere of the ISS, as all of the procedures are done with sealed apparatus.

• At the end of the space flight and one to two hours prior to departure from the ISS, the 12 tubes must be removed from the incubator and placed in an insulated polystyrene container (of about 10x10x10 cm) for the 8-hour flight back to earth.

• On arrival at earth, the 12 tubes with their contents will be transferred to a second incubator, and then immediately flown to our laboratory at the university of Stellenbosch.

• Evaluation of embryo and cellular development and growth will be done in our laboratory in Stellenbosch on arrival.

• If there is still any doubt or uncertainty about the highly improbable wastage of medium or cells into the atmosphere of the ISS during any of the procedures on board the ISS, the incubator can be put into a transparent plastic bag, and all of the procedures done from the outside, through the wall of this plastic bag.  Alternatively the incubator can be placed in a hermetic glove box if available on board the ISS.

Assignment and principles of the equipment:

The portable incubator will create a suitable and sterile environment for the embryos and cells to develop inside the tubes for the duration of the space mission.

Performance of the equipment:

The total weight of all the equipment will not exceed 3 kg, and the total dimensions not exceed 15,000 cm3 (0.015m2).

Work of the crew with the equipment:

• Connecting the portable incubator to a 12V DC onboard socket in Soyuz TM-33 (if available) and the ISS.

• Changing half the volume of culture/growth medium of each tube every second day after launching (see methods and procedures).