The LifeReactor system is comprised of a 1.5 or 5 liter working volume bioreactor together with accessories for maintaining sterility of the system, and purification and humidification of the aerating gas. It is shown diagramatically in Figure 1. 
 

For Kit 800554	For Kit 800555
Click on the image to enlarge

Referring to Figure 1, the reactor vessel (1) is a presterilized, disposable plastic film, generally V shaped, airlift bubble reactor. It contains an inplace, disposable sparger to produce gas bubbles (see Figure 2 for details). The reactor is charged with medium and plant material through a large inoculation port (2) located near the top of the vessel. A multiple use threaded cap (3), containing either 2 or 4 ports, closes and seals the filling port. 

Air is sparged from the disposable sparger (4), which is held in place by a sparger clamp (5) and is connected to an air inlet port (6) in the wall of the vessel. Air is typically supplied to the LifeReactor at 0.8-1.5 vvm (air volume / medium volume/ min.). All connections through which gas or liquid flow to ports, filters or traps are made with flexible, 6mm inner diameter, autoclavable tubing, for example, silicone tubing. 

The compressed air source (7) is connected to a carbon filter (8) to remove any airborne phytotoxic compounds. The outlet of the carbon filter is connected to two 0.2m sterilizing filters (9 & 10), which are connected in series as a safety precaution. 

The outlet of the second sterilizing filter (10) is connected through a one way check valve (11) to a humidifier vessel (12). The humidifier reduces evaporation of medium from the reactor, and the check valve prevents back flow from the humidifier to the filters if the air supply is interrupted. 

The humidifier is connected to a Y connector (13), to allow simultaneous operation of two LifeReactor vessels. One line contains a pinch clamp control valve (14) to balance the air flow through each reactor. A second one way check valve (15) to the air inlet port (6) prevents backflow from the bioreactor to the humidifier. 

Overpressure in the LifeReactor (1) is exhausted through the air outlet (16), which is one of the ports located in the threaded cap (3). The air outlet (16) is connected to a drying unit (17), which is in turn connected to two 0.2m sterilizing filters (18 & 19). These are again connected in series to prevent contaminated air from entering the reactor vessel in the event that the air supply to the sparger is interrupted. The drying unit (17) removes excess moisture from the air exhausted from the reactor vessel to prevent water droplets from reaching the filters (18 & 19). 

The two port model, cap (3) also contains an access port for medium addition (20). The four port model cap contains an additional two ports for whatever special purpose is required (sampling medium, special adds, etc.), which are supplied connected by a silicone tube to maintain a sealed condition. This tube may be opened and used to access the reactor. 

Finally, the reactor vessel (1) contains a mounting sleeve (22) through which a rod is threaded and secured to a frame or stand to support the reactor vessel. 
 

The LifeReactor system Starter kit contains all the components needed to simultaneously operate two, 1.5 liter reactor vessels. Additional components and LifeReactor vessels are available from Osmotek Ltd. 

Starter Kit Components: 
 

LifeReactor vessel, single use (4)	Part Number 800 502
Vessel Dimensions	Base 34mm x top 170mm x length 570mm
Materials	Low density Polyethylene/Nylon Laminate
Sterilization	Supplied presterilized by Gamma radiation, nonautoclavable
Port	56mm screw threaded port, with dummy cap to maintain sterility
Sparger	Porous Ethylene Polyisobutylene
Multiple Use Vessel Components:
Vessel Cap and accessories (2)	Part Numbers: 800 505 & 800 506
Materials	Autoclavable thermoplastic
Port Channels	2 in the cap - Product No. 800 506  4 in the cap - Product No. 800 505
Check valves (3)	Part Number 800 151
Materials of construction	Polypropylene
Stand (1) and clamps (2)	Part Number 1414005
Air pump - 115V   220V	Part Number 1414015   Part Number 1414016
Silicone tubing	Part Number 1412015
Dimensions	9mm OD; 6mm ID
Carbon filter (1)	Part Number 1414025
Carbon filter housing (1)	Part Number 1414030
Sterilizing filters (4)	Part Number 1414020
Dimensions	0.2 micron average pore size hydrophobic 50mm filters.
Humidifier (1)	Part Number 1412025
Control valve (pinch clamp) (2)	Part Number 1412010
Y connector (1)	Part Number 1412030
Air drying unit (2)	Part Number 1412020

1. Location for Operation

1. Determine the location of the operation of the LifeReactors. The air pump (the air supply in this case) is then secured in this location. Next, place the carbon filter in the carbon filter housing, and screw the housing tightly closed. Cut a length of silicone tubing of about 15 cm, and use it to connect the pump to the inlet of the carbon filter housing. (The length of silicone tubing is generally about 15 cm between components, but may be adjusted according to the needs of the location of the operation for LifeReactors).

1. Assembly of the air inlet components

1. Again, cut a length of silicone tubing, and secure one end of this tubing to the inlet of a first sterilizing filter. The length of this section is determined by the distance between the LifeReactor vessel and the carbon filter, since the other end of this section will later be secured to the outlet of the carbon filter.

1. Connect the outlet of the first sterilizing filter to the inlet of the second sterilizing filter by a 10cm length of silicone tubing. Also connect the outlet of the second sterilizing filter, by a 10cm length of silicone tubing, to the inlet of the first check valve (Follow the direction of the arrows on the side of the check valve. The inlet is at the base of the arrow. Note: One of the check valves which comes without a small piece of tubing mounted on it should be used. ) Connect the outlet of the first check valve with a 10 cm length of silicone tubing, to the inlet of the humidifier (the inlet is the longer tube of the humidifier).

1. Fill the humidifier with distilled water and tightly close and seal it with tape that will withstand autoclaving. Now connect the outlet of the humidifier to the Y connector. Mount the balancing pinch clamp on one leg of the Y connector, between the Y and the check valve. Now connect each leg of the Y connector to the inlet of a second check valve (the unit with the small piece of tubing already mounted) by a 20 cm - 50 cm length of silicone tubing.

1. The humidifier is usually placed in a beaker so that it will not tip over until secured for operation.

1. Next, close the outlet of each second check valve with aluminum foil, and set aside the entire assembly of air inlet components for autoclaving.

1. Assembly of the air outlet components

1. Connect one end of a 15 - 50 cm section of silicone tubing to the inlet of the air drying unit. Fill the air drying unit with absorbent cotton, and tightly close and seal the unit with tape which will withstand autoclaving. The second end of the 15 - 50 cm section will later be secured to the air outlet port located in the cap of the LifeReactor.

1. The outlet of the air drying unit is connected to the inlet of a first sterilizing filter by a 15cm length of silicone tubing and the outlet of the filter is connected by a 10cm length of silicone tubing to the inlet of a second sterilizing filter.

1. Preparation of the Medium Addition Port for Medium Exchange (Optional)

1. In the event that medium addition and removal are anticipated during operation, the outlet of a 0.2 micron hydrophilic membrane filter capsule with hose barb connections, such as the Pall Gelman Sciences spiralpack capsule (not supplied with the kit), must be attached with silicone tubing to the medium addition port in the two port cap. A second filter capsule must be autoclaved separately to be attached during assembly to the medium exit port.

1. Autoclaving of the air inlet and air outlet components and other useful Accessories

1. Cover with aluminum foil all of the open ends of the silicone tubing connected to the air inlet and outlet components, and all open ends* of the two or four port threaded cap. (Note: On the 2 port cap the inner ends come closed with rubber plugs.) A pair of scissors should also be autoclaved for cutting the air inlet tube, where necessary. Place all of the autoclaveable components in an autoclaveable container such as a biohazard bag, and autoclave at 121C for 20 minutes.

1. Several accessories are available from Osmotek Ltd. which facilitate the operation of the LifeReactor. These include a reactor holding stand (# 800 524), a funnel and funnel cover (# 800 522), a pouring tube (# 800 521), a receiving vessel (# 800 520), a long handled spoon (# 800 527) and a vessel gripper (# 800 523). At this stage, part of these accessories are autoclaved and aseptically placed on the laminar flow bench prior to use (see above list and Appendix 1).

1. Final SetUp and Inoculation of the LifeReactor (All Steps to be performed in a Laminar Flow Hood)

1. After autoclaving, place all of the multiple use autoclaveable LifeReactor components and accessories on a laminar flow sterile bench to cool. In the laminar flow bench, set up the apparatus for holding the LifeReactor vessel. (The use of the Reactor Holding Stand #800 524 is recommended. The stand provided with the LifeReactor kit may also be used, instead of the Reactor Holding Stand, but it is less convenient to use for inoculation.)

1. Pour about 1 liter of media and plant material that has been aseptically divided into the LifeReactor vessel. Pouring may be facilitated by the use of the funnel accessory (# 800 522). Screw the funnel into the inoculation port prior to addition of media, and cover with the funnel cap when not in use. After the LifeReactor is inoculated with medium and plant material, remove the funnel, and tightly secure the 2 or 4 port cap in place.

1. Now, set up the stand provided with the kit, in the laminar flow bench (if this has not already been done in step 6.1). Secure bar clamps near the top of the stand so that they can hold the support bar. Thread the support bar through the mounting sleeve (22) of the LifeReactor vessel(s), and secure it to the bar clamps. Now connect the air drying unit to the air outlet port of the cap.

1. The air inlet port in the wall of the vessel is received with a closed silicone rubber tube. Cut this tube at a point between the closure and the vessel with the sterile scissors. Remove the aluminum foil on the second check valve for the air inlet components, and connect it to the air inlet port. (At this point the apparatus is secure for exposure to a nonsterile environment.)

1. If medium exchange is anticipated during operation, the extra 0.2 micron filter capsule is now attached to the medium exit port by cutting the tube with the sterile scissors.

1. Set up in Non Sterile Environment for Operation

1. Move the entire fermenter apparatus to the site of the air supply, and connect the inlet of the first sterilizing filter of the air inlet components to the carbon filter. Turn on the air supply, and adjust the airflow to desired levels using the knob on the air pump supplied and the pinch clamp. Together with appropriate lighting, this situation is now maintained for whatever time period is necessary for growth.

1. Operation with Medium Exchange

1. If medium is to be exchanged during operation, care must be taken with regard to pressure buildup in the vessel. In addition, introduction of air into the filter capsule can cause a problem with exit of the medium. It is therefore recommended that the air supply be temporarily shut off during exchange. Vacuum or suction from a pump should be applied to the medium exit port filter, while the entry of fresh medium can be facilitated by a gravity feed attached to the medium addition port filter. (While the new medium should be sterile in order to reduce the microbiological load on the 0.2 micron filter, this operation does not require a sterile environment, because the filter is aseptic from its exit and into the vessel.)

1. Harvesting

1. For harvesting, disconnect the carbon filter from the sterile filters, and return the remainder of the LifeReactor with its stand to the laminar flow sterile bench. Bend shut the tube from the air inlet port, and keep it closed with an elastic band ( in the manner first received). This allows the check valve to be disconnected from the air inlet . Disconnect the air exit port from the air drying unit, and remove the threaded cap.

1. If the Osmotek Accessories Set is being used (See Appendix 1), all components should have first been autoclaved at 121 C for 30 min. in a biobag or other suitable container. Screw the threaded end of the pouring tube (# 800 521) into the inoculation port, and simply invert the reactor vessel with the open end of the pouring tube over the receiving vessel (# 800 520). Pour the entire contents of the reactor vessel into the receiving vessel. The screen element of the receiving vessel retains the plant material and the spent medium is disposed of through the drain valve of the receiving vessel. ( A tube can be attached to the drain valve which can be led out of the sterile area.) If some plant material is retained in the reactor vessel it can be removed by rinsing with sterile water.

1. In lieu of using the Accessories set, two options are available for harvesting.

1. The medium may be drained from the vessel through the medium exit port. The plant material may then be transferred through the inoculation port into a sterile container.

1. Surface sterilize the outside of the LifeReactor vessel. Place a sterile vessel below the LifeReactor vessel, cut the base of the LifeReactor vessel and collect the plant material which empties into the sterile vessel.

1. Close the receiving vessel with its cover, until the plant material is required.

1. Separation of Plant Material

1. Remove the plant material from the receiving vessel with the long handled spoon (# 500 527), and transfer it to a vessel for hand separation, or place the material directly on the Osmotek VitroCut random mechanical separator. (The most efficient method of separating culture is to use the Osmotek VitroCut random mechanical cutter, which can perform this function at a rate X5 faster than hand separation.)

1. The VitroCut cuts the material into a round tissue culture vessel which can be picked up by the vessel gripper (# 500 523) for transfer. The plant material may then be used for elongation or further multiplication.