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LifeReactor TM - The Presterilized,
Disposable Bioreactor
General Background:
The LifeReactorTM system is a simple, inexpensive, disposable,
airlift bioreactor, which is the result of pioneering collaborative work by Professor
Meira Ziv, of the Hebrew University Faculty of Agriculture, and Dr. Robert Levin,
of Osmotek Ltd. It is used for multiplication in liquid medium of organogenic culture
such as nodules, meristem clumps or compact shoot clusters, somatic embryos and cells,
and for growth of bulbs, corms or microtubers.
The following is a short list of just a few of the crops which have been successfully
cultured:
-
Banana
-
Pineapple
-
Potato microtubers
-
Anthurium
-
Boston Fern
-
Orchid
-
Pine
The Literature section
of this website contains a number of formally published references to plants cultured
in bioreactors, as well as application
notes from Osmotek.
Bringing the Advantages of Large Scale Liquid Culture to the Small
Lab:
Producing up to 1000 plantlets per liter of liquid medium, this
easy to use system allows research and small commercial laboratories to carry out
multiplication on a relatively large scale, in less than a square meter of space,
with minimal manpower and at an easily affordable price. Yet they are capable of performing
in the same manner as glass vessel bioreactors costing tens of thousands of dollars.
The 1.5 liter vessel
[800 502] is
ideally suited for:
-
Research facilities which require large numbers of cloned plantlets for field trials.
-
Pilot experimentation preparatory to commercial scale multiplication in bioreactors.
-
Pilot Scale Secondary Metabolite production.
Restoring the Profitability of Commercial Micropropagation:
The 5 liter vessel
[800 501] is
meant for large pilot and commercial propagation. Each vessel can multiply between
5-6000 plantlets in a 3 – 4 week cycle. Using vertical lighting, and hanging the vessels
in two tiers, 12 vessels can be arranged in less than a 1 square meter space, a growing
density of 60,000 plants per sq.
meter of growth room space using a mobile culture rack [800
525].
Once the reactor is filled and underway, the plants do not require any attention other
than assuring that their source of sterile humidified air and their light source are
operational. Since the vessel contents are isolated from the surroundings by 0.2 micron,
microporous filters, there is no threat of contamination from the growth room.
This means that expenditures such as cleaning and spraying growth rooms can be reduced
to an absolute minimum. [For additional economic analysis, see Commercial
Advantages of the Lifeline System].
Description:
Figure 2 Click To Enlarge
The LifeReactorTM vessel is shown in [Figure
2]. The body (1) is a V-shaped bag fabricated from a
special, heavy duty plastic laminate material. At the bottom of the vessel is a porous
bubbler (4), which is connected to an inlet in the wall. During operation, sterile,
humidified air is supplied through this port.
Near the top of the vessel is a 1.5” diameter innoculation port (2), through which
the plant material is initially added and later withdrawn. In a new, unused LifeReactorTM ,
this port is sealed with a plug which is removed in a sterile area, and replaced by
either a two or four port, reusable and autoclavable cap [800
505]. One port on the cap is used to exhaust excess air,
while the other port(s), which are covered by silicone rubber septums, can be used
to make additions, in aseptic fashion.
At the bottom there is a port for withdrawing medium (21), which has a fine screen
over its inside entrance. The vessel can thus be emptied without allowing plant material
out of the reactor. Finally, to allow the LifeReactorTM to be hung on a
rod in a vertical operating position, there is a mounting sleeve at the very top of
the vessel (22), through which a rod can be slid.
Sterility and Cleanliness:
Each LifeReactorTM vessel is packaged in a separate plastic ziplock
bag and subjected to gamma sterilization. This assures that all parts of the vessel,
both interior and exterior, are completely sterile when the bag is opened in the laminar
flow hood.
The LifeReactor Kit
[800 554]:
The LifeReactorTM kit contains four 800
502 vessels, and is meant to provide all the components (except for lighting)
necessary to operate two of them simultaneously. For the air source, it includes a
carbon filter for removal of airborne toxic compounds, an air pump with plenty of
autoclavable silicone tubing, a one liter humidifier bottle to reduce evaporation
of the medium from the reactor, 0.2 micron sterilizing filters, one way check valves,
and plastic Y connectors. It also includes a portable stand from which to hang the
bioreactors.
The LifeReactorTM Accessories Kit
[800 528]:
Contamination Control– the key to success with liquid culture
in bioreactors
The key to success, and the secret which requires the most operator practice to
learn in liquid culture, is that of contamination control, both endogenous and exogenous.
Only you, the plant culturist can deal with the endogenous contamination.
Clean cultures and/or the presence of chemical inhibitors are a necessity to prevent
contamination.
However, the frequency of exogenous contamination is a matter of technique and equipment.
To reduce it to a minimum, we developed some simple mechanical aids to handle the
culture during the two critical steps when it is exposed and vulnerable – (1) loading,
and (2) recovery, of the culture from the LifeReactorTM vessel.
The accessories kit consists of a set of tools which make these two critical operations
easier to carry out and to distance one's body from the open vessel or culture during
these moments. Even when working in a laminar flow hood, air currents and body movement
can carry airborne contamination.
The following working aids allow the operator to do this work conveniently while working
at a distance:
Reactor Holding Stand [800 524] –
This all stainless steel stand is clamped to the working surface of the laminar flow
hood. It holds the top port of the LifeReactor in a convenient upright position inside
the hood. The port is simply slipped into the collar, and is now held firmly in position
for either the filling or emptying process.
Funnel and Funnel Cap [800
522] –
Both the Funnel and its Cap are made of stainless steel. The Funnel is threaded at
the bottom and attaches to the vessel for the filling operation. During “in between”
moments in the work, the Funnel is covered with its Cap, to prevent any possibility
that airborne contamination will reach the interior of the bioreactor.
Vessel Gripper [800
523] -
The Vessel Gripper is used to carry the beaker containing the culture material, or
any other liquid which may be used to help move all the culture material from the
Funnel into the bioreactor. The point is to distance oneself as much as possible from
the open port.
Pouring Tube [800 521] -
The Pouring Tube screws into the main port and allows culture in the bioreactor to
be efficiently emptied into the Receiving Vessel.
Receiving Vessel [800
520]-
The Receiving Vessel is a three piece, stainless steel and glass assembly for separating
culture from spent medium, washing it and transferring it aseptically to other containers.
The outer stainless steel pot has a bulkhead port to which a piece of sterile tubing
can be attached to drain the spent medium into a waste container. This minimizes the
amount of unecessary operator movement in the clean area.
The separate inner section is a perforated stainless steel basket to separate the
culture from the spent medium and allow it to be washed with fresh medium prior to
transfer to other containers. Either a glass (autoclavable) or stainless cover is
provided to keep the culture material as isolated as possible except for the actual
working operations.
Long Handled Spoon [800
527] -
The Long Handled stainless steel Spoon is used to move the culture from the perforated
basket into sterile containers. It's long handle allows the operator to handle the
culture at a distance.
LifereactorTM and Ebb and Flow BioReactors
In contrast to the conditions in a classical airlift Lifereactor, an ebb and flow
or temporary immersion bioreactor does not seek to constantly bathe the plant tissue
in the liquid medium. Rather, it creates a cycle in which the surface of the tissue
is wetted with a thin film of liquid medium, this film is drained away and the tissue
has access to air, followed again by a wetting cycle.
This strategy has been very successful in promoting the elongation and growth phase
of crops such as pineapple, banana, sugarcane and tea, amongst others. It is part
of the growth in plant tissue automation strategies, and is entirely compatible with
the Lifereactor system.
In the Manuals section of this website, you will find instructions
for construction of an ebb and flow reactor using two Lifereactor vessels. This can
be very advantageous, if the next step after multiplication of the culture is going
to be elongation and growth.
Rather than removing the multiplication culture, two multiplication culture Lifereactor
vessels can joined in an ebb and flow system, without ever exposing the culture material
to potential contamination. The old multiplication medium in each vessel is first
drained out. The culture material is washed free of any residual medium by using the
addition port to aseptically add approximately 500 cc of sterile water, rocking the
tissue in the water and draining the vessel. This procedure is repeated 3-4x.
The two vessels are now aseptically connected to one another as shown in the Manual
instructions for an Ebb and Flow reactor. Fresh elongation and growth medium is now
added to the reservoir of the ebb and flow reactor and the reactor timer is activated
to cycle the medium back and forth from one vessel to the other. With appropriate
preparation of all the necessary equipment and medium, the entire operation need not
take more than two to three hours.
Since each of the 5 liter Lifereactor vessels can hold up to 6000 plantlets, this
means that 10,000 to 12,000 plantlets can be moved from multiplication to growth phase
by a single operator in less than a days work. Moreover, the continuing growth phase
is totally automated, unless the operator wishes to intervene in order to refresh
the medium or make specific additions at different points in the growth phase. This
type of highly efficient plant growth automation system is allowing both large and
small laboratories in western based countries to compete effectively with lower cost
production facilities in low labor cost countries.
References for Ebb and Flow Reactors: -
Akiten-Christie J,Davis H (1988)Development of a semi-automated micropropagation system,
Acta Hort 230:81 87.
Akiten-Christie J,Jones C (1987)Towards automation:Radiata pine shoot hedges in vitro,
Plant Cell Tissue Organ Cult 8:185 196.
Alvard D,Cote F,Teisson C (1993)Comparison of methods of liquid medium culture for
banana micropropagation. Effect of temporary immersion of explants.Plant Cell Tissue
OrganCult 32:55 60.
Cabasson C,Alvard D,Dambier D,Ollitrault P,Teisson C (1997), Improvement of Citrus
somatic embryo development by temporary immersion.Plant Cell Tissue Organ Cult 50:33
37
Escalona M,Lorenzo JC,Gonzlez B,daquinta M,Gonzlez JL, Desjardins Y,Borroto CG (1999)Pineapple
(Ananas comosus L.Merr) micropropagation in temporary immersion systems. Plant Cell
Rep 18:743 748.
Etienne H,Lartaud N,Michaux-Ferrire N,Carron M,Berthouly, M,Teisson C (1997) Improvement
of somatic embryogenesis in Hevea brasiliensis (M?LL.ARG.) using the temporary immersion
technique. In Vitro Cell Dev Biol Plant 33:81 87.
Lorenzo J, Escalona, Teisson C,Espinosa P, Borroto C, (1998) Sugarcane shoot formation
in an improved temporary immersion system. Plant Cell Tissue Organ Cult 54:197 200.
Tisserat B, Vandercook CE (1985)Development of an automated plant culture ystem. Plant
Cell Tissue Organ Cult 5:107 117.
