Replies

Debra,

Can you ask Ken whether this patent below is consistent with: (1) the high concentration of the Daschel product, (2) the EDX finding reported by AFIP in its newsletter, and (3) Ken and Dr. M’s observation of the SEMS (when they did not see a silica coating), taking into account the related patent describing how the silica can be removed from the surface using repeated centrifugation? The related patent involving the removal of the silica from the surface so as to reduce weight / increase purity was invented by the guy who had Al-Timimi’s telephone number.

TrebleRebel, can you ask Dr. Spertzel?

EdLake, can you ask Dr. Meselson?

There are others who are equally knowledgeable about microbiology and would welcome their view also. I’d like to understand, even, the basic words he uses — such as his reference to COATING and COATING VESSEL etc.

United States Patent
6,649,408
Bailey , et al.
November 18, 2003

Microdroplet cell culture technique

Abstract
The present invention comprises a novel culture method and device in which living cells are cultured in a plurality of individual microdroplets that are immobilized and isolated within a matrix of hydrophobic particles. The hydrophobic particles adhere to inoculated microdroplets of media, isolating the microdroplets in an aseptic microenvironmet. The plurality of individual microdroplets provide and optimal environment for the concentrated growth of cultured cells contained therein.

Inventors:
Bailey; Charles L. (Fayetteville, TN), Alibek; Ken (Alexandria, VA)
Assignee:
George Mason University (Fairfax, VA)
Appl. No.:
09/805,464
Filed:
March 14, 2001

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DESCRIPTION

This application claims the benefit of U.S. Provisional Application No. 60/191,771, filed Mar. 24, 2000.

Claims

Having thus described our invention, what we claim as new and desire to secure by Letters Patent is as follows:

1. A cell culture method comprising the steps of: introducing liquid media inoculated with cells to be cultured into a vessel; converting the inoculated liquid media into individual microdroplets; introducing a sufficient quantity of hydrophobic particles in the form of a dry powder into the vessel to coat the individual microdroplets; and growing the cells within the individual microdroplets.

2. The cell culture method of claim 1 further comprising the step of recovering the cultured cells from the individual microdroplets.

3. The cell culture method of claim 1 wherein the converting step comprises: adding ferromagnetic particles to the vessel; applying an electromagnetic field within the vessel, thereby causing the random circulation of the ferromagnetic particles throughout the vessel.

...
6. The process of claim 1 wherein the cells are bacterial cells.

...
9. The process of claim 1 wherein the hydrophobic particles are silicon dioxide particles.
...

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to the cultivation and growth of cells on laboratory, pilot plant, or industrial scales and, more particularly, to the cultivation and growth of cells in a plurality of individual microdroplets of liquid media which are interspersed within a matrix of hydrophobic microparticles.

2. Background Description
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SUMMARY OF THE INVENTION
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In one embodiment of the invention, the inoculated media is converted into microdroplets prior to introduction into the COATING vessel. Such a process is enabled by introducing the inoculated media via a spray nozzle that dispenses individual microdroplets into the vessel. It is not essential to the practice of the invention that the microdroplets be created prior to the introduction of the droplets into the COATING vessel. Thus, in yet another embodiment, the microdroplets are created after the inoculated liquid media is introduced into the COATING vessel. Ferromagnetic particles are sterilized and introduced into a non-magnetic mixing/COATING vessel. Electromagnetic inductors are mounted in parallel on either side of the COATING vessel. Activation of the electromagnetic inductors causes an electromagnetic field to exist within the vessel. Oscillations of this electromagnetic field are induced by the inductors. The ferromagnetic particles orient along and follow the field lines of the electromagnetic field and follow the oscillations of the field. The rapid motion of the field and particles vigorously mixes the hydrophobic particles and liquid media, inducing the formation of droplets.

The size of the microdroplets will vary, with an optimum size for the cultivation of microorganisms, for example, usually being between 0.5 and 2.0 mm in diameter. Sizes within this range have been found to result in high concentrations of microorganisms per microdroplet. It should readily be understood by one skilled in the art, however, that the optimal size of microdroplet will vary, depending on such factors as the growth rate of the cultured cell type, the amount of optimal aeration for a given cell type, the most effective cell density for production of a given metabolite, and the like.

The size of individual microdroplets can be regulated by adjusting such factors as the size of the nozzle or portal delivering the liquid or aerosolized media, the volume of the vessel, the speed at which the various components are added, the power and frequency of electromagnetic induction (in one embodiment of the invention), and the type of hydrophobic particle utilized, for example.

In one particular embodiment of the invention, the vessel is contained in a refrigerated environment to prevent the rapid random motion of the electromagnetic process from destroying the inoculated microdroplets with excessive heat.

Once the microdroplets of inoculated media have formed, the hydrophobic particles can then intercalate between and around individual microdroplets, creating a semi-liquid slurry comprising a matrix of interspersed microdroplets of inoculated culture and hydrophobic particles. In one embodiment of the invention, the particles are pumped into the COATING vessel while the ferromagnetic particles and liquid media are agitated, resulting in the simultaneous agitation and mixing of the hydrophobic particles along with the microdroplets. In another embodiment, the hydrophobic particles are introduced through a second opening positioned such that the particles encounter the aerosolized microdroplets of inoculated media as the droplets enter the vessel.

The hydrophobic particles can be introduced into the vessel by a variety of methods well known within the art, for example, by forced flow with the assistance of an air pump. Introduction of the COATING particles can be through the same opening used for the introduction of the inoculated media or through a second opening. The use of two different openings for the media and COATING particle introduction may have the advantage of allowing for easier process controls.

In one embodiment of the invention, the hydrophobic particles comprises a powder of silicon dioxide....

In a particularly preferred embodiment, the silicon dioxide particles are Aerosil 300, produced by Brenntag N.V. of Belgium. In another preferred embodiment, the silicon dioxide particles are selected from the group comprising the AEROSIL series of powders manufactured by the Degussa-huls Corporation (i.e., AEROSIL R 104, AEROSIL R 106, AEROSIL R 202, AEROSIL R 805, AEROSIL R 812, AEROSIL R812.S, AEROSIL R 972, AEROSIL R 974, and AEROSIL R.8200). Other silicon dioxide particles are contemplated and within the scope of the invention. The choice of silicon dioxide particles will vary depending on the organism to be cultured and the amount of aeration required. In general, silicon dioxide particles that are useful in the practice of the present invention will be hydrophobic and have a surface area between 50 and 380 meters.sup.2 per gram of weight.

It is contemplated within the practice of the invention that the percent composition of COATING particles to inoculated medium will vary, depending on, but not limited to, such factors as the cell type, the size of the individual microdroplets, and the desired final density and phase of growth that is the objective of the particular culture. In one embodiment of the invention that the ratio of individual COATING particles to cultured inoculum may be within a range of 99:1 and 1:99. In one preferred embodiment of the invention, the ratio individual COATING particles to cell inoculum to will be within a range of 1:2 to 2:1.

Once the microdroplets are formed and COATED, they are evacuated from the COATING vessel through narrow slotted openings at the bottom of the vessel. In one particular preferred embodiment, the slotted openings will be between 1.5-2.0 mm wide but may vary depending on the size of the microdroplets formed. The microdroplets can be as little as 10 to 20 microns, so long as the initial inoculum is dense enough to ensure each microdroplet contains inoculated medium. The microdroplets can be much larger, with diameters greater than 2.5 mm, so long as the hydrophobic particles are able to maintain the media in individual droplet form. Accordingly, the slots for removal can also be designed to be the same as whatever size the microdroplets are or slightly larger.

In most cases, the space between the COATED microdroplets provides adequate aeration of the cell culture. It is a particularly useful and beneficial feature of the present invention that the space which exists between individual COATED microdroplets provides an optimum environment for the concentrated growth of cell cultures. The adequate aeration provided with the present invention allows the growing cultures to make optimal use of the liquid media contained within each microdroplet.

It can readily be seen by one skilled in the relevant art, however, that various means can be employed to provide the growing microdroplet culture with supplemental oxygen and/or other gases to optimize the aeration conditions for a given cell culture. For example, a fermentation vessel or zone may be provided with a port opening onto the vessel or zone through which exogenous molecular oxygen may be pumped via conduits and means to transport the gas. Additionally, the fermentation vessel or zone may further be equipped with a second port opening for removal of gases during the fermentation process.

In one embodiment of the invention, the cultured cells will be microorganisms. Fermentation of microorganisms can proceed via a batch process or a continuous fermentation process. In the case of batch fermentation, the microdroplets are collected and grown in a fermentation vessel. In a continuous fermentation process, the COATED microdroplets are collected from the slots at the bottom of the COATING vessel and are grown in long conduits that constitute a fermenting zone. The particular fermentation method used to culture the microdroplets is not critical to the practice of the present invention.

As can readily be appreciated by one skilled in the art, it will not always be necessary or preferable to separate the hydrophobic particles away from the liquid cell culture following cell growth. For example, since silicon dioxide is frequently utilized in soil treatment, there is no need to remove the silicon dioxide from cell cultures that are grown for the purposes of soil treatment. Furthermore, since the hydrophobic particles limit the potential for the spread of contamination, it may be desirable to maintain cultivated cells within the Individual hydrophobic microdroplets for storage purposes.

It is a particularly beneficial feature of a preferred embodiment of the present invention that the enhanced aeration of cultured cells, combined with the efficient removal of metabolites, allow for microbial cultures to divide to a density that consumes all of the available liquid present in a microdroplet. Thus, in a preferred embodiment of the invention there is no need to (1) concentrate cultures or (2) remove the hydrophobic particles from the microdroplet culture. When all of the liquid media is consumed, the hydrophobic particles disassociate from the cell cultures, allowing the cells to interact directly with the surrounding environment.

Alternatively, once cell growth is complete, the liquid media can be isolated away from the hydrophobic particles through a simple centrifugation step. As can readily be appreciated by one skilled in the art, the time and force of centrifugation will vary depending on the organism and hydrophobic particle employed in the process. The silicon dioxide particles can be sterilized and re-used in another microdroplet cultivation process.

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