This is a Continuation-in-Part of United States Patent Application
Serial No. 07/263,049, filed October 26, 1988.
Field of the Invention
The invention relates to separation technology, including membranology
and controlled release of solvents, solutes, or cells.
Background of the Invention
Advances in the selective separation of matter have resulted in numerous
developments in a wide variety of industries. Attention first centered on the science
of membranology, beginning in about 1960, when integrally-skinned cellulose acetate
hyperfiltration membranes were developed for hyperfiltration desalination of salt
water. Developments followed in the areas of hemodialysis, electrodialysis, reverse
osmosis, ultrafiltration, cell harvesting, membrane bioreactors, microfiltration,
gas separation, controlled time release, gel permeation chromatography, hollow
fiber technology, non-cellulosic polymer membranes, ionomer membranes, copolymer
membranes, crosslinkable thermoplastic polymer membranes, emulsion-type liquid
membranes and others. These innovations have gained general acceptance, and separation
materials from the above disciplines are in widespread use in medical processes,
pharmaceutical research and production, industrial processes, research tools and
consumer products including consumer products packaging materials.
Controlled release of pharmaceuticals is now possible due to various
technologies, which include application of slow-dissolving coatings to oral dosage
form drugs. U.S. Patent No. 4,755,180 discloses an oral drug dosage form in which
an erodible material, formed as a film around the drug during manufacture, is eroded
or leached from the wall of the dosage form, such erosion or leaching enabling
controlled release of pharmaceutically active agents to the gastrointestinal environment.
The erodible materials disclosed in U.S. Patent No. 4,755,180 are typical of the
polysaccharide (sugar) coatings common in such applications: poly(glycolic) or
poly(lactic) acid compositions, gelatinous compositions, or leachable polysaccharides,
salts or oxides. Enteric coatings are also known in the art, which do not dissolve
in the stomach but allow enteric delivery of an orally dosed drug.
Although means are known for moving ions or molecules--or solvents--at
simple rates, such as the "zero-order" or "first-order" release kinetics typical
in controlled release pharmaceuticals, no technology has heretofore provided a
noningestible means for complex separation technology in which the separation kinetics
may change over time, in response to an environmental stimulus, in a pre-planned
or pre-programmed manner; moving components into and out of a container to provide
a better environment for the retained materials; or release of cells in a controlled
manner. Accordingly, a need remains for a separation barrier which can provide
complex separation protocols for particular separation applications entirely different
from the oral pharmaceutical dosage forms.
Summary of the Invention
In order to meet this need, the present invention is a noningestible
separation barrier having one or more pores or micropores of one or more sizes
therein, with the pores or micropores being initially plugged with a material
selected for its solubility and/or integrity characteristics relative to certain
environmental conditions. Ordinarily, the pores or micropores of the separation
barrier are initially filled with at least one material having greater erodibility,
under a given environmental condition, than the material constituting the separation
barrier itself. Ordinarily, the separation barrier with its plugged pores is prepared
prior to filling with the materials to be separated. The combination of the release
rate (if any) or other membrane characteristic of the separation barrier, combined
with the release rate and/or erosion life of the plugged pores, enables complex
separations including variable release of cells, colloids, solutes or solvents
over time, such as when the plugged pores remain intact until erosion is triggered
by an environmental change such as solvent addition, pH, or thermal or radiation
(ultraviolet light, etc.) change. Particular applications include specialized
protocols for the preservation of rooster sperm and turkey sperm for use in commercial
artificial insemination applications.
Brief Description of the Drawings
Detailed Description of the Invention
- Figure 1 is a hollow tubule (hollow fiber) separation barrier having plugged
- Figure 2 is a side elevational view of a separation barrier membrane having
small and large pores plugged with two different polymers, respectively;
- Figure 3 illustrates a three-dimensional rectangular construct, closed on five
sides, having plugged micropores on each closed polymer face thereof, with the
pores in each closed polymer face being plugged with materials having different
- Figure 4 is a schematic illustration (plan view) of a chamber which contains
exchange medium and into which are placed six containers according to the present
- Figure 5 is a section taken along lines V-V of Figure 4.
The present invention is a noningestible separation barrier having
one or more pores or micropores of one or more sizes therein, plugged with a material
selected for its solubility or erodibility upon exposure to certain environmental
conditions. The material forming the separation barrier itself may also have erodibility
or solubility, as long as the separation barrier and plugged pores do not consist
of the identical composition of matter. The combination of the release rate (if
any) or other membrane characteristic of the separation barrier, combined with
the release rate and/or erosion life or lives of the plugged pores, enables complex
separations including variable release of cells, colloids, solutes or solvents
Separation barriers having plugged pores, according to the present
invention, have a wide variety of uses in a myriad of industries. Suitable applications
include cell cultures and cryobiology, preingestion preservation and storage of
food and pharmaceuticals, shelf-life extension of polymers, proteins and other
products, and the containing, transporting and dispensing of active agents including
cells, herbicides, pesticides, fertilizers, disinfectants, indoor air fresheners,
and cell growth nutrients or other biologically active agents for use in laboratory
or industrial settings.
Because the scope of the present invention is particularly broad,
in view of the numerous variations in the product and process claimed as well as
widespread applications thereof, a specific example is the best introductory illustration
of the present separation barrier having initially plugged pores. For example,
cryopreservation of spermatozoa for artificial insemination, in certain species,
has heretofore required process steps which can be eliminated by the complex separation
protocols possible with the present invention. Ordinarily, cryopreservation of
spermatozoa requires attention to three considerations. First, cryopreservation
requires selection of an appropriate container, including appropriate volume, dimensions,
material, thermal properties, etc., with the appropriate choices usually including
nonporous glass ampules or vials, plastic vials or straws or metal tubes. Second,
a cryoprotectant must be selected to ensure survival of the cells during cooling
from 38oC to 0-5oC, subsequent cooling to minus 196oC,
and subsequent rewarming above 0oC. Conventional cryoprotectants include
egg yolk, lipoproteins, milk proteins, glycerol, dimethylsulfoxide, polyethylene
glycol, sugar and others. Third, controlled post-thaw modification of the cellular
and intracellular environment is essential for certain species. For example, rooster
sperm must be serially diluted with a medium free from the cryoprotectant, followed
by centrifugation and resuspension in the same cryoprotectant-free medium. This
latter process, although cumbersome, has in the past been essential to avoid the
inevitable contraceptive effect of the presence of the cryoprotectant around and
within the sperm at the moment of artificial insemination.
As is explained in greater detail in Example 1, below, the present
separation barrier having plugged pores simplifies thawing and removal of the
cryoprotectant from the spermatozoa of the rooster. Referring now to Figure 1,
the terminal portion of a sealed polymeric tubule is illustrated, comprising the
tubule 10 having plugged micropores 12 therein and an end seal 14. The end of
the tubule not shown may comprise an open end; alternately, the seal 14 may be
removed or originally omitted to leave an open end to the tubule 10. The tubule
may be prelabelled for identification or may be adapted for insertion of a labelled
plug in the open end. Collected rooster semen, evaluated, pooled, extended with
media and processed by means known in the art, is cooled to 5oC, mixed
with cryoprotectant (if cryoprotectant is absent from the original extension media),
and charged to the interior of the tubule 10 of Figure 1. The tubule 10, a polymeric
barrier having polymer-plugged micropores 12, constitutes the storage container
for cryopreservation of the rooster semen. The open end of the tubule is then sealed.
The sealed tubule and its contents are then cooled at one or more controlled rates,
as known in the art, to -196oC.
Prior to insemination of a group of chickens, a tubule 10 is transferred
from cryopreservation storage at minus 196oC (liquid nitrogen) into
a thawing solution known in the art, and after initial thawing the tubule 10 is
subsequently transferred to a post-thaw treatment solution of appropriate temperature,
composition and oxygenation. In the post-thaw treatment solution, the action of
at least one of the constituents of the post-thaw treatment solution on the plugged
micropores 12 opens the micropores 12 and allows controlled egress of the cryoprotectant
from the environment around and within the rooster sperm and simultaneously permits
controlled ingress of the post-thaw treatment solution into the tubule 10. The
micropores 12 are, of course, smaller than the sperm cells. After a 10-60 minute
treatment period, sperm in the tubule are ready for use. The tubule 10 may be
used, along with appropriate auxiliary mechanical devices, to disperse the sperm
directly into one or more hens for artificial insemination of the chicken flock,
or the sperm may be transferred from the tube into an appropriate auxiliary mechanical
device for insemination. As an illustration of the concept of the present invention,
the tubule 10 is insoluble in the liquid nitrogen at minus 196oC, as
are the polymer plugged micropores 12. Both the tubule 10 and plugged micropores
12 are likewise insoluble or not completely eroded in the thawing solutions known
in the art. Thus, during two periods of use, the tubule 10 undergoes no separation
or change in separation kinetics with respect to its contents or structure. However,
upon contact with the post-thaw treatment solution, the action of at least one
constituent of the post-thaw treatment solution constitutes the environmental factor
which commences or completes dissolution of a selected polymer within the plugged
micropores 12, so that pre-determined time- and rate-controlled egress of cryoprotectant
and ingress of medium is accomplished. If two or more cryoprotectants are used,
they can be removed selectively through pores having different sizes and/or erosion
characteristics (as suggested in Figures 2 and 3).
It will be understood by those skilled in the art that both the plugged
micropores 12 and the tubule 10 (the separation barrier itself) may both be fabricated
of materials soluble or erodible under certain conditions. For example, the plugged
micropores 12 may be moderately soluble in human plasma, with the tubule 10 being
only somewhat soluble in human plasma, so that an entire structure comprising
a small tubule 10 according to Figure 1, filled with a pharmaceutically active
agent, would constitute a human subcutaneous controlled release drug implant which
would not release the agent for some period of time, then release the agent at
a controlled rate, and ultimately completely erode. Likewise, tubules 10 according
to Figure 1, or other structures such as that shown in Figure 3, may be used in
the cryopreservation of a wide variety of cells and cell cultures, with the polymer
or other material for plugging the plugged micropore 12 being selected in reliance
on the reaction or release kinetics required by the particular post-thawing parameters
required for the preserved cells. Additional applications include controlled release
of nutrients to cells or organisms; bacteria; herbicides; and release of alkaline
agents to neutralize water or soil, each triggered by the appropriate change in
Separation barriers according to the present invention may be fabricated
from a wide variety of materials including polymers, ceramics, metals and natural
and semi-synthetic cellulosics. More particularly, the separation barrier may consist
of a wide variety of materials and polymers including polyether compositions,
polyethylene and polypropylene polymers, polyvinyl polymers, moisture vapor permeable
urethanes and other polyurethanes, polycarbonate polymers, cellulosics, semi-synthetic
cellulosics, ceramics, metals, natural resins including rubbers, etc. When appropriate,
the separation barrier may be fabricated of one or more of the compositions also
suitable for use in plugging the pores or micropores in the separation barrier,
which compositions include cellulosics (i.e., hydroxypropylmethylcellulose), polyelectrolyte
complexes, polysulfone compositions, acrylic polymers, cellulose acetates, Dynel®
compositions, polyacrylonitrile compositions, polyvinylpyrrolidone polymers, cellulosic
composites on polyvinyl chloride, and other materials known in the membranologic
arts. Also feasible are selectively soluble salt or polysaccharide crystals as
long as the dissolution of same does not poison adjacent biologic media and is
not toxic to cells in or near the barrier. Selection of one or more materials for
the preparation of the separation barrier having plugged pores will be dictated
directly by the specific application intended and the release kinetics desired.
Also, the separation barrier may be constructed for one-time use, or may be designed
for multiple uses i.e., when the barrier device is returned to the producer for
replugging and refilling.
As an overview of the various applications of the different types
of "plugger" polymers, water-soluble materials included within the pores or micropores
of the separation barrier will have applicability at least to cryobiologic preservation
of spermatozoa (poultry, human, horse, fish, pig, sheep, dog, mouse, rat, etc.);
oocytes (cattle, human, horse, pig, mouse, rat, etc.); embryos (human, cattle,
horse, mice, dogs, etc.) of all species of mammals with physiological need and
economic advantage; human pancreas B-cells; human corneas; primary cell cultures;
and seeds. Controlled release of animal agricultural hormones; plant agricultural
fertilizers, herbicides, and pesticides; seeds; agriculture nutrients, pharmaceuticals
and larvae; and home insecticides, fertilizers and herbicides is also envisioned.
Thermal sensitive polymer plugs have application in frost damage control: erodibility
of the plugs, in the separation barrier pores, can upon decreasing temperature
release frost-protective bacteria onto crops, fields or orchards. Polymer plugs
having pH sensitivity may be applied to the non-oral controlled release of pharmaceuticals
to animals and humans in pH-fluctuating anatomic areas, and likewise have application
in the ecosystem to deliver the appropriate acid or base upon a drop or increase
in environmental pH conditions. Photo-inducible polymer erodibility can release
agricultural materials after sufficient exposure to sunlight. Other environmental
conditions including electricity, sound and magnetism can also be harnessed by
appropriate "plugger" material types, which erode at the appropriate cued time.
Finally, environmental cues can trigger polymerization, instead of erosion, of
the material in the pores or micropores. Other examples will be readily evident
to those skilled in the art.
Referring now to Figure 2, a side elevational view of a membrane
20 is shown having pores of two different diameters. The large pores 22 are filled
with a first polymer composition and the small pores 24 are filled with a second
and different polymer composition. By appropriate selection of the material constituting
the membrane 20 and the material within each of the large pores 22 and the small
pores 24, any one of three or all three separation kinetics attributable to the
respective polymers may be controlled by appropriate changes in the corollary
environmental conditions including temperature, pH, presence of specific solvents,
electrical charge, magnetic field, light waves, or sound energy. For example,
if the largest pores open last, the largest separable particles cordoned by the
membrane will diffuse last. The membrane 20 of Figure 2 may be used in lieu of
other prior art separation membranes, when the sequential or selective separation
kinetics possible with the membrane 20 are desired or necessary for a given application.
Such a membrane can be directly fabricated into a variety of shapes, or used as
a sheet for later insertion into an appropriate container.
Figure 3 illustrates a three-dimensional rectangular construct having
five closed sides, or faces. The rectangular construct 30 is fabricated of a polymer,
and the largest closed face thereof has polymer-plugged pores 32 therein. The
remaining closed faces shown have polymer-plugged pores 34 and 36 therein. Polymer
plugged pores 32, 34, and 36 are filled with a second, third, and fourth polymer,
respectively. As with the device shown in Figure 2, the rectangular construct 30
functions as building-block type unit, and can be used alone or in an array of
a number of identical rectangular constructs 30 for laboratory or production use
in a variety of configurations. Complex sequential or selective separations are
possible due to the varied polymer plugs in the polymer-plugged pores 32, 34, and
36. The remaining closed faces not shown may also contain plugged pores. The rectangular
construct 30 is representative of a commercial embodiment of the present separation
barrier having plugged pores, in which units may be assembled by the user as needed.
Referring once again to the present separation barrier generally,
the plugged pores may be true plugged pores, i.e., the pores in the separation
barrier may be filled to the extent of the pore cavity only, or the plugged pores
may be plugged by means of a continuous coating on one or both sides of the separation
barrier to plug the pores by coating over them. Also, with respect to either the
separation barrier itself or the pore-plugging material, or both, additional treating
materials may be used to alter the chemical and physical properties of the polymers,
such as for altering toxicity, adapting polar and non-polar materials for their
specific intended applications, or increasing bonding properties.
Although the structures represented in Figures 1-3 are illustrative,
numerous other containers may be fashioned in view of the concept of the invention.
A container need not be homogeneous, for example, but could have sides each of
different materials. A six-sided construct could include six different materials
in the different-sized pores of each respective side, for example, which construct
would then effect unique separation kinetics by means of each of its separate
Dimensions for the tubule of Figure 1, for cryopreservation of sperm,
range from 1-80 mm. in diameter or more, with 1-5 mm. diameters being preferred.
The tubules may range in length from about 5 mm. to 10 centimeters or more, with
about 50-200 mm. being preferred. Pores or micropores may be of any size. For
many separations, pores of 0.2 to 0.6 micrometers effective diameter are preferred,
but for some applications only pores greater than 0.6 micrometers are appropriate.
Often pores can be larger than the pores in prior art separation membranes due
to the nature of the materials to be retained and the initial plugged configuration
of the pores. Other embodiments of the invention may have widely varied dimensions
depending upon the intended application.
Polymers which can provide a persistent plug in a number of environments
include ethyl cellulose, among others. For applications requiring water erodible
plugs, methyl cellulose plugs are appropriate. The use of polyvinylpyrrolidone
enables thermal transition opening of the pores containing it, and carboxymethylcellulose
allows pores to open in response to pH shift. Detergent activated, photoactivatable,
and other compositions known in the art can be selected as needed.
A further particular application of the present invention is in the
production of fertilized turkey eggs, which eggs can be produced commercially
only by artificial insemination (AI). Prior art methods permit holding
of turkey sperm for only six to eight hours after collection, which allows only
about two hours for transport of the turkey sperm. In the laboratory, oxygenation
of turkey sperm has increased the holding time to about 24 hours, but the laboratory
techniques have been impractical on a commercial basis. By applying the products
and methods according to the present invention, turkey sperm viability and quality
should be maintained for up to 48 hours. Preservation of turkey sperm is discussed
further below and in Example IV herewith.
The present invention provides for the use of unique containers and
storage conditions for preservation of turkey sperm. The container is uniquely
constructed with pores that are ordinarily initially impermeable (allowing easy
loading and handling) but which open rapidly after the container of turkey semen
is placed in contact with exchange solutions. Precise control of temperature and
oxygen content and continuous exchange of nutrients and antioxidants into the
sperm suspension, without damage to the sperm, is possible using the container
along with an optional microprocessor controlled system. This will allow the industry
to create "super stud farms" with the potential for total reorganization of the
distribution of germ plasm.
Despite current limitations of semen holding/storage, the industry
exists only because of use of AI for reproduction. Passage from the breeder to
the commercial producers is by sale of eggs of the individual male and female
lines as needed for the construction of the commercial genetic product. If held
or stored semen were available, industry needs could be better satisfied and several
problems would be eliminated. First, geographical separation of males and females
is desirable to allow optimum management of each. Increasing the holding time
makes possible wider use of stud farms. Second, coordinating purchase of male line
with female eggs to meet future needs is difficult. Shortages of one line are
costly. Held semen would alleviate this problem. Third, selection for production
traits of individual males could be increased, thus decreasing the cost per pound
of meat to the consumer. Fourth, purchase of semen instead of eggs removes the
cost and nuisance of offsex disposal. Fifth, the breeder will have greater control
over the quality of the commercial meat bird, again to the benefit of the consumer.
Finally, the breeder will have greater protection of the male line germ plasm.
The general approach to preservation of turkey sperm is as follows.
Turkey toms (e.g., male line meat strain) are ordinarily housed in a room with
14:10 hour light:dark cycle. Semen is collected by massage every 3-4 days from
a group of toms and pooled within a few minutes. Sperm quality is determined by
several tests, as are known to the art. Several extenders have been developed
for holding turkey sperm and 5oC and are known in the art, and these
extenders may be admixed with acceptable turkey semen and placed within plugged-porous
containers according to the present invention.
The appropriate plugged porous container is ordinarily fabricated
from nylon "frames" and specially prepared polysulfone membranes with 0.22 µm pores,
with the pores initially sealed using methyl cellulose and ethyl cellulose. Identical
membranes are fastened to both large faces of the container using UV-activated
glue. These containers are nontoxic to turkey sperm and have pores that open in
a controlled and repeatable manner. Transmembrane flux of small molecules ( 10,000
kD) in the internal or external diluent has a half-time of about 2-5 min.; sperm
cannot pass through the membrane. The above-identified polymers are exemplary.
The containers are incubated in special environmental control units.
Chambers are fabricated and fitted with circulation pumps, miniature oxygen sensors,
small air pumps, electronically controlled air nitrogen valves, and a small oxygenation
reservoir. The total volume of the chamber is about 30 ml; the chamber is preferably
fitted with control circuits. Alternatively, the desired oxygen content in the
medium can be achieved using appropriately blended gas mixtures and a constant
rate of addition with a diffuser, as are known in the art. When the containers
(containing suitable diluents and extenders along with the turkey semen) are cooled
and held within the appropriate exchange solutions in the controlled chamber,
turkey sperm viability and quality can be maintained for> 18, and possibly>
Although the use of a porous container held within a chamber containing
dialyzing fluid is novel, the appropriate extenders, diluents and exchange solutions
are known in the art. The environmental control chamber regulates oxygen tension
and temperature of turkey sperm to which have been added diluents and/or extenders;
the present invention provides the heretofore unknown means by which cooling,
selective oxygenation, antioxidant treatment, etc., of turkey sperm may be scaled
up to a commercially feasible level using porous containers. A particularly noteworthy
aspect of the cooling/selective oxygenation preservation of turkey sperm is the
option whereby extended and/or diluted turkey semen may be charged to a porous
container in which the pores are not
initially plugged, simultaneous with
immersion of the porous container in the exchange solution. (The pores have a
diameter smaller than the diameter of the turkey sperm cells, of course.)
Referring now to Figures 4 and 5, an apparatus suitable for preservation
of turkey semen is illustrated schematically in plan and sectional views, respectively.
A five-walled chamber 40 has control means for controlling temperature and oxygenation
(for example, oxygen concentration can be monitored with a miniature oxygen sensor
connected to a multiplex A/D converter, with optional electronic data storage)
and acts as a bath-type receptacle for turkey semen receiving containers 42. Exchange
fluid 44 selectively enters and exits the chamber 40 via inlet 46 and outlet 48
as desired, and the exchange fluid 44 can enter and exit the containers 42 depending
upon the constituents of the fluid and the pore size of the container(s).
The invention will be more particularly described by means of the
following illustrative examples.
Frozen rooster semen is not successfully used commercially because
the cryoprotectant most effective for spermatozoa survival during freezing and
thawing, glycerol, also demonstrates contraceptive properties after artificial
insemination. By prior art methods this cryoprotectant is removed by serial dilution
followed by centrifugation or conventional dialysis. Such processes successfully
reduce cryoprotectant concentration but are commercially impractical.
Because of the contraceptive effect of the glycerol, therefore, the
glycerol must be removed from the avian spermatozoa prior to artificial insemination.
However, rapid removal of glycerol from avian spermatozoa damages the spermatozoa
cells, probably as a consequence of rapid movement of glycerol across the cell
membrane, which alters the characteristics of the membrane and the viability of
the cell. Slow removal of glycerol from cryopreserved rooster semen is therefore
a necessity, notwithstanding the typical high cost of achieving it according to
prior art techniques.
Use of the present separation barrier having plugged pores, for the
controlled removal of cryoprotectant from rooster spermatozoa, is described below.
A group of roosters raised by an integrated breeder is selected,
which roosters possess a virtually identical genetic background and therefore transmit
the same phenotype. Semen is collected from this group of roosters, a collection
unit, with the semen then being evaluated, pooled, extended, and cooled by means
known in the art. (Oxygenation is required if the cells are held above 15oC
because avian spermatozoa are highly dependent on oxidative metabolism unless they
are cooled to near 5oC or frozen.) Initial cooling proceeds to 5oC.
After the extended semen achieves 5oC, glycerol is added as a cryoprotectant
(if not present in the original extender), and the cryoprotected extended semen
then is immediately charged to hollow tubules having plugged micropores such as
the tubule illustrated in Figure 1.
More particularly, the hollow tubule is a cylindrical polymeric separation
barrier open at one end and having plugged pores therein, having the following
features: 1) impermeable for 20-30 minutes to aqueous solutions placed inside
at 5oC; 2) impermeable to viruses or bacteria coming in contact with
the outside of the tubule, while the pores remain plugged; 3) resistant to immersion
and long-term storage in liquid nitrogen; 4) resistant for 6-300 seconds to warming
in aqueous solutions at 2-75oC; 5) has pores partially soluble upon
5-20 minutes' further immersion in aqueous post-thaw treatment solutions at 5-38oC;
and 6) develops pores of a size which permit only molecules below a certain size
to pass. These features are accomplished by means of a microporous hollow fiber
having the following characteristics: 1) wall thickness, 0.5 mm; length, 60 mm.;
and diameter, 4 mm. (other possible configurations include: a right cylinder (30-40
mm. diameter and 5-10 mm. in length) of a nonporous material, such as polystyrene,
to which is attached a "lid" of the microporous membrane described herein, or a
flattened tube 2 mm. x 20 mm. x 60 mm.); 2) separation barrier constructed of
polyethylene (or other acceptable polymer); and 3) 0.2 micrometer micropores plugged
with methyl cellulose.
If a three-dimensional rectangular device is used, to provide greater
capacity or an alternative packaging process, it would have five impermeable sides,
fabricated from polystyrene or another material, forming a square or rectangular
shape (25 mm. x 25-40 mm. x 5-10 mm.) and the sixth surface would be a microporous
membrane with plugged pores, applied and sealed as a lid.
Cryopreservation of the semen in the hollow tubules (or tubes or
other containers described above) is effected by means known in the art. Shortly
before insemination of a flock of chickens, a tubule is removed from liquid nitrogen
in storage at minus 196oC and is transferred to a thawing solution.
After thawing, the hollow tubule is transferred to an aqueous post-thaw treatment
solution including additives necessary for the optimal survival of the avian spermatozoa
(with oxygenation, if appropriate). Upon immersion in the post-thaw treatment
solution, the plugged pores open to permit controlled and relatively slow egress
of glycerol cryoprotectant from the solution within the container, and hence from
the spermatozoa, and simultaneous controlled ingress of the external medium to
surround the thawed rooster sperm. The thawed and treated rooster sperm then are
ready for use in artificial insemination.
A second example is provided to illustrate the concept of two different
rates of release, as well as an extended time for initiation of pore opening.
For human sperm, both glycerol and crude lipid micelles (e.g., egg
yolk lipoprotein particles) are used in the art of cryopreservation. It is desirable,
after thawing, to remove the glycerol slowly (as for rooster sperm) and then rapidly
remove the egg yolk lipoprotein particles. To accomplish this objective, the general
process of Example I is followed except that the initial extender contains egg
yolk and is centrifuged (20,000 x g for 30 minutes, for example) or filtered (through
a 0.2 or 0.4 micrometer filter) to remove material larger than the dimension desired
for the colloidal particles, and glycerol is added after the extended sperm are
cooled to 5oC.
The container is a three dimensional rectangular construct (although
the shape is not important) with four nonporous sides. One of the other sides is
a microporous membrane with pores (about 0.2 micrometers diameter) plugged with
a material (i.e., high ratio of methyl cellulose to ethyl cellulose) having erodibility
characteristics suitable for the controlled removal of glycerol. These pores open
soon after immersion of the container into the thawing solution. The last face
consists of a microporous membrane with different size pores (0.3-0.5 micrometers)
plugged with a different material (i.e., low ratio of methyl cellulose to ethyl
cellulose) that is rapidly eroded after a time delay (30-40 minutes) to allow
rapid egress of the egg yolk lipoproteins and further entrance of the treatment
A third example is provided to illustrate release of soluble materials
to the environment and an alternative mode of release in response to changing environmental
Nutrients may be provided to cultured cells held within "bioreactors".
Continuous culture of cells for purpose of production of pharmaceuticals and other
bioproducts requires (at a minimum): removal of the desired products as they are
excreted into the growth medium; removal of toxic side products from the medium;
and selective means for replenishing nutrients and regulatory substances necessary
for continued culture of the cells.
Controlled release of nutrients and regulatory substances is achieved
by the following process. A hollow tube, composed of one or more pores plugged
with one or more materials chosen to allow selective opening under environmental
stimulus, is filled with a sterile suspension (solid or liquid) or solution of
the materials needed for continuous growth and production by the specific cells
under culture. Examples include vitamins, hormones, trace metals, etc. The surface
of the tube then is sterilized, yielding a container that allows long term storage
of the materials to be added to the culture, and facile insertion into the bioreactor
network as needed.
The tube is inserted into the culture (directly or into circulating
media) as needed. In response to this new environment, the pores open to release
the materials to the cells. The environmental signal is either a passive (water
based erosion of the pore plug, as outlined for the treatment of rooster sperm)
or an active response to the changing culture conditions (pH shift as induced
by production of acid by-products (e.g., lactic acid) by the cells). Such a container
can also be used in a large scale agriculture environment, i.e., addition of nutrients
for growth of fish on a commercial scale.
Note: We have never done all the parts of this example.
A plastic chamber holds six rectangular plugged-pore containers plus
exchange solutions. The chamber is fitted with control circuits, an "oxystat,"
a control means which enables maintenance of constant O&sub2; tension by way of
differential addition of air or N&sub2;, and with means known in the art for temperature
control, pumping and valving.
Turkey semen is collected, pooled and extended by means known in
the art; the extended turkey sperm is cooled at a linear rate to temperatures between
5oC and 20oC and ≧ 1.5 ml aliquots are charged to each
of the six rectangular plugged-pore containers. The containers have inside dimensions
of 3.5 x 17 x 36 millimeters, although containers having inside diameters of 3.0
x 17 x 22 millimeters could be substituted. The two largest walls of the containers
are constructed of polysulfone membranes having 0.22 micrometer pores, with the
pores initially sealed with methyl cellulose and ethyl cellulose. The containers
are placed in the chamber containing exchange fluid at the desired temperature
(5-20oC); the pores of the containers are no longer plugged after several
minutes. Cooling and dialysis can continue for the holding period.
Although the invention has been described particularly above, the
invention is only to be limited insofar as is set forth in the accompanying claims.