U.S. patent number 4,649,109 [Application Number 06/580,854] was granted by the patent office on 1987-03-10 for methods for isolating mutant microorganisms from parental populations.
This patent grant is currently assigned to Brandeis University. Invention is credited to Daniel Perlman.
United States Patent |
4,649,109 |
Perlman |
March 10, 1987 |
Methods for isolating mutant microorganisms from parental
populations
Abstract
A method for isolating a mutant microorganism is described. The
method comprises the steps of: (a) separately microencapsulating in
a semi-permeable membrane each or a small number of microorganisms
from a microorganism population containing said mutant; (b) growing
said microencapsulated microorganisms including treating to induce
a detectable difference between microcapsules containing mutant
microorganisms and those containing non-mutant microorganisms; and
(c) separating said microcapsules containing mutant microorganisms
from those containing non-mutant microorganisms based on said
difference.
Inventors: |
Perlman; Daniel (Arlington,
MA) |
Assignee: |
Brandeis University (Waltham,
MA)
|
Family
ID: |
24322851 |
Appl.
No.: |
06/580,854 |
Filed: |
February 16, 1984 |
Current U.S.
Class: |
435/30; 435/182;
435/243; 435/39 |
Current CPC
Class: |
C12N
7/00 (20130101); C12N 11/04 (20130101); C12Q
1/24 (20130101); C12N 15/00 (20130101); C12N
2710/00051 (20130101) |
Current International
Class: |
C12N
11/00 (20060101); C12N 7/02 (20060101); C12N
11/04 (20060101); C12N 15/00 (20060101); C12Q
1/24 (20060101); C12P 001/24 () |
Field of
Search: |
;435/5,30,32,33,40,178,182,243,261 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Lehinger, Biochemistry, 2nd ed., 1975, Worth Publishing, New York,
pp. 157-159..
|
Primary Examiner: Rosen; Sam
Assistant Examiner: Herald; William J.
Attorney, Agent or Firm: Neuner; George W.
Claims
What is claimed is:
1. A method for isolating a mutant microorganism, which method
comprises the steps of:
a. separately microencapsulating in a semi-permeable membrane each
or a small number of microorganisms from a microorganism population
containing said mutant;
b. growing said microencapsulated microorganisms and treating them
to induce a difference in cell number between microcapsules
containing mutant microorganisms and those containing non-mutant
microorganisms; and
c. separating said microcapsules containing mutant micoorganisms
from those containing non-mutant microorganisms by a method based
on said difference in cell numbers.
2. The method of claim 1, wherein said mutant microorganism is a
naturally occuring mutant of a wild type microorganism.
3. The method of claim 1, wherein the mutation in the mutant
microorgahism is artificially induced.
4. The method of claim 3, wherein said artificially-induced mutant
is the result of genetic engineering by a biological, biochemical
or biophysical process.
5. The method of claim 1, wherein said microcapsules containing
said single microorganisms are formed by:
a. forming a dilute suspension of said microorganisms in a liquid
diluent capable of forming a gel upon said dilute suspension having
a dilution selected so that there is a high probability that each
microcapsule produced from said suspension contains one
microorganism;
b. converting said suspension into gel droplets, forming said
microcapsules.
6. The method of claim 1, wherein said treatment comprises growing
said microencapsulated microorganisms under conditions restrictive
to said mutant whereby said microcapsules containing said mutants
have fewer microorganisms per microcapsule than microcapsules
containing non-mutants.
7. In the method of claim 6, prior to said separation step,
incubating said microcapsules in a high-density medium of
appropriate diffusion rate, thereby causing the mutants in the
microcapsules which have fewer microorganisms to have a higher
density than the non-mutants in the microcapsules which have larger
numbers of microorganisms.
8. The method of claim 1 wherein in said growing step (b) the
microencasulated mutants have a capacity to accumulate a particular
material resulting in an increase in mass and number greater than
that of said microencapsulated non-mutants.
9. The method of claim 1, wherein said separation is based on the
difference in mass between said microcapsules containing mutant and
non-mutant microorganisms and comprises (a) equilibrium density
centrifugation of a suspension containing said microcapsules or (b)
velocity sedimentation of a suspension containing said
microcapsules.
10. The method of claim 1, wherein said microorganisms comprise
eucaryotic cells.
11. The method of claim 1, wherein said microorganisms comprise
procaryotic cells.
12. The method of claim 1, wherein said microorganisms comprise
viruses.
13. The method of claim 1, wherein said microorganisms comprise
hybridoma cells.
14. The method of claim 1, wherein mutant microorganisms are
selected from chemically complex and non-sterile agricultural,
industrial or other commercial process environments.
15. The method of claim 1, wherein said microencapsulated
microorganisms are grown in a laboratory culturing medium.
16. The method of claim 1, wherein said microencapsulated
microorganisms are grown in an agricultural or industrial process
medium wherein one or more species of said microorganism are found
in the native environment.
Description
FIELD OF THE INVENTION
The present invention relates to a method of isolating mutant
microorganisms from a population containing the same by
microencapsulation techniques and to kits for practicing the
methods of this invention.
BACKGROUND OF THE INVENTION
The enormous size of microbial populations has proved to be a great
asset in a variety of studies, but only because it is possible to
effectively select certain kinds of rare gene type or mutant
microorganisms. Mutant varieties of a single strain of
microorganism (procaryotic, eucaryotic or viral) have classically
been isolated by a variety of methods including positive cell
"selection" and differential "screening".
Selection is used to isolate mutant varieties of microorganisms
when a genetic alteration provides the microorganism with a
positive growth advantage over its parental population. For
example, acquisition of antibiotic resistance can be used to select
such mutants on a nutrient agar surface containing the antibiotic.
Another example is the acquisition of a biosynthetic gene enabling
the organism to grow in a culture medium that would not otherwise
support growth. There are, however, other genetic alterations such
as additions, substitutions or deletions of the microorganism's
genome which affect the primary or secondary metabolism of the
microorganism, causing a small change or negative change (decrease)
in the rate of growth. Such alterations may result in a beneficial
increase or decrease in the synthesis or the breakdown of chosen
biochemicals. Under such circumstances, screening must generally be
utilized to isolate the mutant colony. Screening may involve
examination of tens of thousands of individual colonies to
determine the presence of mutants. Replica plating is one such
screening technique. In general, it can be said that screening
techniques, although highly effective in achieving the desired
result, are labor and material-intensive requiring examination of
many individual colonies usually in petri dishes; replica plating
and tedious visual comparison of petri dish pairs are required as
well as relatively large amounts of selective and/or restrictive
materials which serve to differentiate the mutant from its
parent.
Recently, a technology has emerged which provides methods of
encapsulating biological material such as living tissue, individual
cells, viruses, and biological macromolecules within a
semipermeable membrane. The basic approach in this technique
involves suspending the biological material to be encapsulated in a
physiologically compatible medium containing a water soluble
substance that can be made insoluble in water, that is, a gel, to
provide a temporary environment for the biological material. The
medium is formed into droplets containing the tissue and gelled by
changing any one of the variety of ambient conditions. These
temporary capsules are then subjected to a treatment which results
in the production of membranes with a desired permeability
(including impermeable membranes). One such technique, is
exemplified in U.S. Pat. No. 4,352,883 entitled "Encapsulation of
Biological Material", the disclosure of which is incorporated
herein by reference.
A description of a technique for separating cells having desired
properties from a larger population is found in U.S. Pat. No.
4,401,755 entitled "Process for Measuring Microbiologically Active
Material" which discloses a method for measuring an unknown
quantity of microbiologically active material utilizing a
microencapsulation techniques similar to U.S. Pat. No. 4,352,883.
The disclosure of U.S. Pat. No. 4,401,755 is also incorporated
herein by reference. After preparing a suspension of gel
microdroplets, the suspension is processed in an apparatus having
the capability of sensing a physical characteristic of individual
gel microdroplets to determine the presence or absence of a desired
physical characteristic of the biological material in such a
droplet.
Microencapsulation technology, as described in the above referenced
patents, provides the potential for solving a variety of problems
including the labor and cost excesses of prior art mutant
microorganism isolation techniques. It is apparent that a need to
develop new isolation techniques exists which will reduce the costs
and time spent in selection and screening processes used to isolate
mutants from their respective parent populations.
SUMMARY OF THE INVENTION
The present invention provides a method of isolating a mutant
microorganism from it's parent population in the laboratory or in a
mixed multispecies population such as encountered in agricultural
or industrial environment, or in fermentation. Single or small
numbers of microorganisms from the parent population (containing
the mutant which is desired to be isolated) are encapsulated in a
semi-permeable membrane by means of microencapsulation techniques.
Thereafter, the cells are cultured and the microcapsules containing
substantially pure clones of microorganisms are treated to induce a
detectable difference (e.g. change in number of microorganisms per
microcapsule) between microcapsules containing the desired mutant
microorganisms and those containing non-mutant microorganisms. This
detectable difference (such as provided by a difference in cell
number) serves to enable the discrimination and/or separation of
microcapsules containing mutants from those with non-mutants.
Finally, the microcapsules containing mutant microorganisms are
isolated from those microcapsules containing non-mutant
microorganisms by separation techniques based directly or
indirectly on the detectable difference, e.g. a difference in
microcapsule density or mass resulting from a change in cell number
per microcapsule.
A non-inclusive list of characteristics which can be the basis of
identifying and/or separating microencapsulated mutant cells from
the parental population are increased or decreased cell growth
rate, cell density, cell size, level of synthesis of a detectable
primary or secondary metabolite, level of accumulation of chemical
elements or compounds containing these elements, rate of breakdown
of designated chemicals, antibiotic resistance and various
combinations of these properties.
For instance, the difference in cell number between microcapsules
containing mutant and non-mutant cells permits any one of a variety
of separation techniques. These include both simultaneous (bulk or
in toto) separation techniques as well as sequential (or serial)
separation techniques. Examples of bulk separation include
equilibrium density centrifugation, velocity or gravity
sedimentation, separation in electrical or magnetic fields,
chromatography, etc. Examples of serial techniques include
detection of individual microcapsules via radioactive, luminescent,
fluorescent or colorimetric labels, etc.
The present invention overcomes many of the prior art problems
associated with isolating mutant microorganisms from parent
populations in that, inter alia, it provides in some instances for
separation of microcapsules containing the mutants from the
microcapsules containing non-mutants without the necessity for
screening techniques which involve individual examination of
microorganisms and therefore can eliminate the labor and cost
excesses of prior art isolation techniques. In other instances, by
the increasing number of cells in microcapsules containing mutant
cells a particular physical characteristic is amplified so that the
mutant can more easily be separated from a parental population
(where the parental population also exhibits that characteristic,
but to a lessor degree).
DESCRIPTION OF THE INVENTION
In accordance with the present invention, a method is provided for
isolating mutant microorganism from a parent microorganism
population containing this same mutant. The method utilizes the
relatively new technique of microencapsulation of biological
material.
In one embodiment, the method of the present invention comprises
first, microencapsulating in a semi-permeable membrane individual
microorganisms in the microorganism population which contains the
mutant (desired to be isolated). This enables single mutant
microorganisms to divide within a physical envelope so that
whatever physical, chemical and/or biological identity they possess
as single mutants (either a constant identity or an inducible
property) can be amplified so as to facilitate the physical
separation and recovery of the desired mutant.
Encapsulation of single microorganisms can be accomplished by
techniques well known to those skilled in the art and, as will be
appreciated, results in microcapsules which are clonally pure. In
particular, single microorganism encapsulation can be readily
achieved by controlling the concentration of the microorganism in
suspension such that each microcapsule will receive, on average,
one microorganism. Generally, the microorganisms to be encapsulated
are prepared in accordance with well known prior art techniques, as
individual (disaggregated) cells, and suspended in an aqueous
medium suitable for maintaining viability and for supporting the
ongoing metabolic processes of the particular microorganism
involved. Media suitable for this purpose are available
commercially. Similarly small numbers of microorganisms can be
encapsulated in one microcapsule, if so desired.
The microcapsules are formed so that there is a high probability
that each microcapsule contains a small number or one unit of
microbiologically active material (i.e. microorganism or cell).
This can be effected by regulating the dilution of the liquid
composition to be used to produce the microcapsules using knowledge
of the size of the microbiologically active material and the
predetermined size of the microcapsule to be produced. The
regulation of these factors can be determined by conventional
Poisson statistical analyses so that the number of microcapsules
containing more than the desired number of microbiologically active
materials is more than two standard deviations from the mean. It is
desirable, for example, to encapsulate zero to one
microbiologically active cell per microcapsule in mutant screening
and in recombination DNA research (where the object is generally to
isolate desirable spontaneous mutant microorganisms or genetically
engineered microorganisms from a large parental population of such
microorganisms).
The preferred encapsulation technique is that described in the
above-referenced U.S. Pat. No. 4,352,883 (Lim). In brief, this
approach involves suspending the microorganism to be encapsulated
in a physiologically compatible medium containing a water soluble
substance that can be made insoluble in water (gelled) to form a
temporary protective environment for the microorganisms so
encapsulated. The medium is next formed into droplets containing
the single microorganism and gelled, for example, by changing
ambient conditions such as temperature, pH or the ionic
environment. The "temporary capsules" thereby produced are then
subjected to a treatment that results in the production of a
membrane of a controlled permeability about the shape-retaining
temporary capsules.
The temporary capsules can be fabricated from any non-toxic, water
soluble substance that can be gelled to form a shape-retaining mass
by a change of conditions in the medium in which it is placed, and
that also comprises plural groups which are readily ionized to form
anionic or cationic groups. The presence of such groups in the
polymer enables surface layers of the capsule to be cross-linked to
produce the desired membrane when exposed to polymers containing
multiple functionalities of the opposite charge.
The presently preferred material for forming the temporary capsules
is a polysaccharide gum, either natural or synthetic, of the type
which can be (a) gelled to form a shape-retaining mass by being
exposed to a change in conditions such as a pH change or by being
exposed to multivalent cations such as Ca.sup.++ ; and (b)
"cross-linked" or hardened by polymers containing reactive groups
such as amine or imine groups which can react with acidic
polysaccharide constituents. The presently preferred gum is alkali
metal alginate. Other water soluble gums which can be used include
guar gum, gum arabic, carrageenan, pectin, tragacanth gum, zanthan
gum or acidic fractions thereof. When encapsulating thermally
refractory materials, gelatin or agar may be used in place of the
gums.
The preferred method of formation of the droplets is to force the
gum-nutrient-tissue suspension through a vibrating capillary tube
placed within the center of the vortex created by rapidly stirring
a solution containing a multivalent cation. Droplet ejected from
the tip of the capillary immediately contact the solution and gel
as spheriodal shaped bodies.
The preferred method of forming the desired semi-permeable membrane
about the temporary capsules is to "cross-link" surface layers of a
gelled gum of the type having free acid groups with polymers
containing acid reactive groups such as amine or imine groups. This
is typically done in a dilute solution of the selected polymer.
Generally, the lower the molecular weight of the polymer, the
greater the penetration into the surface of the temporary capsule,
and the greater the penetration, the less permeable the resulting
membrane. Cross-links are produced as a consequence of salt
formation between the acid reactive groups of the cross-linking
polymer and the acid groups of the polysaccharide gum. Within
limits, semipermeability can be controlled by selecting the
molecular weight of the cross-linking polymer, its concentration,
and the duration of reaction. Cross-linking polymers which have
been used with success include polyethylenimine and polylysine.
Molecular weight can vary, depending on the degree of permeability
required, between about 3,000 and 100,000 or more. Good results are
obtained using polymers having an average molecular weight on the
order of 35,000.
Optionally, with certain materials used to form the temporary
capsules, it is possible to improve mass-transfer within the
capsule after formation of the desired membrane by re-establishing
the conditions under which the material is liquid, e.g., removing
the multivalent cation. This can be done by ion exchange, e.g.,
immersion in phosphate buffered saline or citrate buffer. In some
situations, such as where it is desired to preserve the
encapsulated tissue, or where the temporary gelled capsule is
permeable, it may be preferable to leave the encapsulated gum in
the cross-linked, gelled state.
An alternative method of membrane formation involves an interfacial
polycondensation or polyaddition reaction. This approach involves
preparing a suspension of temporary capsules in an aqueous solution
of the water soluble reactant which includes a pair of
complementary monomers which can form a polymer. Thereafter, the
aqueous phase is suspended in a hydrophobic liquid in which the
complementary reactant is soluble. When the second reactant is
added to the two-phase system, polymerization takes place at the
interface. Permeability can be controlled by controlling the makeup
of the hydrophobic solvent and the concentration of the reactants.
Still another way to form a semi-permeable membrane is to include a
quantity of protein in the temporary capsule which can thereafter
be cross-linked in surface layers by exposure to a solution of a
cross-linking agent such as gluteraldehyde.
The second step of the method of the present invention preferably
comprises growing the microencapsulated microorganism under
conditions which induce a difference in the number of
microorganisms per capsule between microcapsules containing mutant
microorganisms and those containing non-mutant microorganisms. The
detectable difference can be a result of the difference in cell
number, per se, or the cells can be treated to amplify the
difference between mutant clones and parental clones. That is,
within the last three generations of growth or, in some cases,
after the microorganisms within the microcapsules have been grown,
they can be further treated in order to amplify particular
characteristics in the material to facilitate identification and
isolation of microcapsules containing mutants. Methods of treatment
include incubation, incubation with heavy isotope or radioactive
isotope metabolites, staining with fluorescent stains, labeling
with magnetically tagged substances or immunological agents,
etc.
In one embodiment of the present invention the microorganisms are
grown under non-restrictive non-selective conditions within the
microcapsules for several generations to a predetermined
microorganism density to establish the microorganism's viability
and the appropriate population number within each microcapsules. By
way of example, single microorganisms are grown, using complete
medium, to no more than about 5-10% of the final density within the
microcapsule. At this point, it is possible, if desirable, to
eliminate empty microcapsules and those containing non-viable or
very slow growing organisms by for example differential
density-sedimentation, because microorganisms are heavier than
water. If the desired mutant is slow growing, the separation is
accomplished at this point. Thereafter, the microcapsules are grown
under conditions which would induce a difference in mass between
mutant and non-mutant containing microcapsules. For example,
restrictive and/or selective conditions could be employed during
this stage of growth to induce the desired difference in cell
number per capsule. If the mutant desired to be isolated, for
example, is characterized in that it has acquired antibiotic
resistance, then the cells within microcapsules can be cultured in
a medium containing the specific antibiotic. This results in mutant
growth within microcapsules while further growth of non-mutant
strains in microcapsules would be prevented. If, on the other hand,
the mutant requires a particular amino acid to grow, restrictive
conditions can be used which will result in mutant-containing
microcapsules possessing fewer microorganisms than
non-mutant-containing microcapsules, hence providing the desired
cell number differentiation.
This has obvious industrial importance. In isolating mutants of
yeasts, molds, single cell bacteria, and actinomycetes, in which
the mutants overproduce valuable enzymes and primary metabolites,
it has been noted that growth of such mutants is usually slower
than the parental cells. This is because unbalanced metabolism, if
not producing cell toxicity, results in energy waste and nutrient
limitations. These mutants, which are normally hidden in the
population, can be selected in accord with the present invention
by, for example, a microcapsule density centrifugation protocol.
Moreover, the growth rate difference and resulting difference in
cell number (which is the basis for the separation) between
microcapsules containing mutant and non-mutant microorganisms may
be accentuated further by a chosen feeding regimen. Depending on
the metabolic process desired to be de-regulated in the mutant, an
appropriate nutrient source (i.e., carbon, nitrogen, phosphate, or
other essential nutrient) can be made limiting. This same growth
rate selection protocol can be used to functionally differentiate
mutations in genetically engineered organisms in which, out of a
spectrum of mutations, very few result in enzyme or metabolite
overproduction.
In another embodiment, mass differentiation between mutant and
non-mutant-containing microcapsules can be enhanced by transferring
the microcapsules to culture medium containing heavy isotope
metabolites including, for example, deuterium oxide and/or .sup.15
N-labeled compounds for an appropriate period of time.
Subsequently, the microcapsules are washed in normal culture medium
such that the cells within the microcapsules selectively retain
heavy isotopes. This procedure serves to increase the overall
density difference between microcapsules containing many as
compared to those containing few cells.
In still another embodiment, mass differentiation between mutant
and non-mutant-containing microcapsules can be established by
inversion of the density of the microcapsules. This is accomplished
by incubating the microcapsules in any non-toxic high density
medium of appropriate diffusion rate. The microcapsules containing
fewer microorganisms will exchange more volume of the medium than
microcapsules containing the greater number of microorganisms, thus
becoming more dense and establishing the desired difference in
mass.
Another distinct basis for density separation of encapsulated
mutant microorganisms from the encapsulated parental microorganism
population is the difference in the intrinsic density of the
microorganism itself. If, for example, a mutant microorganism
accumulates a heavy metal in elemental or ionic form more
efficiently than the non-mutant then, after growth in the presence
of such a metal, the microcapsule containing the mutants would be
more dense. Similarly, other metabolites, periodic elements, or
compounds thereof, which accumulate either in the microorganism or
in the microcapsule and change the microcapsule density, can
provide the basis of separation based on difference in mass between
the various microcapsules.
The final step required in practicing the present invention
comprises separating the microcapsules containing mutants from
those containing non-mutants based on the detectable difference,
such as the difference in number of cells per capsule therebetween.
Any one of a variety of techniques well-known to those skilled in
the art may be employed to effect the desired separation. In
particular, where applicable, it is preferable to use equilibrium
density centrifugation, or alternatively velocity sedimentation.
Electrical or magnetic field separation protocols, or a combination
thereof can also be used to effect separation. Automated laser cell
sorting devices can similarly be utilized to sort microcapsules
containing differing numbers of cells. If cells are, for example,
uniformly labeled with a fluorescent dye then microcapsules
containing greater numbers of cells will fluoresce with a greater
intensity than microcapsules containing fewer cells. Thus,
microcapsules containing mutant cells are separable from
microcapsules containing non-mutant cells.
The above approach to mutant isolation is revolutionary in several
senses. Once microcapsule size and porosity are chosen, the
isolation of mutant microorganisms becomes a simple task. In a
preferred embodiment, centrifugation replaces visual scanning of a
field or any other collection of microorganism colonies. Isolation
of single microcapsules containing desirable mutants is
accomplished by, for instance, equilibrium density centrifugation.
Once the centrifugation parameter is established for a species of
microorganism, the isolation of almost any mutant of that species
is facilitated.
Conditional lethal mutations can be difficult to isolate, however,
because the microencapsulation method relies on non-growth, yet
survival, of such mutants under restrictive growth conditions for
recovery of the mutants. Many conditional lethal mutations may,
however, still be isolated by the method of the present invention,
because only one microorganism out of the many present in each
microcapsule at the time of shift to restrictive growth conditions
need survive.
Microorganisms for which the method of the present invention is
useful include procaryotic cells--such as, for example,
microorganisms including single cell bacteria, spores, and
actinomycetes, eucaryotic cells--such as yeasts, molds and higher
plant and animal cells including fused cell hybrids such as
antibody-producing hybridoma cells, and virally-infected cells. A
non-inclusive list of cell identities which can be the basis of
identifying mutant cells are increased or decreased growth rate,
cell density, cell size, level of synthesis of a detectable primary
or secondary metabolite, level of accumulation of chemical
elements, or compounds of these elements, rate of breakdown of
designated chemicals, antibiotic resistance and various
combinations of these properties.
In another embodiment. a hybridoma cell mixture (which may have
been preselected for fused (hybridoma) cells with antibiotic or
nutrient regimen to eliminate unfused parental cells) is suspended
at a cell concentration such that each microcapsule will receive,
on average, one hybridoma cell. For extremely rare monoclonal
antibody selection it may be desirable to originally encapsulate a
"pool" of cells, i.e., 2-50 hybridoma cells per microcapsule.
Following growth and antibody expression within the microcapsules
(suspended in appropriate nutrient medium) the microcapsules are
screened for specific antibody production by employing a challenge
antigen.
The antigen may be fluorescent or radioistope-labeled such that
following incubation of the microcapsules in the presence of
microcapsule-diffusible (small) antigen and thorough rinsing to
remove unbound antigen, the specific binding of labeled antigen to
antibody can be easily monitored. In the case of antigens larger
than the effective microcapsule pore size and therefore not
microcapsule-diffusible, the labeled antigen is first broken down
by mechanical or enzymatic cleavage to a diffusible size and then
employed in the screening.
This has enormous potential as a research tool for the following
reasons. For example, if an objective is to generate monoclonal
antibodies against surface determinants on a patient's cancer
cells, the cancer cells or their outer membranes would be injected
into a mouse to elicit antibody response. Later the individual
mouse spleen cell-myeloma cell hybrids would be microencapsulated
and grown to appropriate density within the microcapsules.
Hybridoma cell-monoclonal antibody within these microcapsules
cannot be challenged with whole cancer cells. Rather, the surface
components of the intact cancer cells would be made fluorescent or
radiolabeled. The cancer cells would then be broken, the labeled
membrane pelleted, and this membrane then broken and/or
enzymatically digested to reduce the labeled surface components to
microcapsule-diffusible size. These labeled components would then
be utilized for microcapsule monoclonal antibody screening. The
microcapsules containing different monoclonal antibodies optionally
can be pre-incubated (pre-competed) with the unlabeled
(non-fluorescent and non-radioactive) cell surface components of
the respective non-cancerous cells. Such pre-competing reduces the
"false-positive" microcapsules, i.e. those not producing
cancer-specific antibodies.
One can also differentially label (by isotope and fluorescent
derivatives) different classes of macromolecules on the cancer cell
surface such as protein, carbohydrate or lipid. One can then
distinguish microcapsules containing antibodies directed against
the different classes of cellular macromolecules.
Following the binding of labeled antigen to antibody within the
microcapsule, residual unbound antigen is washed from the
microcapsules using appropriate buffer or culture medium. The
desired microcapsules containing radioactive and/or fluorescent
bound antigen are physically separated from the gross population of
microcapsules. Most easily separated are fluorescent spheres which
can be harvested using an automated laser cell sorter typically
employed to separate T and B lymphocyte cells. Rapid screening of
radioactive (as well as fluorescent) microspheres can also be
accomplished by a very different and less expensive method. First,
an 8.times.10 inch "monolayer" of spheres can be deposited on a
plate (each sphere covers approximately 0.01 mm.sup.2). This layer
will contain approximately 5.times.10.sup.6 microspheres.
Photographic emulsion-autoradiography can be utilized to locate the
radioactive microspheres. Since these microspheres are on the order
of 100 microns (0.1 mM) in diameter their autoradiographic image
should be visible on film. A luminescent reference grid included in
the microsphere support surface helps the investigator to align the
film with the microsphere support surface and identify a small
region containing the radioactive microsphere. Suction aspiration
of this region yields a small number of microspheres. These small
groups of microspheres are spread out and again autoradiographed to
yield individual desired microspheres. For fluorescent antigen
applications, the microspheres are briefly exposed during
photography to UV light. Again a luminescent or fluorescent spacial
reference grid is included to align the film with the microsphere
support surface. The support surface between microspheres and film
is preferably sufficiently thin to minimize angular dispersal of
microsphere source radiation.
Current methods for selecting new and commercially important
strains of microorganisms for industrial process usually involve
screening single cells for altered physical or biochemical
properties (under the microscope) or screening multicellular
colonies derived from single cells for new biochemical properties,
or for increases or decreases in existing biochemical abilities.
This is particularly difficult to do when the microorganism is a
member of a complex culture or ecosystem consisting of more than
one species of live organism (said complex culture being also
termed "non-sterile"). Furthermore, the property or properties of
the desired strain (i.e. "mutant") may depend upon concerted growth
of more than one microorganism in the non-sterile medium. Some
examples of such non-sterile culture environments include
activated-sludge process for sewage treatment, fruit and grain mash
process, biomass fermentation, dairy process, petrochemical
process, mineral leaching process, soil microorganism growth
(nitrogen fixation, etc.), swamp and lake eutrification process,
etc. In accord with the method of the present invention in order to
obtain a mutant of a particular microorganism strain for industrial
process, the microorganism is microencapsulated as described above.
After incubating the microcapsules in the complex culture
environment, the microcapsules are recovered and those containing
the desired mutant cells are selected as described herein. Valuable
new mutants of desired species can thus be isolated from such
complex environments. One can select, screen, and recover mutant
cells which have grown to microcolonies within the microcapsules
while they are in "communication" via diffusion with the complex
environment.
It is easy to see that state of the art isolation of plant or
animal cell mutants, e.g. mammalian cells in tissue culture, is
revolutionized by the method of present invention. This envisions
encapsulation of single cells, growth of all cells for a few
generations without selection, and finally completion of growth
under the restrictive and/or selective conditions. This procedure
is preferably followed by the density-centrifugation separation of
mutant from wild-type cells. Nutritional cross-feeding of
procaryotic or eucaryotic mutants by wild-type cells is not a
problem. To the contrary, the mutant and wild-type clones are
physically separated from each other by the microcapsule membrane
as are colonies on agar. More importantly, because the selective
phase typically lasts about five to seven generations using
microcapsules, any microcapsule colony cross-feeding problems are
reduced compared with conventional colonies experiencing longer
incubations.
In still yet another embodiment, other substances may be included
along with the microorganism during the microencapsulation step.
For instance, it may be desirable to include growth hormones such
as fibroblast growth factor and/or epidermal growth factor, other
non-diffusible growth factors (typically proteins or glycoproteins)
or adhesion surfaces, such as fibrinogen, collagen, etc.
For example, adhesion surfaces (or substratum), such as
microcarrier beads, may be encapsulated together with the
microorganisms. Certain cells require an adhesion surface for
growth. The encapsulation of microcarrier beads as the substratum,
each bead carrying a single cell, prevents cross-contamination of
microorganisms which would otherwise occur between non-encapsulated
carrier beads. The encapsulation of microorganisms on beads is
particularly useful in growing microorganisms such as normal and
malignant mammalian cells which often require the presence of solid
substratums. For example, in screening potential anti-cancer drugs,
such microencapsulation allows one to follow the inhibition of
growth and development of individual cancer cells in the presence
of chosen drug regimens.
The present invention also provides the means for solving other
prior art problems. For example, conventional agar methods of
selecting mutations conferring positive growth advantage have been
previously mentioned. When, however, the parental cells exhibit
"leaky" growth or grow at a rate just 10-20% slower than the
desired mutant, the selection may become problematic. In such a
case repeated serial "passage" of a culture may be attempted to
enrich for the faster growing mutant. However, if positive
selection is performed in microcapsules and in accord with the
present invention, the following would be expected. A 200 um
diameter microcapsule (4.times.10.sup.-6 ml volume) is formed
containing one mutant cell. This cell is a bacterium which in
normal medium grows to a cell density of 10.sup.10 cells/ml or
about 4.times.10.sup.4 cells/microcapsule. This represents 15 cell
doubling (generations). If the mutant's growth is only 10% faster
than the parent, then after 14 generations, the "mutant"
microcapsules will contain twice as many cells as the other
microcapsules. This can provide a sufficient basis for physical
separation. The same mutant cells grown by serial passage selection
for the same number of generations would simply be enriched
two-fold within the bulk parental population. Using calculations,
one can rapidly estimate the ability to isolate mutants of other
microorganisms given a modest growth rate differential. Thus, the
advantages of the present invention are readily apparent.
The present invention also provides a kit for practicing the
methods described above. The kit comprises the ingredients or
components required to form microcapsules on a laboratory or
research scale to screen for mutants in accord with the present
invention. As such, the kit comprises a container or package having
quality-controlled reagents therein: sterile alginate solution,
sterile 2-(cyclohexylamino)ethane sulfonic acid, sterile solution
of polylysine having a predetermined molecular weight and a sterile
solution of polyethylenimine (PEI) having a predetermined molecular
weight. The molecular weights of the polylysine and PEI are
predetermined to produce microcapsules having a desired
permeability in accord with the known technology as described in,
for instance, U.S. Pat. No. 4,352,883. In addition, sterile
CaCl.sub.2 and mechanical devices for forming microdroplets can be
supplied as part of the kit. Preferably, the solutions are provided
in sealed sterile vials having sufficient quantities for one
experiment. Further, it is preferred that the solutions comprise
physiological saline and the polylysine and PEI solutions also
contain 0.2M MOPS [3-(N-morpholino)propanesulfonic acid] buffer
(pH6).
From the foregoing it will be apparent that isolation of mutant
microorganisms in accordance with the present invention can be
practiced on a wide variety of organisms, using: (1) a wide variety
of techniques to induce the difference in number of microorganisms
per capsule between mutant and non mutant-containing microcapsules,
and (2) a wide variety of separation techniques to isolate the
desired mutant without departing from the scope and spirit of the
invention.
It is appreciated that those skilled in the art, upon consideration
of this disclosure, may make modifications and improvements within
the spirit and scope of this invention.
* * * * *