GB2159171A - Process for screening or selecting cells - Google Patents
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- GB2159171A GB2159171A GB08428320A GB8428320A GB2159171A GB 2159171 A GB2159171 A GB 2159171A GB 08428320 A GB08428320 A GB 08428320A GB 8428320 A GB8428320 A GB 8428320A GB 2159171 A GB2159171 A GB 2159171A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
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- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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Abstract
An improved process for screening cell cultures, e.g., cultures of genetically modified cells or hybridomas, for cells secreting a specific substance involves encapsulating cells within membranes permeable to the substance of interest but substantially impermeable to the cells. The cell-containing capsules are cultured in an apparatus having plural compartments, e.g. a microtiter tray, each compartment containing an extracapsular medium and capsules containing cells. The encapsulated cell producing the substance of interest synthesizes and secretes the substance into the intracapsular volume of the capsule and the secreted substance passes through the membrane into the extracapsular medium. The extracapsular medium of each compartment is assayed for the substance and the process is repeated for the capsules from all compartments yielding a positive assay. Thus, by separating the capsules in a positive-assaying compartment from one another and placing them anew in separate compartments, the assay can be repeated to locate that compartment containing the cell which secretes the substance of interest, thereby to identify the cell and differentiate it from other cells.
Description
SPECIFICATION
Process for screening or selecting cells
The present invention relates to a process for screening or selecting cells.
More particularly, this invention concerns the selection of a cell secreting a particular substance from a heterogeneous cell population. For example, the invention provides a process for simpler screening of cell cultures, including recombinant DNA and hybridoma fusion products, for viable cells secreting a specific substance.
Recent advances in the producton and modification of biological material, e.g., genetic engineering and monoclonal antibody hybridoma technology, have led to a need for the identification of individual cells producing specified substances. For example, monoclonal antibody production involves fusing a heterogeneous cell culture, including at least one cell producing the specific antibody, with an immortal cell line, e.g., a myeloma. Even in the best of conditions, the fusion technique is only partially successful; some cells do not fuse, some cells from the culture fuse with each other rather with the myeloma, and some myeloma cells fuse with each other. Even assuming that all the cells of the cell culture fuse with myeloma, few of the fusion products would produce the substance of inerest.
Accordingly, a screening technique is needed to select specific fusion products. One currently used screening technique consists of culturing the cells from a fusion mixture in microtiter plates, assaying the fluid of each compartment of the microtiter plates for the substance of interest, and subculturing the cells in each compartment yielding a positive assay. Problems associated with this procedure include the possibility of causing physical damage to the cells upon separaton for subculturing, the di,' :ulty of separating the cells, the possibility of contaminating the cultures, and the effect of extraneous matter, e.g., cell fragments and high mole cularweight contaminants, on the specificity of the assay for the substance of interest. A further difficulty is that certain types of cells do not grow well in microtiter plates.
Recombinant DNA technology also requires screening for viable recombinants. Standard recmbinant formation tecnology includes the introduction of a plasmid or other vector into a cell, normally a prokaryotic organism. The plasmid or vector contains a gene which can be transcribed within the cell to form the substance of interest. As with hybridoma technology, a large percentage of the fusion products of the plasmids or vectors and the cells do not form viable cultures which produce the substance of interest, so a screening process is necessary. Techniques similar to those used for the identification of hybridomas are common in recombinant technology and similar attendant problems are presented.
are presented.
U.S. Patent No. 4,352,883, the disclosure of which is incorporated herein by reference, teaches a method for encapsulating viable cells, including hybridomas or genetically modified cells, within a semipermeable membrane. After encapsulation, the cells are healthy, viable, capable of ongoing normal metabolism, secrete the materials they normally secrete, and can undergo mitosis when disposed within the capsules. Recent experiments have indicated, in accordance with the broad disclosure of the said U.S. patent, that it is possible to control the dimensions of the pores of the capsule membrane while precluding transit of cells and high molecular weight contaminants. In this manner, it is possible to use the membrane of the capsule as a filtering agent which facilitates assay for the substance of interest.
This prior encapsulation tecnique helps prevent mechanical damage of the cells and simplifies cell handling.
It is an object of the present invention to provide a new screening technique for identifying cells secreting a substance of interest from a mixed cell culture.
By way of example, the invention aims to provide a process for selecting bybridomas producing a monoclonal antibody of interest and a method of identifying genetically modified cells which secrete a substance of interest. Further, the invention seeks to provide a process for identifying or selecting hybridomas producing a substance of interest from a cell containing at least one cell producing the substance.
According to one aspect of the invention, there is a process for screening a cell culture for a selected cell secreting a substance of interest, comprising the steps of:
A. encapsulating the cells of the cell culture within a plurality of capsules, each capsule comprising an intracapsularvolume defined buy a membrane permeable to the said substance but impermeable to the cells;
B. dispensing the encapsulated cells within a plurality of compartments each of which contains an extracapsular medium;
C. allowing the selected cell to synthesize and secrete the said substance within the intracapsular volume;
D. allowing the secreted substance to traverse the membrane;
E. assaying the extracapsular medium of each compartment for the said substance; and
F. repeating Steps B to E with the capsules from a compartment which yields a positive assay in Step
E until a capsule containing the said selected cell is identified.
According to another aspect of the invention, there is a process for identifying a selected cell secreting a substance of interest from a heterogeneous cell population, comprising the steps of:
1. fusing a plurality of cells, comprising at least one cell producing the said substance of interest, with an immortal cell line to produce a heterogeneous population of cells comprising fused cells;
2. encapsulating the said population within a plurality of capsules, each capsule comprising a membrane defining an intracapsularvolume and being permeable to the said substance but impermeable to the fused cells;
3. dispensing the encapsulated cells within a plurality of compartments each of which contains an extracapsular medium capable of supporting ongoing metabolism of a cell secreting the said substance;;
4. allowing the selected cell to synthesize and secrete the said substance within the intracapsular volume;
5. allowing the secreted substance to traverse the membrane; 6. assaying the extracapsular medium of each compartment for the said substance; and
7. repeating Steps 3 to 6 with the capsules from a compartment which yields a positive assay in Step 6 until a capsule containing the selected cell is identified.
The invention is now described in more detail in the following description, which is given only by way of example of the invention.
The invention features a process for screening or identifying a selected cell which secretes a substance of interest. In practising the invention, a heterogeneous cell culture containing a plurality of cells is encapsulated within a plurality of capsules.
The cell culture may be any culture which includes a cell secreting the substance of interest, e.g., a hybridoma formed by fusing a lymphocyte and an immortal cell line such as a myeloma, or a genetically modified cell such as a fusion product of a prokaryotic cell and a vector containing a gene coding for the production of the particular substance by intracellular transcription. The capsules have membranes defining their intracapsular volume, the membrane being permeable to the substance of interest and impermeable to cells. The membrane may also limit passage of molecules having a molecular weight greater than a predetermined value. In the case of hybrodimas, the cells can be encapsulated art a density that yields no greater than one hybridoma in every 10 capsules; this, then, would constitute a clone.The encapsulated cells are cultured within an apparatus having a plurality of compartments, each compartment containing an extracapsular medium which is preferably a differential growth medium. The substance of interest is synthesized and secreted within the intracapsular volume by the appropriate cell and the secreted substance traverses the membrane into the extracapsular medium. The extracapsular medium of each compartment is assayed for the substance of interest and the process is repeated with the capsules from each compartment which yield a positive assay for the substance until the selected cell is identified. The compartments are preferably in the form of wells which can retain the extracapsular medium and capsules.In the case of hybridomas, the ability to produce a clone from the initial fusion procedure facilitates screening, since no capsule would contain more than a single hybridoma, and greatly accelerates the isolation of stable, antibodysecreting hybridomas.
Capsules identified in this process can be used as an immunizing agent. The capsules are injectedinto an animal which then produces antibody against the secreted substance. Our U.K. applicaton No.
(Agent's ref: 6176), filed simultaneously with the present applicaton is directed to immunization with encapsulated cells.
The identified capsules can also be implanted into a host animal, e.g., into the peritoneal vacity of a mouse. The secreted substance can then be recovered from the implantation site, preferably from fluid produced by the host in response to the implantation.
The process according to the present invention, for screening a cell culture for a particular cell secreting a substance of interest, is particularly useful in hybridoma technology. Fusion products of lymphocytes and an immortal cell line, e.g. a myeloma, must be screened to determine which cell is producing the monoclonal antibody of interest.
Similarly, the process is well suited for recombinant
DNA technology. The present screening process can be carried out in a simpler, less destructive manner than conventional techniques allow. The invention is particularly well suited to identifying anchorage dependent cells producing a particular substance.
To perform the process, first, the heterogeneous cell culture containing the cell to be selected is obtained. A previously prepared cell culture, e.g., a hybridoma culture or genetically modified cell line, may be used, or a new culture may be prepared specifically for the process. An exemplary hybridoma forming technique, as reported by G. Kohler and
C. Milstein, "Continuous Culture of Fused Cells
Secreting Antibody of Predetermined Specificity",
Nature 256:495 (1975), starts with a culture, e.g., lymphocytes from a spleen at least some of which are producing the substance of interest. The culture is divided into individual cells using conventional techniques, e.g. by forcing the spleen through a wire mesh. The cells are mixed together with an immortant cell line, e.g., a myeloma, and the hybridoma is formed by polyethylene glycol (PEG) fusion.More particularly, the lumphocytes and myeloma are incubated in a fusion medium such as Dulbecco's modified Eagle's medium (DMEM) and about 40% PEG is added. The cells are removed from the PEG solution and grown as a culture before screening.
Preferably, the myeloma is antibiotic sensitive or requires additives to the medium so a differential growth medium, e.g. a medium containing drugs which kill non-fused cells, can be used as a separating device. Once viable hybridomas have been identified, further screening may be necessary to determine if they are producing the substance of interest.
If the cell to be selected is genetically modified, one can use a formation procedure such as disclosed by F. Bolivar et al., Construction and Characterization of New Cloning Vehicles, II, a multipurpose cloning system. Gene 2, 95-113 (1977). A plasmid or vector is inserted into a cell using restriction enzymes and the cell is cultured using standard procedures. The plasmid or vector contains a gene that can be transcribed by the cell to produce the substance of interest. As in the case with the hybridomas, a differential growth medium is helpful for cell selection.For example, the plasmid containing the gene coding for the substance of interest may also contain a gene which allows the fusion productto be differentiated from non-fusion products, e.g., the plasmid may provide rifamycin resistane to a rif sensitive cell, thereby permitting the use of a rif-containing differential growth media for screening.
After the cell colony has been formed, the cells are encapsulated. The general technique set forth in the aforesaid U.S. patent No. 4,352,883 is preferred. The dimensions of the pores of the microcapsules may be controlled as disclosed in the patent and as further disclosed herein. In accordance with teachings herein, it is possible to encapsulate cells that secrete both IgG (molecular weight about 160,000 daltons) and IgM (molecular weight about 900,000 daltons) within capsules that permit free passage of IgG through the membrane pores while substantially precluding passage of IgM. The capsule membrane acts as a filter, precluding passage of cells and high molecular weight contaminants.
As previously noted, the preferred encapsulation technique is disclosed in the aforesaid U.S. patent.
The cell culture is first prepared in a finely divided form (in accordance with well known techniques) and suspended in aqueous medium suitable for maintaining and supporting ongoing metabolic processes. Suitable maintenance media, e.g., Dulbecco's PBS (Ca/Mg free), are known to those skilled in the art. Awater-soluble substance, physiologically compatible with the cell, which may be rendered water-insoluble to form a shape-retaining, coherent temporary capsule or gelled mass is added to the medium. The resulting solution is then formed into droplets containing the cells together with their growth medium and the droplets are immediately rendered water-insoluble, forming the temporary capsules.The gellable material may be non-toxic, water-soluble material which by change in temperature, pH, or ionic environment is rendered waterinsoluble without affecting the metabolic processes of the cells. Preferably, the gellable material contains plural, easily ionizable groups, e.g., carboxyl or amino groups, which can react with polymers containing plural groups of the opposite charge by salt-bond formation.
The presently preferred gellable materials are natural or synthetic polysaccharides, preferably sodium alginate. Other polysaccharides, useful in practising the invention include acidic fractions of guar gum, gum arabic, carageenan, pectin, tragacanth gum, and xanthan gum. These materials can be "cross-linked" by reaction with cations to form shape-retaining masses and can be resolubilized upon removal of the cross-linking ions by ion exchange or by means of a sequestering agent.
A gellabie solution which has been used successfully contains about 106 cells/ml in 1.0 - 1.5% (w/v) sodium alginate solution. The solution is formed into droplets, e.g., by forcing the solution through a jet head apparatus. The jet head apparatus consists of a housing having an upper air intake nozzle and an elongate hollow body friction fitted into a stopper. A syringe, e.g., a 10 cc syringe, equipped with a stepping pump is mounted atop the housing with a needle, e.g., a 0.01 inch (0.25 mm) I.D. Teflon coated needle, passing through the length of the housing.
The interior of the housing is designed such that the tip of the needle is subjected to a constant laminar airflow which acts as an air knife. In use, the syringe full of the solution containing the material to be encapsulated is mounted atop the housing, and then the stepping pump is activated to incrementally force drops of the solution to the tip of the needle.
Each droplet is "cur-off" by the air stream and falls approximately 2.5-3.5 cm into a gelling solution where it is immediately gelled by absorption of cross-linking ions. The preferred gelling solution is a solution containing calcium ions, e.g., 1.2% (w/v) calcium chloride in saline. The distance between the tip of the needle and the calcium chloride solution is great enough to allow the sodium alginate/cell solution to assume the most physically favoured shape, i.e. a sphere (maxuimum volume/surface area). Air within the tube bleeds through an opening in the stopper. The gel led, shape-retaining spheroidal masses or temporary capsules, which preferably are between 50 microns and a few millimeters in diameter, collect in solution as a separate phase and can be recovered by aspiration.
A permanent membrane is then formed about each temporary capsule by cross-linking surface layers of the temporary capsule. Preferably, an aqueous solution of a polymer having plural groups of the opposite charge is used to cross-link the gelled masses. If carboxylated or other acidic polysaccharides are used to form the temporary capsule, polycationic materials containing acid reactive groups such as amines, imines or amides are useful as cross-linking agents. Presently preferred polycationic materials include polylysine, polyethyleneamine, and polyvinylamine. The polycationic materials react with acidic groups on the polyanionic material of the gelled mass to form sale bobd-type cross-links.Depending on the system used, it may be preferable to react the permanent capsule formed by this reaction with a solution which occupies free cationic groups, e.g., by reacting the capsule with a solution of low concentration sodium alginate.
Capsules designed for the use in the present invention have membranes tailored to provide the requisite filtering by molecular weight needed to allow the substance of interest to traverse the membrane while maintaining higher molecular weight contaminants and cells within the membrane, As previously noted, capsules can be formed to permit free passageof IgG while substantially precluding passage of IgM. Two new techniques have been developed to help control pore dimensions, one based on a multiple layer technique and the second based on a hydration phenomenon. In the first of these techniques, the gelled spheres are immersed in polycationic solutions of different ionic strength or different concentration. Layers of materials deposited about the gelled spheres form multiple layer structures. For example, temporary capsules can be immersed in a solution of high molecular weight polylysine followed by a low molecular weight polysyline or polyvinylamine solution. The effect of the second immersion is to form a second membrane which decreases the pore size.
The second pore dimension control technique exploits the observation that alginate gels vary in volume depending on their hydration, which in turn depends on the ionic strength of the metal ion slution in which they are equilibrated. A membrane formed about an expanded alginate gel mass is more uniform than those formed about unexpanded gel masses. This phenomenon, coupled with the observation that membranes formed about dense gels tend to remain intact when the gel is expanded, leads to a method of increasing pore dimensions and forming improved membranes.For example, a membrane with a pore size of about 200,000 daltons (sufficient to release IgG while retaining IgM) can be formed by using a moderate molecular weight polycationic cross-linker about a high density, cellcontaining gel after equilibrating and expanding the gelled masses with a low ionic strength monovalent cation. The monovalent cation solution removes calcium ions and increases the degree of hydration of the gel, causing the pores to enlarge.
While the techniques disclosed herein can improve membrane permeability control, membranes formed following these teachings do not have a sharp molecular weight permeability cut-off. The pores of each particular membrane vary in size and do not absolutely prohibit passage of high molecular weight molecules. Rather, the passage of such moleculersthrough the membrane is so slowthat they are effectively precluded from traversing the membrane. The pores are believed to have the form of tortuous paths defined by interstices among the cross-linked materials forming the membrane. Molecules having higher molecular weight and hence higher effective volumes than other moleculars will collide more often with the membrane structure and their passage through the membrane will thus be hindered.All of the quantitative permeability data disclosed herein are based on empirical observations of transmembrane diffusion of molecules having known molecular weight.
The encapsulated cells are grown for several days in a growth medium and then piaced in a screening apparatus having a plurality of compartments, e.g., a ninety-six well microtiter plate or soft agar. Preferably, approximately 10-100 capsules are placed in each compartment. The cell producing the desired substance synthesizes and secretes the substance, which traverses the membrane of the capsule into the extracapsular medium. The extracapsular medium of each compartment is assayed for the substance using conventional techniques e.g., RIA or enzyme assay. The process can be repeated with capsules from each compartment yielding a positive assay until the selected cell is identified. If possible, the repeating step should take place at a one capsule per well density.At this density, any positive assay shows that the encapsulated cell secreting the substance of interest is within the microcapsule.
The invention is now illustrated further by the following Example.
A cell line secreting IgG antibodies againstAzotobacter nitrogenase was established by polyethylene glycol fusion of the spleen cells of a BALB/c mouse immunized with Azotobacter nitrogenase and a
BALB/c mouse myeloma cell line, GM 3570. The myeloma cell line, which does not produce IgG, was purchased from the Human Genetic Mutant Cell
Repository.
The BALB/c-hybridoma (designated C25) was suspended in 1.34% (w/v) sodium alginate (NaG-Kelco) in 150 mM sodium chloride solution. The viscous suspension was transferred into a 10 cc syringe and then onto a jet head droplet forming apparatus as previously described. The droplets were gelled by contact with a 1.2% (w/v) calcium chloride in 150 mM sodium chloride solution. The gelled spheres formed a separate phase and were collected by aspiration.
The gelled spheres were washed three times with 150 mM sodium chloride then incubated for six minutes at room temperature in a 1.875 mg/ml solution of poly-L-lysine (Sigma, molecular weight 65,000 daltons). After incubation, the capsules were washed with 25 ml of 5 mM CHES buffer, (2-Ncyclohexylamino ethane sulfonic acid - Sigma in 0.2% (w/v) calcium chloride and 150 mM sodium chloride ), pH 7.5, once in 0.2% (w/v) calcium chloride in 150 mM sodium chloride, and once in 150 mM sodium chloride solution. The capsules were then incubated for an additional four minutes at room temperature in 0.6% (w/v) NaG in 150 mM sodium chloride, washed once in 150 mM sodium chloride and incubated for an additional fifteen minutes at room temperature in a 55mM sodium citrate in 150 mM sodium chloride solution.The solution was decanted and replaced with a fresh 55 mM sodium citrate in sodium chloride solution. The capsules were incubated at room temperature for an additional six minutes then washed twice with 150 mM sodium chloride, once with Dulbecco's modified
Eagle's medium (high glucose), and once with
Dulbecco's modified Eagle's medum (high glucose) containing 20% fetal calf serum (FCS-Flow Laboratories) and pennicillin, streptomycin, 1 mM glutamine, and 5 x 10-5 M mercaptoethanol. Capsules formed by this procedure were permeable to IgG but substantially impermeable to IgM.
The cells within the capsules were grown for several days in a culture flask and then were dispersed in a 24 well microtiter plate together with the growth medium at a concentration of about 20% by volume capsules and 80% by volume growth medium. After about three days incubation, samples of each well were tested for anti-Azotobacter nitrogenase using the procedure set forth below. Wells showing positive assay were recultured into new 24 well microtiter plates with the growth medium until viable hybridomas producing anti-Azotobacter nit- rogenase were identified.
The following assay used for determination of anti-Azotobacter nitrogenase production. The wells of a 96 well Linbro EIA plate were coated with a 100 > 1 of a 10 llg/ml solution of Azotobacternitrogenase in phosphate buffered saline (PBS). After incubation overnight at 4"C, the excess PBS solution was shaken off and the wells were postcoatedwith a 1% BSA in PBS solution and standards were made by diluting a 1 pg/ml solution of purified antinitrogenase solution in BSA-PBS. The samples were allowed to incubate for one hour at 37"C and the wells were then washed three times with PBS.A 1:1000 diiutionof goat anti-mouse IgG conjugated with peroxidase in BSA-PBS was added to each well.
After a two hour incubation at room temperature, each well was washed five times with PBS and 100 iil of a 4 mg/ml orthophenylenediamine, 0.02% (v/v) hydrogen peroxide in 55 mM sodium citrate, pH 4.5, was added to each well. After 15 minutes, the reaction was stopped by the addition of 100 ui of 4N
HCL. The plates were read on a plate reader for colour determination of anti-nitrogenase activity.
As is evident from the foregoing example, the process disclosed herein permits simplified selection of a cell producing a substance of interest.
Those skilled in the art will appreciate that variations and modifications of the techniques described herein can be employed for practising the invention, within the scope of the following claims.
Claims (28)
1. A process for screening a cell culture for a selected cell secreting a substance of interest, comprising the steps of:
A. encapsulating the cells ofthe cell culture within a plurality of capsules, each capsule comprising a intracapsular volume defined by a membrane permeable to the said substance but impermeable to the cells;
B. dispensing the encapsulated cells within a plurality of compartments each of which contains an extracapsular medium;
C. allowing the selected cell to synthesize and secrete the said substance within the intracapsular volume;
D. allowing the secreted substance to traverse the membrane;
E. assaying the extracapsular medium of each compartment for the said substance; and
F. repeating Steps B to E with the capsules from a compartment which yields a positive assay in Step
E until a capsule containing the said selected cell is identified.
2. The process according to claim 1, wherein statistically, the density of viable cells within the intracapsular volume is no greater than one cell per capsule.
3. The process according to claim 1 or claim 2, wherein the selected cell comprises a fusion product of a lymphocyte and an immortal cell line.
4. The process according to claim 3, wherein the immortal cell line comprises a myeloma.
5. The p ocess according to claim 3, wherein the selected cell comprises a hybridoma
6. The process according to claim 5, wherein the hybridoma comprises a cross-species fusion product.
7. The process of claim 5, wherein the said substance comprises a monoclonal antibody.
8. The process according to claim 1 or claim 2, wherein the selected cell comprises a geneticallymodified cell.
9. The process according to claim 8, wherein the genetically modified cell comprises a fusion product of a prokaryotic cell and a vector containing an expressable gene coding for the said substance.
10. The process according to any preceding claim, wherein the extracapsular medium comprises a growth medium for the selected cell.
11. The process according to any preceding claim, wherein the plurality of compartments comprise wells capable of retaining the capsules and the extracapsular medium.
12. The process according to any preceding claim, wherein the capsule membrane is permeable to the said substance and substantially impermeable to molecules having a molecular weight greater than a predetermined value.
13. The process according to any preceding claim, additionally comprising the step of injecting the identified capsule into an animal to induce an immune response against the said substance of interest.
14. The process according to any of claims 1 to 12, further comprising the step of implanting the identified capsule within a host animal and collecting fluid containing the substance of interest from the host animal.
15. A process for identifying a selected cell secreting a substance of interest from a heterogeneous cell population, comprising the steps of:
1. fusing a plurality of cells, comprising at least one cell producing the said substance of interest, with an immortal cell line to produce a heterogeneous population of cells comprising fused cells;
2. encapsulating the said population within a plurality of capsules, each capsule comprising a membrane defining an intracapsularvolume and being permeable to the said substance but impermeable to the fused cells;
3. dispensing the encapsulated cells within a plurality of compartments each of which contains an extracapsular medium capable of supporting ongoing metabolism of a cell secreting the said substance;
4. allowing the selected cell to synthesize and secrete the said substance within the intracapsular volume;;
5. allowing the secreted substance to traverse the membrane;
6. assaying the extracapsular medium of each compartment for the said substance; and
7. repeating Steps 3 to 6 with the capsules from a compartment which yields a positive assay in Step 6 until a capsule containing the selected cell is identified.
16. The process according to claim 15, wherein statistically, the density of cells within the intracap sular volume is no greater than one cell per capsule.
17. The process according to claim 15 or claim 16, wherein the immortal cell line comprises a myeloma.
18. The process according to claim 17, wherein the plurality of cells comprises lymphocytes.
19. The process according to claim 18 wherein the fused cells comprise a hybridoma.
20. The process according to any of claims 15 to 19, wherein the plurality of compartments comprise wells capable of retaining the capsules and the extracapsular medium.
21. The process according to any of claims 15 to 20, wherein the extracapsular medium comprises a growth medium for the selected cells.
22. The process according to claim 21,wherein the growth medium comprises a differential growth medium.
23. The process according to any of claims 15to 22, wherein the membrane is permeable to the said substance and substantially impermeable to molecules having a molecular weight greater than a predetermined value.
24. The process according to any of claims 15 to 23, further comprising the additional step of injecting the identified capsule into an animal to induce an immune response against the substance of interest.
25. The process according to any of claims 15 to 23, further comprising the additional step of implanting the identified capsule witin a host animal and collecting fluid containing the substance of interest from said host animal.
26. The process of claim 1, and substantially as herein described by way of example.
27. The process of claim 15, and substantially as herein described by way of example.
28. Aselected substance when produced by adoption of the process claimed in any of claims 1 to 27 and harvested from a culture of the identified cell or from a host animal.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US61323584A | 1984-05-24 | 1984-05-24 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB8428320D0 GB8428320D0 (en) | 1984-12-19 |
| GB2159171A true GB2159171A (en) | 1985-11-27 |
Family
ID=24456442
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB08428320A Withdrawn GB2159171A (en) | 1984-05-24 | 1984-11-09 | Process for screening or selecting cells |
Country Status (11)
| Country | Link |
|---|---|
| JP (1) | JPS60250256A (en) |
| AU (1) | AU3450684A (en) |
| BE (1) | BE902457A (en) |
| DE (1) | DE3509210A1 (en) |
| DK (1) | DK57885A (en) |
| FR (1) | FR2564858A1 (en) |
| GB (1) | GB2159171A (en) |
| IL (1) | IL73344A0 (en) |
| IT (1) | IT1179899B (en) |
| NL (1) | NL8500451A (en) |
| SE (1) | SE8405105L (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2192171A (en) * | 1986-06-06 | 1988-01-06 | Univ Ramot | Production of polymetric beads having alginate shells |
| EP0975969A1 (en) * | 1997-03-18 | 2000-02-02 | Chromaxome Corp. | Methods for screening compounds using encapsulated cells |
| WO2012103516A1 (en) * | 2011-01-28 | 2012-08-02 | Amyris, Inc. | Gel-encapsulated microcolony screening |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4130272A (en) * | 1977-09-22 | 1978-12-19 | Emmie George W | Flexible picket fence |
| CA1232849A (en) * | 1983-12-01 | 1988-02-16 | The Washington University | Circulating antigens of dirofilaria immitis, monoclonal antibodies specific therefor and methods of preparing such antibodies and detecting such antigens |
| US4686098A (en) * | 1984-05-14 | 1987-08-11 | Merck & Co., Inc. | Encapsulated mouse cells transformed with avian retrovirus-bovine growth hormone DNA, and a method of administering BGH in vivo |
| US4680174A (en) * | 1984-05-24 | 1987-07-14 | Damon Biotech, Inc. | Induction of immune response by immunization with encapsulated antigen-producing cells |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4352883A (en) * | 1979-03-28 | 1982-10-05 | Damon Corporation | Encapsulation of biological material |
| US4663286A (en) * | 1984-02-13 | 1987-05-05 | Damon Biotech, Inc. | Encapsulation of materials |
-
1984
- 1984-10-12 SE SE8405105A patent/SE8405105L/en not_active Application Discontinuation
- 1984-10-19 AU AU34506/84A patent/AU3450684A/en not_active Abandoned
- 1984-10-29 IL IL73344A patent/IL73344A0/en unknown
- 1984-11-09 GB GB08428320A patent/GB2159171A/en not_active Withdrawn
- 1984-12-25 JP JP59281927A patent/JPS60250256A/en active Pending
- 1984-12-28 IT IT68287/84A patent/IT1179899B/en active
-
1985
- 1985-02-07 DK DK57885A patent/DK57885A/en not_active Application Discontinuation
- 1985-02-18 NL NL8500451A patent/NL8500451A/en not_active Application Discontinuation
- 1985-03-14 DE DE19853509210 patent/DE3509210A1/en active Granted
- 1985-05-20 BE BE0/215039A patent/BE902457A/en not_active IP Right Cessation
- 1985-05-21 FR FR8507641A patent/FR2564858A1/en not_active Withdrawn
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2192171A (en) * | 1986-06-06 | 1988-01-06 | Univ Ramot | Production of polymetric beads having alginate shells |
| GB2192171B (en) * | 1986-06-06 | 1990-07-11 | Univ Ramot | Production of alginate beads. |
| EP0975969A1 (en) * | 1997-03-18 | 2000-02-02 | Chromaxome Corp. | Methods for screening compounds using encapsulated cells |
| EP0975969A4 (en) * | 1997-03-18 | 2002-07-31 | Chromaxome Corp | Methods for screening compounds using encapsulated cells |
| WO2012103516A1 (en) * | 2011-01-28 | 2012-08-02 | Amyris, Inc. | Gel-encapsulated microcolony screening |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS60250256A (en) | 1985-12-10 |
| FR2564858A1 (en) | 1985-11-29 |
| DK57885D0 (en) | 1985-02-07 |
| SE8405105L (en) | 1985-11-25 |
| DE3509210C2 (en) | 1988-03-31 |
| BE902457A (en) | 1985-09-16 |
| IT1179899B (en) | 1987-09-16 |
| IT8468287A0 (en) | 1984-12-28 |
| DE3509210A1 (en) | 1985-11-28 |
| SE8405105D0 (en) | 1984-10-12 |
| DK57885A (en) | 1985-11-25 |
| IL73344A0 (en) | 1985-01-31 |
| NL8500451A (en) | 1985-12-16 |
| AU3450684A (en) | 1985-11-28 |
| GB8428320D0 (en) | 1984-12-19 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |