CA2476237A1 - Methods and apparatus for whole cell panning - Google Patents
Methods and apparatus for whole cell panning Download PDFInfo
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Abstract
A new method and apparatus for whole cell panning employs homegenous populations of cells separated by a membrane(s) permeable to phage/phagemid particles.
Description
METHODS AND APPARATUS FOR WWOLE DELL PANNING
BACKGROUND
Technical Field This disclosure relates to methods and apparatus for whole cell panning using phage/phagemid libraries against homogeneous cell cultures, Background of Related Art In the technique of phage or phagemid particle display, pre-constructed antibody or antibody fragment libraries are attached to phage coat protein genes of phagelphagemid. The antibody fragments are then expressed and displayed on the surface of the phagelphagemid particle. Antibodies with an affinity for a target antigen are captured, propagated in bacteria, and further enriched through successive rounds of selection. This technique, as reviewed~in Phage Display of Proteins and Peptides: A
Laboratory Manual (Siegel, 2009), is referred to as panning. In most cases, panning methods use purified antigen immobilized to a solid support. Purification of the Target antigen is often not possible either because the antigen is unknown, as with putative stem-cell markers or tumor-specific antigens, or the antigen cannot be produced in sufficiently large quantities. Also, pu~~cation of some membrane targets (i.e, seven-transmembrane receptors) will likely cause improper folding in solution. In addition, purification procedures may affect a.. antigen's native binding properties, ror these reasons, it is desirable to pari for antibody-bearing phagelphagemid particles that bind an antigen in its native state, such as on intact cell surfaces.
Non-specific binding of phagelphagemid tends to be a problem in all types of panning. This is due to a variety of reasons including using complex antigens wifih "sticky surfaces". In whole cell based panning techniques, this problem is amplified by the presence of undesirable non-specific antigens or ligands presented an the cell surface. These antigens may take the form of endogenous proteins, carbohydrates, and lipids. The problems associated with non-specific binding in cell based panning techniques can be partially circumvented by employing a negative selection, in which the phag.e/phagemid libraries are pre-incubated with cells lacking the antigen of interest The unbound phage/phagemid is then used as the starting library for panning the target cells. Negative selection schemes are rather inefficient due to the low affinity of the non-specific binding interaction, Additionally, weak specific interactions between phagelphagemid and target antigen can be masked by negative selection, quickly depleting phage/phagemid libraries and reducing the chances of selecting for novel antibody-antigen interactions.
Recently, methods that employ simultaneous positive and negafiive selection have been developed. These methods employ a heterogeneous mixture of antigen-positive'cells and an excess of antigen-negative adsorbent cells. The antigen-negative cells serve as a non- speciFc phagelphagemid sink while the target cells capture any phagelphagemid exhibiting specific binding properties. The fidelity of specific binding interactions can be controlled by adjusting the ratios of antigen-positive to antigen-negative cells. Methods employing these heterogeneous mixtures must also employ some scheme for separating the two celG types after incubation with phagelphagemid, A number of methods exist to effect this separation. For example, one methods employs flow cytometry and fluorochrome-labeled antibodies that recognize an orthogonal antigen on the target cell (See,.U.S. Pat. No. 6,265,150 BI). This method is predicated on the existence of a marker antigen on the target cell which distinguishes it from the adsorber cells and a fluorochrome-labeled antibody specific for. it, a requirement that is not~always easily attained, especially in tumor cell fiargets.
Another method commonly used labels the target cells with magnetic beads followed by mixing with adsorber cells (See, U.S. Pat. No. 5,876,925).
Bacteriophage encoding Ig fragments are panned against a mixture of antigen-positive, biotinylated-~cell targets pre-coated with streptavidin-conjugated magnetic microbeads and an excess of unlabeled antigen-negative cells. After phage incubation, the cells are separated through the use of a magnetic affinity column. One disadvantage with this technique is that the panning of Ig fragments is done against a non-native cell surface, which displays biotinlstreptavidinlmagnetic bead complexes.
In yet another method, target cells with bound phage can be agglutinated from a heterogeneous mixture using immunoprecipitation (See, U.S. Pat. No.
5,985,543).
Whole cell panning using the above methods has several disadvantages. After panning with the phage library, the target cell populations must be separated from the non-specific adsorber cells. This requires that the target cells in the heterogeneous sample have markers on the cell surface which distinguish them from adsorber cells and that secondary antibodies that recognize these markers are available.
Second, the act of adding components to detect the markers on the cell surface of the.
target cells could perturb the native environment of the antigens on the cell surface, resulting in selection for antibodies that bind antigens displayed on tumor cells with a magnetic bead complex attached. Finally, the antibody-antigen interaction may be disrupted due to the process of separation (e.g. shear forces).
A more general method for whole cell panning that retains the benefits of the target/adsorber cell heterogeneous mixture, but eliminates the need for a cell separation step to obtain the phage/phagemid of interest would be advantageous.
SUMMARY
A new method and apparatus for whole cell panning employs homogenous populations of cells separated by a mernbrane(s) permeable to phage/phagemid particles. ane population of cells.(e.g., target cells) is maintained on a first side of the membrane and 'consists of target cells displaying possible antigens of interest. . A
second population of cells is maintained on a second side of the membrane and consists of non-specific absorber cells. At no point in the protocol do the two cell populations ~...ix.
The cell culture apparatus maintains the separation of two or more cell fines in two or more different chambers by one or more removable barriers. The isolated chambers allow the separate growth of non-specific cells and target antigen-positive cells. The semi-permeable membrane can be sandwiched between two solid barriers. If the different cells lines require different growth media, they can be placed into different chambers and grown with the solid pieces in place, preventing the mixture of any material, between the chambers. After growth is completed, the solid barriers are removed. When the barrier between the chambers is removed, the cell populations both contact a semi-permeable membrane that allows passive lateral diffusion among adjacent chambers. The membrane has a pore size that prevents cells from passing therethrough, but allows smaller particles (e.g., phage or phagemid) to pass through. A
cell-type specific library of phage/phagemid particles is then added. The membrane allows the lateral diffusion of the phage/phagemid parkicles carrying antibody fragments between the chambers of the apparatus while keeping the cell lines separate.
The entire apparatus can be loosely covered with a lid to allow air circulation.
In another embodiment, individual culture chambers having openings at both the top and side. The side opening accommodates a solid or semi-permeable insert.
During cell growth, the individual chambers are provided with solid side inserts allawi,Og for isolated cell growth. The solid side inserts can be removed and the two'chfi~i~tbErs can be attached onto either side of a semi-permeable membrane insert. The phage/phagemid library can then be added to one or both of the cell populations. The semi-permeable~membrane allows the passive diffusion of the phage/phagemid particles between the chambers while keeping the adsorber and target cell types separate. The entire apparatus can be loosely covered with a lid to allow air circulation.
Brief Description Of The Drawings Fig. 1 is a cross-sectional view of a whole cell panning apparatus in accordance with this disclosure having the solid barriers in place.
Fig. 2 is a cross-sectional view of the whole cell panning. apparatu of Fig. 1 having the solid barriers removed.
Fig. 3 is a cross-sectional view of two individual chambers useful in assembling a whole cell panni~~g apparatus in accordance with ibis disclosure.
Fig. 4 is a cross-sectional view of the two individual chambers of Fig. 3 as assembled to provide.a whole cell panning apparatus in accordance with this disclosure.
Fig. 5 is a cross-sectional view of an alternative embodiment of two individual chambers useful in assembling a whole cell panning apparatus in accordance with this disclosure having the solid barriers in place.
Detailed Description Of Preferred Embodiments As seen in Fig. 1, a new apparatus 10 for whole cell panning includes a container 11 defining a first chamber 12 and a second chamber 22. The container l1can be made from any inert material capable of supporting a solution of cells.
Suitable materials of construction include, but are not limited to, glass and polymers;
such as, for example, polycarbonate or polystyrene. Preferably the material of construction is transparent. Chamber 12 holds a first suspension 13 containing a population of cells (e.g., target cells). Chamber 22 holds a second suspension containing a population of cells (e.g_, absorber cells). A loosely fitting cover 16 prevents contamination of suspensions 13, 23. One side of chamber 12 is defined by a solid, removable insert 14. One side of chamber 22 is defined by a solid, removable insert 24. . Solid, removable inserts 14, 24 create a water tight seal so that mixing of the contents of chambers 12, 22 is prevented. Sandwiched between solid, removable inserts 14, 24 is a semi-permeable membrane 15. The semi-permeable membrane is designed to have openings that are too small to allow cells to move across the membrane. The pores of membrane 15 must be large enough, however to allow the passage of the display library through the membrane. In the case of a phage or phagemid library, the pore size of the membrane should be in the range of 0.2 to 7 microns, preferably about 5 microns. The membrane can be made of any inert material to which pores of the appropriate size can be imparted.
Upon removal of the solid, removable inserts 14, 24, the first and second suspensions 13 and 23, respectively are permitted to come into contacfiwith the membrane 15. Because the pores of the membrane are too small to permit the passage of cells, the two populations of cells remain distinct. A display library is added to one or both of containers 12, 22. The individual members of the library are free to pass through the membrane 15. Certain members of the display library will bind preferentially to the target cells in the first suspension 13. After a period of time, any binding that will take place has already taken place and the first suspension 13 can be removed and the. binding members of the library can be removed from the target cells and recovered. The second suspension 23 and non-bound members of the library can be discarded.
An alternative embodiment of a whole cell panning apparatus is shown in Fig.
3, wherein chambers 112 and 122 are defined by separate containers 111, 121 each of which has a cover 116, 126. Chamber 112 holds a first suspension 113 containing a population of cells (e.g., target cells). Chamber 122 holds a second suspension 123 containing a population of cells (e.g., absorber cells). The top of each coritainer 111, s 121 has a threaded portion 117, 127 respectively. Tha covers 116, 126 can be screwed on tightly to avoid spillage during handling or transport, but can be left on loosely to allow air circulation during cell culturing. After adequate cell growth has occurred and the cell suspensions 113, 123 are to be used for panning, covers 116, 126 are removed, a display library is added to one or both suspensions 113, 123, and a common cover 130 is applied to containers 111, 121. The common cover 130 has a first threaded portion 135 which can be screwed tightly to threaded portion 1'17 of container 111 and a second threaded portion 137 which can be screwed tightly to threaded portion 127 of container 121. Membrane 115 defines the center portion of the common cover 130. Once the assembled apparatus is placed on ifis side as shown in dig. 4, bofih suspensions 113 and 123 come into contact with membrane 115.
As in the previous embodiment, because the pores of the membrane are too small to permit the passage of cells, the two populations of cells remain distinct. Th~
individual members of the library, however, are free to pass through the membrane 115.
Certain members of the display library will bind preferentially to the target cells in the first suspension 113. After a period of time, any binding that will take place has already taken place and the first suspension 113 can be removed and the binding members of the library can be removed from the target cells and recovered. The second suspension 123 and non-bound members of the library can be discarded.
In yet another embodiment {shown in Fig. 5), chambers 212 and 222 are defined b; separate containers 211, 221 in the shape of rectangulartest~tubes. Each container 211, 221 has cover atop 216, 226, and a water tight side cover 218, 228 which snap fits onto side flanges 219, 229, respectively, so as to cover membranes 215, 225.
Chamber 212 holds a first suspension 213 containing a population of cells (e.g., target cells). Chamber 222 holds a second suspension 223 containing a population of cells (e.g., absorber cells). In this embodiment, suspension 213 is in direct contact with membrane 216 and suspension 223 is in direct contact with membrane 225. After adequate cell growth has occurred and the cell suspensions 213, 223 are to be used for panning, a display library is added to one or both suspensions 213, 223. Side covers 218, 228 are removed and a joining sleeve 230 is applied to containers 211, 221 in place of side covers 218, 228. The joining sleeve 230 connects the two containers~211, G
221 so that filuid and library members (but not cells) can pass from one chamber 212 to the other 222. As in the previous embodiment, because the pores of the membranes 215 and 225 are too small to permit the passage of cells, the two populations of cells .
remain. distinct. The individual members of the library, however, are free to pass through the membranes 215, 225. Certain members of the display library will bind preferentially to the target cells in the first suspension 213. After a period of time, any binding that will take place has already taken place and the first suspension 213 can be removed and the binding members of the library can be removed from the target cells and recovered. The second suspension 223 and non~bound members of the library can be discarded.
It is possible, in a variation of the embodiment shown in Fig. 5, to employ only a single membrane on joining sleeve 230 instead of a membrane on each oontainer.
Also, it may be advantageous to employ stirring (e.g., via a magnetic stirrer) in one or both of the chambers of any of the embodiments described above. In addition, pumping can be advantageously employed to assist in moving members of the library through the membrane in any of the previously described embodiments. Pumping may also help in the event a washing step is to be performed while the cell suspensions are still located within the chambers of the apparatus. Suitable types of pumps include, buff are not limited to, peristaltic pumps. It is also contemplated that the two syringe-like containers maybe employed in which case one or more syringe plungers can be used manually to pro~!ide a pumping action.
The method of whole cell panning described herein retains the benefits of the existing methods while eliminating the major drawbacks. Because the target cells and absorber cells are kept as separate homogenous populations in different chambers of the apparatus at all times, a cell separation step is not required, therefore no cell markers are needed. In addition, cell lines can be grown in different media using solid separators. These solid separators can be removed exposing a semi-permeable membrane. I?hage/phagemid libraries are then added. The phage/phagemid particles are able to move freely through the membrane to both chambers while the different cell types are kept separate.
While the above description contains many specific details of methods in accordance with this invention, fihese specific details should not be construed as limitations on the scope of the invention, but merely as exemplifications. of preferred embodiments thereof. Those skilled in the art will envision many other possible variations that all within the scope and spirit of the invention as defined by the claims appended hereto. For example, instead of threaded covers, the containers may be covered with what are commonly known to those skilled in the art as "snap caps".
These "snap caps" have a first position in which the cap is loosely on the container and a .second snapped-down position which forms a water-tight seal, As. another example, the water tight side covers in the embodiment shown in Fig, 5 can be replaced with peel-away covers to provide a one time use device for cell panning. Thus, the foregoing description should be viewed as illustrative, not limiting.
BACKGROUND
Technical Field This disclosure relates to methods and apparatus for whole cell panning using phage/phagemid libraries against homogeneous cell cultures, Background of Related Art In the technique of phage or phagemid particle display, pre-constructed antibody or antibody fragment libraries are attached to phage coat protein genes of phagelphagemid. The antibody fragments are then expressed and displayed on the surface of the phagelphagemid particle. Antibodies with an affinity for a target antigen are captured, propagated in bacteria, and further enriched through successive rounds of selection. This technique, as reviewed~in Phage Display of Proteins and Peptides: A
Laboratory Manual (Siegel, 2009), is referred to as panning. In most cases, panning methods use purified antigen immobilized to a solid support. Purification of the Target antigen is often not possible either because the antigen is unknown, as with putative stem-cell markers or tumor-specific antigens, or the antigen cannot be produced in sufficiently large quantities. Also, pu~~cation of some membrane targets (i.e, seven-transmembrane receptors) will likely cause improper folding in solution. In addition, purification procedures may affect a.. antigen's native binding properties, ror these reasons, it is desirable to pari for antibody-bearing phagelphagemid particles that bind an antigen in its native state, such as on intact cell surfaces.
Non-specific binding of phagelphagemid tends to be a problem in all types of panning. This is due to a variety of reasons including using complex antigens wifih "sticky surfaces". In whole cell based panning techniques, this problem is amplified by the presence of undesirable non-specific antigens or ligands presented an the cell surface. These antigens may take the form of endogenous proteins, carbohydrates, and lipids. The problems associated with non-specific binding in cell based panning techniques can be partially circumvented by employing a negative selection, in which the phag.e/phagemid libraries are pre-incubated with cells lacking the antigen of interest The unbound phage/phagemid is then used as the starting library for panning the target cells. Negative selection schemes are rather inefficient due to the low affinity of the non-specific binding interaction, Additionally, weak specific interactions between phagelphagemid and target antigen can be masked by negative selection, quickly depleting phage/phagemid libraries and reducing the chances of selecting for novel antibody-antigen interactions.
Recently, methods that employ simultaneous positive and negafiive selection have been developed. These methods employ a heterogeneous mixture of antigen-positive'cells and an excess of antigen-negative adsorbent cells. The antigen-negative cells serve as a non- speciFc phagelphagemid sink while the target cells capture any phagelphagemid exhibiting specific binding properties. The fidelity of specific binding interactions can be controlled by adjusting the ratios of antigen-positive to antigen-negative cells. Methods employing these heterogeneous mixtures must also employ some scheme for separating the two celG types after incubation with phagelphagemid, A number of methods exist to effect this separation. For example, one methods employs flow cytometry and fluorochrome-labeled antibodies that recognize an orthogonal antigen on the target cell (See,.U.S. Pat. No. 6,265,150 BI). This method is predicated on the existence of a marker antigen on the target cell which distinguishes it from the adsorber cells and a fluorochrome-labeled antibody specific for. it, a requirement that is not~always easily attained, especially in tumor cell fiargets.
Another method commonly used labels the target cells with magnetic beads followed by mixing with adsorber cells (See, U.S. Pat. No. 5,876,925).
Bacteriophage encoding Ig fragments are panned against a mixture of antigen-positive, biotinylated-~cell targets pre-coated with streptavidin-conjugated magnetic microbeads and an excess of unlabeled antigen-negative cells. After phage incubation, the cells are separated through the use of a magnetic affinity column. One disadvantage with this technique is that the panning of Ig fragments is done against a non-native cell surface, which displays biotinlstreptavidinlmagnetic bead complexes.
In yet another method, target cells with bound phage can be agglutinated from a heterogeneous mixture using immunoprecipitation (See, U.S. Pat. No.
5,985,543).
Whole cell panning using the above methods has several disadvantages. After panning with the phage library, the target cell populations must be separated from the non-specific adsorber cells. This requires that the target cells in the heterogeneous sample have markers on the cell surface which distinguish them from adsorber cells and that secondary antibodies that recognize these markers are available.
Second, the act of adding components to detect the markers on the cell surface of the.
target cells could perturb the native environment of the antigens on the cell surface, resulting in selection for antibodies that bind antigens displayed on tumor cells with a magnetic bead complex attached. Finally, the antibody-antigen interaction may be disrupted due to the process of separation (e.g. shear forces).
A more general method for whole cell panning that retains the benefits of the target/adsorber cell heterogeneous mixture, but eliminates the need for a cell separation step to obtain the phage/phagemid of interest would be advantageous.
SUMMARY
A new method and apparatus for whole cell panning employs homogenous populations of cells separated by a mernbrane(s) permeable to phage/phagemid particles. ane population of cells.(e.g., target cells) is maintained on a first side of the membrane and 'consists of target cells displaying possible antigens of interest. . A
second population of cells is maintained on a second side of the membrane and consists of non-specific absorber cells. At no point in the protocol do the two cell populations ~...ix.
The cell culture apparatus maintains the separation of two or more cell fines in two or more different chambers by one or more removable barriers. The isolated chambers allow the separate growth of non-specific cells and target antigen-positive cells. The semi-permeable membrane can be sandwiched between two solid barriers. If the different cells lines require different growth media, they can be placed into different chambers and grown with the solid pieces in place, preventing the mixture of any material, between the chambers. After growth is completed, the solid barriers are removed. When the barrier between the chambers is removed, the cell populations both contact a semi-permeable membrane that allows passive lateral diffusion among adjacent chambers. The membrane has a pore size that prevents cells from passing therethrough, but allows smaller particles (e.g., phage or phagemid) to pass through. A
cell-type specific library of phage/phagemid particles is then added. The membrane allows the lateral diffusion of the phage/phagemid parkicles carrying antibody fragments between the chambers of the apparatus while keeping the cell lines separate.
The entire apparatus can be loosely covered with a lid to allow air circulation.
In another embodiment, individual culture chambers having openings at both the top and side. The side opening accommodates a solid or semi-permeable insert.
During cell growth, the individual chambers are provided with solid side inserts allawi,Og for isolated cell growth. The solid side inserts can be removed and the two'chfi~i~tbErs can be attached onto either side of a semi-permeable membrane insert. The phage/phagemid library can then be added to one or both of the cell populations. The semi-permeable~membrane allows the passive diffusion of the phage/phagemid particles between the chambers while keeping the adsorber and target cell types separate. The entire apparatus can be loosely covered with a lid to allow air circulation.
Brief Description Of The Drawings Fig. 1 is a cross-sectional view of a whole cell panning apparatus in accordance with this disclosure having the solid barriers in place.
Fig. 2 is a cross-sectional view of the whole cell panning. apparatu of Fig. 1 having the solid barriers removed.
Fig. 3 is a cross-sectional view of two individual chambers useful in assembling a whole cell panni~~g apparatus in accordance with ibis disclosure.
Fig. 4 is a cross-sectional view of the two individual chambers of Fig. 3 as assembled to provide.a whole cell panning apparatus in accordance with this disclosure.
Fig. 5 is a cross-sectional view of an alternative embodiment of two individual chambers useful in assembling a whole cell panning apparatus in accordance with this disclosure having the solid barriers in place.
Detailed Description Of Preferred Embodiments As seen in Fig. 1, a new apparatus 10 for whole cell panning includes a container 11 defining a first chamber 12 and a second chamber 22. The container l1can be made from any inert material capable of supporting a solution of cells.
Suitable materials of construction include, but are not limited to, glass and polymers;
such as, for example, polycarbonate or polystyrene. Preferably the material of construction is transparent. Chamber 12 holds a first suspension 13 containing a population of cells (e.g., target cells). Chamber 22 holds a second suspension containing a population of cells (e.g_, absorber cells). A loosely fitting cover 16 prevents contamination of suspensions 13, 23. One side of chamber 12 is defined by a solid, removable insert 14. One side of chamber 22 is defined by a solid, removable insert 24. . Solid, removable inserts 14, 24 create a water tight seal so that mixing of the contents of chambers 12, 22 is prevented. Sandwiched between solid, removable inserts 14, 24 is a semi-permeable membrane 15. The semi-permeable membrane is designed to have openings that are too small to allow cells to move across the membrane. The pores of membrane 15 must be large enough, however to allow the passage of the display library through the membrane. In the case of a phage or phagemid library, the pore size of the membrane should be in the range of 0.2 to 7 microns, preferably about 5 microns. The membrane can be made of any inert material to which pores of the appropriate size can be imparted.
Upon removal of the solid, removable inserts 14, 24, the first and second suspensions 13 and 23, respectively are permitted to come into contacfiwith the membrane 15. Because the pores of the membrane are too small to permit the passage of cells, the two populations of cells remain distinct. A display library is added to one or both of containers 12, 22. The individual members of the library are free to pass through the membrane 15. Certain members of the display library will bind preferentially to the target cells in the first suspension 13. After a period of time, any binding that will take place has already taken place and the first suspension 13 can be removed and the. binding members of the library can be removed from the target cells and recovered. The second suspension 23 and non-bound members of the library can be discarded.
An alternative embodiment of a whole cell panning apparatus is shown in Fig.
3, wherein chambers 112 and 122 are defined by separate containers 111, 121 each of which has a cover 116, 126. Chamber 112 holds a first suspension 113 containing a population of cells (e.g., target cells). Chamber 122 holds a second suspension 123 containing a population of cells (e.g., absorber cells). The top of each coritainer 111, s 121 has a threaded portion 117, 127 respectively. Tha covers 116, 126 can be screwed on tightly to avoid spillage during handling or transport, but can be left on loosely to allow air circulation during cell culturing. After adequate cell growth has occurred and the cell suspensions 113, 123 are to be used for panning, covers 116, 126 are removed, a display library is added to one or both suspensions 113, 123, and a common cover 130 is applied to containers 111, 121. The common cover 130 has a first threaded portion 135 which can be screwed tightly to threaded portion 1'17 of container 111 and a second threaded portion 137 which can be screwed tightly to threaded portion 127 of container 121. Membrane 115 defines the center portion of the common cover 130. Once the assembled apparatus is placed on ifis side as shown in dig. 4, bofih suspensions 113 and 123 come into contact with membrane 115.
As in the previous embodiment, because the pores of the membrane are too small to permit the passage of cells, the two populations of cells remain distinct. Th~
individual members of the library, however, are free to pass through the membrane 115.
Certain members of the display library will bind preferentially to the target cells in the first suspension 113. After a period of time, any binding that will take place has already taken place and the first suspension 113 can be removed and the binding members of the library can be removed from the target cells and recovered. The second suspension 123 and non-bound members of the library can be discarded.
In yet another embodiment {shown in Fig. 5), chambers 212 and 222 are defined b; separate containers 211, 221 in the shape of rectangulartest~tubes. Each container 211, 221 has cover atop 216, 226, and a water tight side cover 218, 228 which snap fits onto side flanges 219, 229, respectively, so as to cover membranes 215, 225.
Chamber 212 holds a first suspension 213 containing a population of cells (e.g., target cells). Chamber 222 holds a second suspension 223 containing a population of cells (e.g., absorber cells). In this embodiment, suspension 213 is in direct contact with membrane 216 and suspension 223 is in direct contact with membrane 225. After adequate cell growth has occurred and the cell suspensions 213, 223 are to be used for panning, a display library is added to one or both suspensions 213, 223. Side covers 218, 228 are removed and a joining sleeve 230 is applied to containers 211, 221 in place of side covers 218, 228. The joining sleeve 230 connects the two containers~211, G
221 so that filuid and library members (but not cells) can pass from one chamber 212 to the other 222. As in the previous embodiment, because the pores of the membranes 215 and 225 are too small to permit the passage of cells, the two populations of cells .
remain. distinct. The individual members of the library, however, are free to pass through the membranes 215, 225. Certain members of the display library will bind preferentially to the target cells in the first suspension 213. After a period of time, any binding that will take place has already taken place and the first suspension 213 can be removed and the binding members of the library can be removed from the target cells and recovered. The second suspension 223 and non~bound members of the library can be discarded.
It is possible, in a variation of the embodiment shown in Fig. 5, to employ only a single membrane on joining sleeve 230 instead of a membrane on each oontainer.
Also, it may be advantageous to employ stirring (e.g., via a magnetic stirrer) in one or both of the chambers of any of the embodiments described above. In addition, pumping can be advantageously employed to assist in moving members of the library through the membrane in any of the previously described embodiments. Pumping may also help in the event a washing step is to be performed while the cell suspensions are still located within the chambers of the apparatus. Suitable types of pumps include, buff are not limited to, peristaltic pumps. It is also contemplated that the two syringe-like containers maybe employed in which case one or more syringe plungers can be used manually to pro~!ide a pumping action.
The method of whole cell panning described herein retains the benefits of the existing methods while eliminating the major drawbacks. Because the target cells and absorber cells are kept as separate homogenous populations in different chambers of the apparatus at all times, a cell separation step is not required, therefore no cell markers are needed. In addition, cell lines can be grown in different media using solid separators. These solid separators can be removed exposing a semi-permeable membrane. I?hage/phagemid libraries are then added. The phage/phagemid particles are able to move freely through the membrane to both chambers while the different cell types are kept separate.
While the above description contains many specific details of methods in accordance with this invention, fihese specific details should not be construed as limitations on the scope of the invention, but merely as exemplifications. of preferred embodiments thereof. Those skilled in the art will envision many other possible variations that all within the scope and spirit of the invention as defined by the claims appended hereto. For example, instead of threaded covers, the containers may be covered with what are commonly known to those skilled in the art as "snap caps".
These "snap caps" have a first position in which the cap is loosely on the container and a .second snapped-down position which forms a water-tight seal, As. another example, the water tight side covers in the embodiment shown in Fig, 5 can be replaced with peel-away covers to provide a one time use device for cell panning. Thus, the foregoing description should be viewed as illustrative, not limiting.
Claims (17)
1. An apparatus comprising:
a first chamber capable of containing a first population of a first type of cells;
a second chamber capable of containing a second population of a second type of cells; and a membrane separating the two chambers, the membrane being permeable to phage and phagemid particles, while preventing mixing of the first and second population of cells.
a first chamber capable of containing a first population of a first type of cells;
a second chamber capable of containing a second population of a second type of cells; and a membrane separating the two chambers, the membrane being permeable to phage and phagemid particles, while preventing mixing of the first and second population of cells.
2. An apparatus as in claim 1, further comprising at least one removable solid insert adjacent to the membrane.
3. An apparatus as in claim 1, further comprising first and second removable, solid inserts, sandwiching the membrane.
4. An apparatus as in claim 1, further comprising a lid.
5. An apparatus comprising:
a first container capable of containing a suspension of a first population of cells and having a open end;
a second container capable of containing a second population of cells and having an opening end; and a coupling adapted to receive the open ends of both the first and second containers, the coupling having a membrane disposed therein to maintain the first population of cells separate from the second population of cells while allowing the passage of phage and phagemid particles therethrough.
a first container capable of containing a suspension of a first population of cells and having a open end;
a second container capable of containing a second population of cells and having an opening end; and a coupling adapted to receive the open ends of both the first and second containers, the coupling having a membrane disposed therein to maintain the first population of cells separate from the second population of cells while allowing the passage of phage and phagemid particles therethrough.
6. An apparatus as in claim 5, wherein the first and second containers each have a threaded portion adjacent to the open ends.
7. An apparatus as in claim 6, wherein the coupling includes first and second threaded portions to receive the threaded portions of the first and second containers.
8. An apparatus as in claim 5, further comprising a first lid capable of sealing the open end of the first container in a water tight manner and a second lid capable of sealing the open end of the second container in a water tight manner.
9. An apparatus as in claim 8, wherein the first and second lids are screw caps.
10. An apparatus as in claim 8, wherein the coupling receives the first and second containers in a snap fit relation.
11. An apparatus comprising:
a first container capable of containing a first suspension of a first population of cells in a medium and having an open end;
a semi-permeable membrane covering the open end of the first container, the semi-permeable membrane being impermeable to the passage of cells;
a second container capable of containing a second suspension of a second population of cells in a medium and having an open end;
a semi-permeable membrane covering the open end of the second container, the semi-permeable membrane being impermeable to the passage of cells;
and a coupling adapted to receive the open end of both the first and second containers in a manner that permits contact of the media of the first and second suspensions.
a first container capable of containing a first suspension of a first population of cells in a medium and having an open end;
a semi-permeable membrane covering the open end of the first container, the semi-permeable membrane being impermeable to the passage of cells;
a second container capable of containing a second suspension of a second population of cells in a medium and having an open end;
a semi-permeable membrane covering the open end of the second container, the semi-permeable membrane being impermeable to the passage of cells;
and a coupling adapted to receive the open end of both the first and second containers in a manner that permits contact of the media of the first and second suspensions.
12. An apparatus as in claim 11, wherein the first and second containers each have a threaded portion adjacent to the open ends.
13. An apparatus as in claim 12, wherein the coupling includes first and second threaded portions to receive the threaded portions of the first and second containers.
14. An apparatus as in claim 11, further comprising a first lid capable of sealing the open end of the first container in a watertight manner and a second lid capable of sealing the open end of the second container in, a water tight manner.
15. An apparatus as in claim 14, wherein the first and second lids are screw caps.
16, An apparatus as I claim 11, wherein the coupling receives the first and second containers,in a snap fit relation.
17. A method for screening an antibody library for antibodies that bind to an antigen comprising:
providing a first suspension containing a population of antigen positive cells;
providing a second suspension containing a population of antigen negative cells;
adding a display library containing a plurality of members to at least one of the first or second suspensions;
maintaining the population of antigen positive cells separate from the population of antigen negative cells by a semi-permeable membrane while allowing passage of the members of the display library into both the first and second suspensions; and recovering cells from the antigen positive population to identify members of the display library bound thereto.
providing a first suspension containing a population of antigen positive cells;
providing a second suspension containing a population of antigen negative cells;
adding a display library containing a plurality of members to at least one of the first or second suspensions;
maintaining the population of antigen positive cells separate from the population of antigen negative cells by a semi-permeable membrane while allowing passage of the members of the display library into both the first and second suspensions; and recovering cells from the antigen positive population to identify members of the display library bound thereto.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US35608802P | 2002-02-11 | 2002-02-11 | |
| US60/356,088 | 2002-02-11 | ||
| PCT/US2003/004001 WO2003068916A2 (en) | 2002-02-11 | 2003-02-10 | Methods and apparatus for whole cell panning |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2476237A1 true CA2476237A1 (en) | 2003-08-21 |
Family
ID=27734605
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002476237A Abandoned CA2476237A1 (en) | 2002-02-11 | 2003-02-10 | Methods and apparatus for whole cell panning |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1570053A2 (en) |
| JP (1) | JP2005526498A (en) |
| AU (1) | AU2003210949A1 (en) |
| CA (1) | CA2476237A1 (en) |
| WO (1) | WO2003068916A2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006055371A2 (en) * | 2004-11-16 | 2006-05-26 | Alexion Pharmaceuticals, Inc. | Functional screening of antibody libraries |
| DE102016119391B3 (en) * | 2016-10-12 | 2018-01-18 | Yoen Ok Roth | Microbioreactor module |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6737266B1 (en) * | 1999-10-01 | 2004-05-18 | 3M Innovative Properties Company | Devices and methods for microorganism detection |
-
2003
- 2003-02-10 EP EP03739727A patent/EP1570053A2/en not_active Withdrawn
- 2003-02-10 CA CA002476237A patent/CA2476237A1/en not_active Abandoned
- 2003-02-10 WO PCT/US2003/004001 patent/WO2003068916A2/en not_active Application Discontinuation
- 2003-02-10 JP JP2003568031A patent/JP2005526498A/en not_active Withdrawn
- 2003-02-10 AU AU2003210949A patent/AU2003210949A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| WO2003068916A2 (en) | 2003-08-21 |
| WO2003068916A3 (en) | 2005-07-14 |
| EP1570053A2 (en) | 2005-09-07 |
| JP2005526498A (en) | 2005-09-08 |
| AU2003210949A1 (en) | 2003-09-04 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 20060210 |