EP0114756A2
Expired / Lapsed
EP
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Isolation and culture of mammalian cells secreting acetylcholinesterase and recovery of acetylcholinesterase therefrom
Assignee: Bio Response Inc
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Filing: 1984-01-20
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Granted: —
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Expires: 2004-01-20
Abstract
Abstract There is disclosed a method for isolating living mammalian cells secreting acetylcholinesterase (AChE) from a population of cells secreting AChE at varying rates, in which the cells of the population are encapsulated in a semi-permeable layer (10) along with excess of a material which captures and immobilizes within the capsule all AChE secreted by the encapsulated cell, and then a product having an enzyme affixed to it is added to the population to bind only to the AChE or AChE complex i.e. in only those capsules containing an AChE-secreting cell. Thereafter, a substrate is added to the population for the purpose of establishing a physical difference between enzyme-containing and enzyme-free cells thereby permitting physical separation of these two categories of capsule. Subsequently the cells producing AChE can be recovered for culturing. The procedure can be performed again, on the recovered cells to isolate from them the cells with the highest rate of production of AChE.
Inventors
- 👤 Sam Rose
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-24 06:40
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/02; C12Q1/24; C12N; C12N9/00; C12N9/14; C12N9/16; C12N9/18; A; A61; A61K; A61K38/00
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
261 words
A method for isolating living mammalian cells secreting acetylcholinesterase, characterised by the steps of: (a) obtaining a population of cells containing cells secreting acetylcholinesterase at varying rates; (b) encapsulating the individual cells of said population within a semi-permeable material along with a capture material capable of specifically binding to acetylcholinesterase, said semi-permeable material being sized such that neither the capture material nor capture material bound to acetylcholinesterase can permeate therethrough; (c) allowing to elapse a predetermined period of time sufficient for acetylcholinesterase-producing cells to secrete acetylcholinesterase to bind to said capture material; (d) adding to the entire cell population a product having an enzyme affixed thereto, the product being permeable through said semi-permeable material and capable of binding specifically to acetylcholinesterase or the acetylcholinesterase lowered to the capture material, said product having affixed thereto an enzyme, said product and enzyme becoming immobilized only within the semi-permeable encapsulating material in which a cell producing acetylcholinesterase is contained, the amount of enzyme and product so immobilized being directly proportional to the amount of acetylcholinesterase secreted by the cell in a given period of time; (e) removing from the cell population any non-immobilized product and enzyme; (f) adding to the entire cell population a substrate capable of being acted upon by said enzyme to bring about a measurable or identifiable change only at those encapsulated cells where enzyme is immobilized, the magnitude of such change being directly proportional to the quantity of immobilized enzyme present in a capsule; and (g) recovering those encapsulated cells in which a predetermined degree of identifiable change has occurred.
Claim 2
Independent Claim
261 words
A method for isolating living mammalian cells secreting acetylcholinesterase, characterised by the steps of: (a) obtaining a population of cells containing cells secreting acetylcholinesterase at varying rates; (b) encapsulating the individual cells of said population within a semi-permeable material along with a capture material capable of specifically binding to acetylcholinesterase, said semi-permeable material being sized such that neither the capture material nor capture material bound to acetylcholinesterase can permeate therethrough; (c) allowing to elapse a predetermined period of time sufficient for acetylcholinesterase-producing cells to secrete acetylcholinesterase to bind to said capture material; (d) adding to the entire cell population a product having an enzyme affixed thereto, the product being permeable through said semi-permeable material and capable of binding specifically to acetylcholinesterase or the acetylcholinesterase lowered to the capture material, said product having affixed thereto an enzyme, said product and enzyme becoming immobilized only within the semi-permeable encapsulating material in which a cell producing acetylcholinesterase is contained, the amount of enzyme and product so immobilized being directly proportional to the amount of acetylcholinesterase secreted by the cell in a given period of time; (e) removing from the cell population any non-immobilized product and enzyme; (f) adding to the entire cell population a substrate capable of being acted upon by said enzyme to bring about a measurable or identifiable change only at those encapsulated cells where enzyme is immobilized, the magnitude of such change being directly proportional to the quantity of immobilized enzyme present in a capsule; and (g) recovering those encapsulated cells in which a predetermined degree of identifiable change has occurred.
Claim 3
Dependent Claim
61 words
A method according to claim 1 for isolating living mammalian cells secreting acetylcholinesterase characterised in that the capture material of step (b) is chosen from the group consisting of acetylcholine analogs which specifically bind to acetylcholinesterase and which generally remain in the ester group configuration in the presence of acetylcholinesterase, and specific antibodies to any particular determinant of the acetylcholinesterase molecule.
Claim 4
Dependent Claim
50 words
A method according to claim 1 or 2 for isolating living mammalian cells secreting acetylcholinesterase characterised in that the capture material of step (b) is in an excess amount such that the acetylcholinesterase secreted by the cell over a given period of time is specifically bound by the capture material.
Claim 5
Dependent Claim
69 words
A method according to claim 1, 2 or 3 for isolating living mammalian cells secreting acetylcholinesterase which is further characterised by the step of suspending the encapsulated individual cells of the population in a medium in which the cells remain viable and which is of a density such that the encapsulated cells remain in relatively uniform, fixed vertical position within the suspension for the duration of the isolation process.
Claim 6
Independent Claim
40 words
A method according to any one of the preceding claims for isolating living mammalian cells secreting acetylcholinesterase characterised in that the complex of acetylcholinesterase and capture material of step (d) is too large to permeate through the semi-permeable encapsulating material.
Claim 7
Independent Claim
80 words
A method according to any one of the preceding claims for isolating living mammalian cells secreting acetylcholinesterase characterised in that the substrate of step (f) is a soluble material which is converted to an insoluble precipitate in the presence of the enzyme such that the insoluble precipitate is formed at those encapsulated cells containing the enzyme and acetylcholinesterase which causes those encapsulated cells to increase in mass and physically separate from other encapsulated cells not containing the enzyme and acetylcholinesterase.
Claim 8
Independent Claim
55 words
A method according to any one of claims 1 to 5 for isolating living mammalian cells characterised in that the substrate of step (f) is converted by the enzyme into gaseous products such that the encapsulated cells at which the enzyme is present levitate in the medium and separate from other cells in the population.
Claim 9
Independent Claim
81 words
A method according to any one of claims 1 to 5 for isolating living mammalian cells secreting acetylcholinesterase characterised in that the substrate of step (f) is a material toxic to the cells of the population which, in the presence of the enzyme is converted to a non-toxic material such that the encapsulated cells containing a sufficient quantity of enzyme will survive, divide and eventually cause those encapsulated cells to become heavier than encapsulated cells in which cells have been destroyed.
Claim 10
Independent Claim
60 words
A method according to any one of the preceding claims for isolating living mammalian cells secreting-acetylcholinesterase which is further characterised by the step of repeating the steps for isolating for resubjecting to isolation the encapsulated cells in which a predetermined degree of identifiable change has occurred so as to further cull the cells producing acetylcholinesterase at the very highest rates.
Claim 11
Dependent Claim
103 words
A method according to claim 1 characterised in that said encapsulated cells produced in step (b) are suspended in a medium in which the cells therein are viable and which maintains such encapsulated cells in a relatively fixed position within said medium, and wherein said substrate is a soluble material which is converted by said enzyme into an insoluble precipitate, whereby the precipitate formation at encapsulated cells containing enzyme causes the encapsulated cells to become heavier and separate from all other encapsulated in the suspension at a rate and to a degree proportional to the amount of enzyme present at the encapsulated cells.
Claim 12
Dependent Claim
185 words
ll. A method for isolating living mammalian cells secreting acetylcholinesterase, characterised by the steps of: (a) obtaining a population of cells containing cells producing and secreting acetylcholinesterase at varying rates; (b) arranging the cells of the population in a medium which permits the cells to remain viable while the cells secreting acetylcholinesterase are being isolated and which limits the diffusivity and mobility of products secreted by the cells such that acetylcholinesterase secreted by any cells in the population will remain in the immediate vicinity of the cell secreting it and cells not secreting acetylcholinesterase will have no acetylcholinesterase in their immediate vicinity; (c) allowing to elapse a predetermined period of time for cells producing acetylcholinesterase to secrete acetylcholinesterase; and (d) adding to the entire cell population a material which specifically binds to acetylcholinesterase and has bound to it a poison which is toxic to the cells of the population only if it penetrates the cells such that cells secreting a large quantity of acetylcholinesterase will be protected from the poison and cells producing little or no acetylcholinesterase cannot similarly be protected and will be destroyed.
Claim 13
Dependent Claim
185 words
ll. A method for isolating living mammalian cells secreting acetylcholinesterase, characterised by the steps of: (a) obtaining a population of cells containing cells producing and secreting acetylcholinesterase at varying rates; (b) arranging the cells of the population in a medium which permits the cells to remain viable while the cells secreting acetylcholinesterase are being isolated and which limits the diffusivity and mobility of products secreted by the cells such that acetylcholinesterase secreted by any cells in the population will remain in the immediate vicinity of the cell secreting it and cells not secreting acetylcholinesterase will have no acetylcholinesterase in their immediate vicinity; (c) allowing to elapse a predetermined period of time for cells producing acetylcholinesterase to secrete acetylcholinesterase; and (d) adding to the entire cell population a material which specifically binds to acetylcholinesterase and has bound to it a poison which is toxic to the cells of the population only if it penetrates the cells such that cells secreting a large quantity of acetylcholinesterase will be protected from the poison and cells producing little or no acetylcholinesterase cannot similarly be protected and will be destroyed.
Claim 14
Dependent Claim
51 words
A method according to claim 11 for isolating living mammalian cells secreting acetylcholinesterase characterised in that the material which specifically binds to acetylcholinesterase of step (d) is chosen from the group consisting of an acetylcholine analog, an acetylcholinesterase inhibitor, and an antibody to acetylcholinesterase and in which the poison is ricin.
Claim 15
Dependent Claim
58 words
A method according to claim 11 or 12 for isolating living mammalian cells secreting acetylcholinesterase which is further characterised by the step of repeated isolations using increasing concentrations of the material which specifically binds to acetylcholinesterase and its bound poison of step (d) such that only those cells secreting acetylcholinesterase at a very high rate will be isolated.
Claim 16
Independent Claim
62 words
A method according to any one of claims 11, 12 and 13 for isolating living mammalian cells secreting acetylcholinesterase which is further characterised by the step of individually encapsulating the cells of the population containing surviving cells such that the capsules containing surviving cells will eventually descend due to the increased weight brought about by the division of the living cells therein.
Claim 17
Independent Claim
127 words
A method for isolating living mammalian cells secreting acetylcholinesterase which is characterised by the steps of: (a) obtaining a population of cells containing cells producing and secreting acetylcholinesterase and which require choline in order to remain viable; (b) suspending the cells in a medium deficient in choline which limits the diffusivity and mobility of products secreted by the cells; (c) allowing to elapse a predetermined period of time sufficient for cells producing acetylcholinesterase to produce and secrete acetylcholinesterase; and (d) adding to the entire system a substance which is hydrolyzable in the presence of acetylcholinesterase to form choline such that the substance is converted to choline in the vicinity of those cells secreting acetylcholinesterase to the extent necessary for their survival in an otherwise choline free medium.
Claim 18
Independent Claim
127 words
A method for isolating living mammalian cells secreting acetylcholinesterase which is characterised by the steps of: (a) obtaining a population of cells containing cells producing and secreting acetylcholinesterase and which require choline in order to remain viable; (b) suspending the cells in a medium deficient in choline which limits the diffusivity and mobility of products secreted by the cells; (c) allowing to elapse a predetermined period of time sufficient for cells producing acetylcholinesterase to produce and secrete acetylcholinesterase; and (d) adding to the entire system a substance which is hydrolyzable in the presence of acetylcholinesterase to form choline such that the substance is converted to choline in the vicinity of those cells secreting acetylcholinesterase to the extent necessary for their survival in an otherwise choline free medium.
Claim 19
Dependent Claim
54 words
A method for isolating living mammalian cells secreting acetylcholinesterase according to claim 15, which is further characterised by the step of treating the cell population with a predetermined dose of an acetylcholine inhibitor such that only those cells making acetylcholinesterase at a predetermined rate will survive after addition of the inhibitor and the substance.
Claim 20
Dependent Claim
23 words
A method according to claim 15 or 16 for isolating living mammalian cells secreting acetylcholinesterase characterised in that the substance is acetylcholine analog.
Claim 21
Dependent Claim
48 words
A method according to claim 15, 16 or 17 for isolating living mammalian cells secreting acetylcholinesterase characterised in that the cells are individually encapsulated in a semi-permeable encapsulating material, such that the capsules containing dividing living cells will eventually separate from other cells due to their increased weight.
Claim 22
Dependent Claim
140 words
-A method for a preventative treatment against the effects of the cholinergic nervous system on a subject of an invading acetylcholinesterase inhibitor characterised by the steps of: (a) administering acetylcholinesterase to the subject; (b) permitting the acetylcholinesterase to pass into the circulatory system of the subject, the amount of acetylcholinesterase administered to the subject (i) being an amount sufficient to establish a concentration thereof in the circulatory system of the subject which is substantially below the concentration of acetylcholinesterase present on or near cells involved in the cholinergic nervous system; and (ii) being at least equal to the amount necessary to establish a concentration in the circulatory system of the subject to intercept and tie up the invading acetylcholinesterase inhibitor before the inhibitor can act upon the acetylcholinesterase involved in the functions of the cholinergic nervous system of the subject.
Claim 23
Dependent Claim
140 words
-A method for a preventative treatment against the effects of the cholinergic nervous system on a subject of an invading acetylcholinesterase inhibitor characterised by the steps of: (a) administering acetylcholinesterase to the subject; (b) permitting the acetylcholinesterase to pass into the circulatory system of the subject, the amount of acetylcholinesterase administered to the subject (i) being an amount sufficient to establish a concentration thereof in the circulatory system of the subject which is substantially below the concentration of acetylcholinesterase present on or near cells involved in the cholinergic nervous system; and (ii) being at least equal to the amount necessary to establish a concentration in the circulatory system of the subject to intercept and tie up the invading acetylcholinesterase inhibitor before the inhibitor can act upon the acetylcholinesterase involved in the functions of the cholinergic nervous system of the subject.
References Cited (Backward Citations)
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