JPS59151886A - Cell isolation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for mediating M11 cells. More specifically, the present invention relates to a method for immediately searching for desired cells from a cell population in which "desired" cells and "undesired" cells exist.

In the context of this specification, a "desired" cell is a living cell that is producing and releasing a specific substance (e.g. an antibody), and a specific molecule is bound to the surface of the cell rather than being released. means a living cell that is In either case, "unwanted" cells are also 4+1 cells that do not have the hallmark properties of a desired cell.

flower arrangement? In various fields of biotechnology, such as immunology, genetics, etc., researchers often identify cells with specific properties, particularly cells that produce and release desired substances or that produce specific molecules, from a large number of cellular ports. It may be desired to form a CP cell and bind it to the surface of the cell. Usually, the relative migration of these desired cells to undesired cells in the M11 cell population is very small.

For example, the production of antibodies against a particular anti- The isolated cell population is isolated from an animal or human infected with the disease in such a manner as to have the antigen on the cell surface. Contains a large number of "unwanted" cells that do not occur. It is troublesome and difficult to extract antibody-producing cells from this cell population.

Following recently developed techniques such as cell fusion, NO.
- Similar national difficulties will be faced when producing cloned antibodies. In cell fusion, a cell population is collected from an animal or human to which an antigen has been administered, or from an animal or human that is diseased and has a II+ cell surface. What?!1 The cell population identified is J9r, which is abundant in many cells that do not produce Jr antibodies.
f! antibody Jf containing live cells. This cell population is combined with can cells such as myeloma cells in the presence of a fusing agent such as polyethylene glycol. Then, the myeloma cells and the cells in the 7+2 cell population combine to form a fused cell. After fusion, cells can be dissociated from the cell population by conventional methods. However, it is difficult and troublesome to describe the desired fused cells, ie, the fused cells of myeloma cells and living antibody Jr cells, which produce and release antibodies.

Alternatively, the desired cell may be one that is producing and releasing substances other than antibodies, such as antigens, and the desired cell may be one that is bound to the surface of the cell rather than being released from the cell. may produce molecules or substances that Kaji in any case! It is difficult to extract desired cells from the cells in the tea room q.

Due to the difficulty of isolating desired cells from a cell population containing both desired and undesired cells, there are currently only a few techniques in use in this field. In one method, to isolate cells that produce and release the desired substance, individual cells in a cell population are cloned to obtain a clone 4+1 cell population with a size suitable for testing. Check whether the desired substance is present in the culture solution of the cloned cell population, and if a positive result of 1 is found, the cells that make up the cloned cell population produce and release the desired substance. There will be. However, it is clear that the application of such so-called "handcrafted" methods, especially to typical cell populations containing millions of cells, is extremely time-consuming.

Tokoro e! 1 cell or antibody of I! Another method (plaque analysis) that is suitable for use when cells are living and emitting Antigens against antibodies released from cells are plated on gel-1 along with red blood cells bound to the surface. The desired antibody released from the cells diffuses through the cell and binds to the antigen on the surface of the red blood cells. When complement is added to the entire population of cells, the red blood cells are lysed by the antigen-antibody complexes formed on the surface of the blood cells. The lysed regions in the population appear transparent (usually when viewed under a microscope). Therefore, the desired antibody-producing cells are found near (usually in the center) of the clear area. This method has the limitation that it can only be used for CI'# of desired cells of one type, it is also limited in the number of cells that can be seen microscopically during a given 11f period, and it It is also difficult to physically remove desired cells from the designated 9'j region.

Lively produced by cells and attached to their surfaces! II
Another method employed to enrich cells using characteristic molecules involves adding to the cell population a ligand that binds to the characteristic molecules. Fluorescein is covalently bound S to the added coordination t, and therefore the coordination -r
The bound cells (i.e., the desired cells) are located in the light-generating region of the cell population. Suitable automatic liquid phase sorters exist that are capable of sorting out regions in a cell population based on bone quality or quality. however,
Such a sorter can detect the existing background "noise" (
1), it is not possible to effectively sort out the desired cells if they represent less than 3% of the cell population to be sorted.Furthermore, these sorters
Less than 1,000 cells can be sorted, and therefore, a complete scan and classification of a typical cell population containing a large number of cells is extremely time consuming.

Another force V, which is employed to isolate cells by making use of 4,ν characteristic molecules produced by 4+1 cells and bound to their surface, is The cell population is flowed onto a surface containing suitable particles or substrates containing ligands that have an affinity for the molecules present. Theoretically, the desired cells bind to surfaces bearing 111 molecules and are picked up from the flowing 4+1 cell population. However, it is extremely difficult to perform this mid-range sound satisfactorily. If the force is less specific or strong in the affinity of the ligand for molecules present on the surface of the desired cells, many unwanted cells will also be captured by the coordination r-. - If the affinity between the force, the ligand and the molecule is high, it results in cells in the circulating cell population being picked off and unwanted cells being retained.

An object of the present invention is to provide a method for extracting desired cells from a cell population containing a mixture of desired cells and undesired cells.

Another object of the present invention is to provide a method that can be applied to a wide range of cell populations and a wide range of desired cell types.

Yet another object of the present invention is to provide a j-dispatch method that is relatively uncomplicated and can be performed quickly.

Accomplishment of these and other objects will become apparent from the following description of the invention.

In this invention, a substance produced and released by a desired cell, or a molecule produced by a desired cell and bound to its surface, is transferred to an environment in a selected area that indicates the location of the desired cell in a population of cells. Use it to bring about. This environment preferably results in selective killing of all unwanted cells in the cell population, but also selective staining,
It may also be one that results in fluorescent labeling or insolubilization of desired or undesired cells, thereby indicating the location of the desired M11 follicle within the cell population.

According to one aspect of this invention, a specific substance is produced
A method is provided for separating desired MII cells from a population of MII cells containing undesired cells (cells that produce and do not release the 41r-specific substance). The specific substance produced and released by the desired cells is, for example, an antigen or an antibody. In this method, the specific substance protects the desired cells from the lethal effects of an agent applied to the entire cell population; Alternatively, the -1-notation r'↓ can be used to bring about an environment in the vicinity of the desired cells that protects the desired cells from the lethal environment imposed on the entire cell population. Illll superposition is used.-
IIJ can also be used to immobilize the enzyme in the vicinity of cells at qJ 0'), and then this enzyme is only present in the vicinity of +9i eI cells in a certain environment. used to form. J-Specific substances are also listed. In the vicinity of the cell of female J, the enzyme 1. ';I
It can also be used to prevent iJization, and the enzyme is then used to create a specific environment in all areas where q+ cells are absent. Finally, the specific substances described above can be used to release the agent only in the vicinity of the desired cells, which in turn brings about a specific environment only in the vicinity of the desired cells.

According to another aspect of the invention, desired cells that produce a specific molecule and are immobilized on their surface, including these desired cells and undesired cells (cells that do not have the specific molecule on their surface). A method of isolating from a cell population is provided. Molecules present on the surface of the desired cells can be used to immobilize enzymes in the immediate vicinity of the desired cells, which enzymes can then be used to bring about a specific environment only in the vicinity of the desired cells. It will be done. Alternatively, the amount present on the surface of the desired cell
can also be used to release the environment-forming agent only in the vicinity of the 9J cells.

Next, the embodiments of the present invention described in 1-1 will be explained in detail with reference to some drawings and specific situations. 1j Since many of the concepts and techniques employed in the isolation of foam can be applied to some or all embodiments,
1\, do not repeatedly explain the details of these divination powers and techniques.

1) of the desired substance emitting 1) of the desired substance producing 4i of the substance 1).
tlf, L, M, which releases the substance of IW.
There are 11 cells. Tokoro 9! The substances released by the m cells (hereinafter referred to as raw materials) are, for example, anti-
, antibodies or immunoglobulins.

In each case to incubate the desired cells of this type, a substance (hereinafter referred to as "antiproduct") is used that is capable of specifically binding to the product. for example,
When it is a product or an antibody.

Anti-products are antigens that associate with the antibody at the -. region of the antigenic determinant. When the product is an antigen, the anti-product has a specific insert or advantage. In addition, it is an antibody against that antigen. When the product is an immunoglobulin, the anti-product may be, for example, a grape T! that binds to the Fc region of the immunoglobulin. It may also be a bacterium.

In this type of method of separating desired cells by Q'L, a specific environment is formed for the product only in the vicinity of the desired cells (or the formation of such an environment is prevented).
used for Therefore, in these methods, it is essential that the product, that is, the substance produced by the desired cell and released from it, has the property of hardly diffusing outside from the vicinity of the desired cell. It is. A method for providing such limited diffusivity will be described later.

According to a first embodiment for isolating desired III+ vesicles that produce and release a specific product, all +1111 vesicles are located at a fixed relative position. Upon immobilization, a population of 4+1 cells is distributed into the receptor bed, which allows for limited diffusivity of the biological products produced and released by the desired cells.

FIG. 1A shows a live cell gyrus T1 after sufficient time has elapsed for the desired cells (D) to produce and release the product (indicated by -→). (The scale has been expanded significantly).

1. of the receptor bed in which the cells are placed.
9. Since the products cannot diffuse very much, the raw Jr. products remain only in the immediate vicinity of the desired cells and cannot migrate to other areas in the cell population. Therefore, since production and release of the raw product by the desired cells continues, the concentration of the product is maximum at or near the surface of the desired cells. As can be seen from Figure 1A, since the undesired cells do not produce products and the products can only diffuse away from the desired cells, they are deposited at four locations on or near the surface of cell 9J (U). There are no products.

The next step in this case is to add antibiotic rL to the entire 411 cell population. This anti-product is bound by a suitable bonding method to a drug or chemical that will kill any living A++ cells that invade it. The state of the system at this point is shown in Figure 1B. In Figure 1, the anti-product is represented by (n-1), and the drug bound to it is represented by (o).
It's public. Because antibiotic drugs act specifically with the products of the desired cells, the drug cannot enter the desired cells due to the product barrier surrounding the desired cells.

-PJ, No such barrier exists around the surface of the undesired Mll+. Therefore, the drug-antiproduct complex enters the unwanted cells unhindered and kills them. As a result, only the cells that are producing in the cell population are the desired cells, and their identification and mourning can be achieved.

A second embodiment of centralizing desired cells that produce and release specific products is shown in Figures 2A and 2B.

In this specific example, desired cells (D) and undesired cells (
Cell populations containing both U) are so-called “scavenging” cells (S
) in the receiving bed. The cells are arranged so that scavenging cells are present throughout the population and xylemally surround all desired and undesired cells. Because a specific medium or receptor bed is used, the relative location of all cells is fixed, and little or no product produced or released by the desired cells will move. Can not.

As a '#' + sweeper cell, its 4H cell membrane changes due to the reaction between the anti-producing cell membrane ()-) present on its surface and the producing cell membrane, increasing its ability to take in molecules from the outside world. You can use any cell that you have. When the product is an antigen, examples of such scavenging cells include biological antibodies or antigens that bind (antibiotics).
Can cells that exhibit physical properties can be mentioned. This one
Because of the 1 coupling, Kan M (1 cell n) changes, increasing the ability of Kan cells to take in molecules from the outside world (pinocytosis).
. Similar phenomena occur in other cells that demonstrate their ability to induce tlI, and the ability to induce tlI has been demonstrated by the method described in more detail below. Can be combined.

Another point about scavenging cells that have 41" anti-inflammatory products is that these will eventually die. It is possible to irradiate the body with radiation at a level of 1f' degrees (1" minute time to accomplish its 17j cleaning function), which is enough to carry out normal catalysis.

Alternatively, mutant scavenging cells that cannot survive in certain media can be employed.

As shown in Figure 2B, when appropriate scavenging cells are selected and placed together with the cells in the cell population, the binding of live Ic (released from the desired cells) to the anti-products of the scavenging cells 1- , occurs only in the vicinity where the product (-→) is present, i.e. in the vicinity of the desired cell. The next step is to synthesize whole cells by adding, for example, methyl trexate (4-amino-butyro-glutamic acid), which irreversibly inactivates folate reductase, which is essential for the production of folinin.
The goal is to put Group F in a harmful (lethal) environment. A harmful environment is one in which anti-products have only scavenging cells associated with the product (i.e. only scavenging cells in the immediate vicinity of the desired cells) or a significantly enhanced scavenging function. It is taken up by scavenging cells at a rate that prevents it from affecting the cells. Unwanted cells surrounded by scavenging cells that do not react with products and therefore do not have an enhanced ability to take in the harmful environment are cleared by harmful substances. After the appropriate amount of time necessary to kill the scavenging cells, the desired cells are the only ones left alive during the cell cycle and are therefore easily separated from other dead cells in the cell cluster. You can cross the border.

What about the desired cells that release special products? i
A third method of #t is shown in Figures 3A-30. In this method, a cell population containing desired and undesired cells is placed in a suitable receiving bed or medium with so-called "carrier" particles or materials. In this case, 111 particles such as crow beads, red blood cells, liposomes, or gelatinous bodies, etc., may be used. One-body material or essentially all desired cells (D) and undesired cells (U) in a cell population
Arrange it so that it surrounds it. This arrangement is also carried out in such a way that the relative positional relationship between the cells and the carrier is fixed and that the 1,000 F produced and released by the desired cells can hardly migrate away from the desired cells.

As shown in FIG. 3A, a suitable anti-product ()-) that specifically binds only to the product produced and released by the desired cells is bound to the carrier. Figure 3B shows the state of the system after sufficient time has elapsed for the desired cells to produce and release biotlr (→). As a result, all the anti-products held in JLj are converted to raw J! in the vicinity of the desired cell area. 1', combine with things. Because the product is not in the vicinity of the undesired cell (because the product is not produced and released by the undesired cell, and the product is not transferred from the undesired cell), the undesired cell In the vicinity of , the supported antiproduct remains unbound.

The next step in this process is to add to the whole cell population what we will hereafter refer to as ``anti-products'' or ``anti-APJ.'' Anti-AP binds specifically to antibiotic products. For example, in the case of a product or an antigen, an anti-product is an antibody of one species (e.g. mouse) and an anti-(anti-4F product) is an antibody from another species (e.g. anti-mouse rabbit). The enzyme (E) may be bound to the anti-AP by an appropriate binding force.

Addition of enzyme-anti-AP to the system is shown in Figure 3c.

In the vicinity of the desired cells, all the antibiotic sites to which anti-AP can bind are occupied by the product, so in the vicinity of q cells, anti-AP (and the enzyme bound to it)
is not fixed. However, anti-AP and its bound enzymes bind to available antibiotic Jt in the vicinity of undesired cells. Unbound enzyme-anti-AP is removed from the system by extensive washing.

By the procedure described above, the enzyme is present only in the vicinity of the undesired cellular areas. Therefore, it is possible to then visually or otherwise undergo an enzymatic reaction, thereby making it possible to distinguish the area in which enzyme reaction did not occur (near vi of cells 9J) from the area in which it did occur. substances can be added to the entire system. For example, a colorless substance that changes to 41 colors in the presence of an enzyme t° can be added to the system. Alternatively, the enzyme I
Colored substances that turn colorless in the presence of water can also be added to the entire system.

Furthermore, for example, a non-fluorescent substance that becomes fluorescent in the presence of an enzyme can be added to the entire system, or a soluble substance that becomes insolubilized in the presence of an enzyme can be added to the entire system. Whichever of these methods is used, it is possible that a specific environment is formed only in the vicinity of the undesired cells by selectively immobilized and immobilized enzymes that enter the vicinity of the undesired cells. You'll understand. In this way, desired cells are identified and separated from undesired cells.

The preferred “
"Environment" is a lethal environment. Thus, one can add to the entire system a free chemical that can be converted by the enzyme into a right jjF substance that is lethal to nearby cells (ie, only the unwanted cells). Then, in the cell population,
The only surviving cells are the desired cells.

A fourth embodiment of separating cells producing and releasing a particular product is shown in Figures 4A-40, and is similar in many respects to the previous embodiment. However, in this method, the enzyme is immobilized only in the vicinity of desired cells and not in the vicinity of undesired cells. - As shown in Figure 4A, the cell population together with the carrier particles or carrier material to which the anti-product is bound, the relative position of the cells and the carrier is fixed, and the desired product is released from the cells. They are placed so that they cannot move at all or at all.

After a suitable period of time, the products from the desired cells bind to the anti-products of the carrier particles-1- surrounding the desired cells, as shown in Figure 4B. Since there are no biological substances in the vicinity of the undesired cells, such binding does not occur.

The next step (Figure 4C) is to add to the entire system a substance that specifically binds only to the product, product complex, or anti-product. This "anti-product 2" is an anti-immunoglobulin that can bind to the Fc or FA6 region of the immunoglobulin, for example, when the product II is an immunoglobulin and the antibiotic Jf product is staphylococcus. Antibiotic It 2 includes:
Enzyme-￥(E) is combined. As a result, the enzyme-antiproduct 2 complex (vomit →) is raw J￥: product, product complex,
Alternatively, the anti-product is immobilized only where it is present (ie, in the vicinity of the desired cells).

After removing unbound enzyme-antibiotics from the system, the system can be provided with an environment that indicates the location of the desired cells by colorimetry, fluorescence, or insolubilization by the action of the enzyme, as described above. Alternatively, the system can be provided with a toxic environment that is detoxified by the enzyme only in the vicinity of the desired cells. Then, the only surviving cells in the cell population will be the desired cells. A somewhat similar method involves making the entire system a lethal environment for all cells (e.g. adding methotrexate to the cell population. For enzymes that are pre-immobilized only in the vicinity of the desired cells, this 'J WA An enzyme is selected that replaces folinic acid and leaves only the vicinity of the desired cells a viable environment.An enzyme suitable for this purpose is folate reductase.

A fifth embodiment of isolating cells that produce a constant product is shown in Figures 5A-5D.

Similar to the method described above, the cell population, together with carrier particles or carrier materials to which anti-products are attached, is fixed in relative position between the cells and the body, and the products released from the desired cells are controlled. Placed so that there is little or no movement.

In this specific example, the carrier particles contain a so-called "incorporated agent" therein. Here, we will proceed with the discussion assuming that the first drug to be used is an enzyme. This encapsulating agent can be incorporated into carrier particles during the manufacturing process (if the carrier is a liposome, for example), or, if the carrier is a liposome, the erythrocytes can be incorporated into the encapsulating agent (enzyme). Hypotonic Fuchu, which contains
The red blood cells can then be contracted back to their original size by returning the solution containing the red blood cells to isotonicity. The aforementioned J
- Similar to the L-body example, it has 11j anti-products and a 111-body particle containing an enzyme and a button! The cell population is all the desired and
The carrier particles are placed in close proximity to, preferably in contact with, the cells of 1 (see Figure 5A).

After a period of incubation 1111, the products released from the desired cells bind to the anti-products on the carrier particles in the vicinity of the desired cells (FIG. 5B). Such binding does not occur because there is no product in the vicinity of the unwanted cells.

One method in this embodiment includes the following step (5C
Figure) binds only to the product-antiproduct complex and is similar to the anti-product 2 described above), and furthermore, through this binding, a substance (←) that destroys the red blood cells or liposomes carrying the complex is released. It is added to the entire system. Complement can be mentioned as an example of such a substance. Disruption of these red blood cells or liposomes (which occurs only in the immediate vicinity of the desired cells) releases the enzymes contained within them. In this system, the movement of everything contained inside (i.e., cells, carriers, products, and released contained agents) is prevented, so the enzyme (E) released from the destroyed carrier is It is fixed at the released location, ie, in the vicinity of the desired cells (see Figure 5D).

Since the enzyme is immobilized in the vicinity of the desired cell, various operations described in 1-1 above can be employed to create a state that can be identified only in the vicinity of the desired cell using the enzyme. By using an appropriate enzyme, the desired ti(I+ cells are colored (or decolored) or insolubilized only in the vicinity, or the iiji mulberry is made FOP-free only in this region,
Only desired cells can be rescued from the f1i environment.

In other methods of this embodiment, the included agent can be a substance that directly rescues the desired cells from the 4i environment. For example, the system can be treated with Met I Rexade, which begins to kill all desired and undesired cells, before adding the substance that binds to the product-antiproduct complex. Mel-trexate is then washed out of the system and the product-antibiotic
1 (A substance that binds to the substance is added. When the red blood cell or ribbon-1 is destroyed by the binding of this substance, it is contained in the red blood cell or liposome and saves the desired cell from the process that leads to death.) Chemicals such as folinic acid are released (only in the vicinity of the desired cells).

In the specific example described in h above, it is preferable to bind an inert polymer to the contained agent (eg, enzyme or rescue substance) contained in the red blood cells or liposomes. By doing so, it is prevented that the contained agent naturally spontaneously leaks out of the red blood cell or liposome 11 before the red blood cell or liposome 11 is destroyed, for example by the action of complement. If the contained agent is spontaneously and substantially released, the enzyme or rescue substance will be present near cells other than the desired cells. In general, the leakage of substances from liposomes is so small that there is no need to use natural release-inhibiting polymers. In addition to preventing the natural release of enzymes and rescue substances before the red blood cells or liposomes are destroyed, the macromolecules attached to the enzymes or rescue substances also prevent the release of enzymes or rescue substances from the destroyed red blood cells or liposomes. Preferably, the diffusion of the enzyme or rescue substance is prevented, thereby ensuring that only cells residing in the vicinity of the destroyed red blood cells or liposort (T, i.e. in the vicinity of the desired cells) have access to the enzyme or rescue substance. be done. However, because
Normally, the geometry of the system will ensure that this length of 11-min.

! In each of the specific examples described above, the cell population (and the 111 particles in the case of ≧11), the cells (and the carrier particles) in the cell population are solid A! It is safe to place it in such a way that it is immobilized in a fixed position.

The system must also be designed to ensure that the products produced and released by the desired cells are not transferred from these cells. This is because there are various methods for selectively creating a specific environment only in the vicinity of desired cells, or selectively not creating it.
This is because the effect of the substance is limited to the vicinity of the desired cell area. Roughly speaking, in these specific examples, the effect of the substance being released into the system is limited. agent or substance r'
Displacement from the point of release must likewise be minimized.

Many techniques can be used to achieve these conditions. In one method, shown in Figure 6A, the cell population is comprised of desired cells (D) and undesired cells (U).
is placed in a gel-like medium such as agar, agarose, alkyne salt-tolerant, collagen, nitrocellulose, or gelatin, which immobilizes the product at a fixed position 1δ and prevents the product from diffusing from the desired cells. Even when carrier particles are employed (see Figure 6B), they fix the position of the desired cells, the undesired cells and the carrier (C) and prevent the diffusion of the product and any substances released from the carrier. The same Kel-like material can be used to restrict. Alternatively, if the anti-product is bound to a carrier, the gel may constitute the carrier for the anti-product.

To achieve this immobilization, a centrifuge can be employed to maintain the cell population in a thin single cell layer on the surface of a rotating centrifuge. When carrier particles (C) are used, as shown in FIG. - Maintained on a regular basis. A filter paper layer 28 containing an immobilized carrier layer 30 is brought into contact with a thin cell population layer. Due to the centrifugal force, the positional relationship between the cells in the thin cell layer and the particles in the filter paper is changed, and the action of particles with anti-product 411 in the vicinity of the 9 cells rotates the desired cell position. It is fixed so that it can be used as a single stage.

Another method of achieving the necessary immobilization is to microscopically stipple the cells 42 such that each cell 42 in the cell population occupies an individual fixed position when the cell population is added. (See Figure 8). 7) However, if anti-product supporting particles are used, a material 48 such as filter paper is used to bring the cells 42 into contact with the anti-product Ifj particles or supporting material in the filter paper.

Yet another girlfriend who achieves immobilization, is the 9th
As shown in Figures A and 9B, the MII cell population (including desired cells 62 and undesired cells 68) is placed inside or outside the hollow fiber. The hollow fiber 60 is formed of a membrane with a permeability of +1, and the size of the pores in n;
4 (which is fixed on the inside or outside of the hollow fiber), but is of such a size that cells and carrier particles cannot pass through the membrane.

In the specific methods described above for isolating desired cells that produce a particular product, there are many instances in which various substances must be combined before these methods can be carried out. For example, in some embodiments, the enzyme may be an anti-producer, an anti-AP
, or must be combined with anti-product 2. This bonding can be done by known covalent bonding methods, or
When an antibody is produced against an enzyme, this antibody is
binds to enzymes by natural affinity, and anti-products,
Anti-AP and anti-product 2 are those that have affinity for the enzyme antibody or enzyme-antibody complex. Methods of conjugating chemicals and drugs, for example to various types of anti-products, are also known.

If anti-111 particles are required, there are a number of known methods for obtaining such particles. For example, when the product is an antibody, the carrier t-j'- is red blood cells or liposomes, and the anti-product or antigen supported on the red blood cells or liposomes, the following method can be adopted. .

(a) Chemically attaching antigens to the surface of red blood cells or liposomes without killing or severely damaging the red blood cells or liposomes.

(b) The red blood cells or liposort and the antigen are placed in an incubator, and the antigen is adsorbed onto the surface of the red blood cell or liposort.

This also allows the erythrocytes or liposomes to be combined with organisms (such as trypanomas) that release antigens and bind them to the erythrocytes or liposomes! By doing i, you can also do something i1.

(C) One stage of crosslinked T is used. That is, cells and cell-like 1
For example, palmito-
It binds antigens to molecules such as fatty acids.

(d) Liposomes that naturally have the property of fusing with cell membranes 1. Alternatively, an antigen may be incorporated therein, and the substance in the liposome may be applied to the cell membrane of red blood cells.

(e) Incubating cells having receptors for the complex with the antigen-antibody complex (or a liquid containing the complex). If the antigen is a multivalent antigen and the complex is present in an area of antigen excess, the bound complex makes available free antigen available for specific antibodies produced by the desired cells. Do 1.

(f) Induce the cells to a) place the antigen on the surface of the cells. This can be done either by making the antigen part of the surface of the cell, or by infecting the cell with an organism (such as a virus or a virus) that induces the cell to produce the specific antigen. I can do it.

(g) Binding the antigen to the surface of the red blood cell or liposome by fusing to the surface of the red blood cell or liposome a membrane fragment obtained from a cell containing the antigen.

(h) as carrier particles when the product produced by the desired cells is an antibody and the anti-product is an antigen;
For example, living cells can be used, such as Kan cells that are sensitive to specific antibiotic J1f agents, such as cancer antigens.

According to another aspect of this invention, there is provided a method for locating desired cells that produce but do not release the product J[9J] on their surface. be done. Rather than not releasing the product, the "product" is bound to the surface of the desired cell or becomes H at the surface of the desired cell. This product or molecule characteristic of the desired cells to be immobilized is found only in the vicinity of the desired M++ cells, in the same way as described above.
It can be used to create (or not create) a certain environment.

The difference from the above-mentioned case where the product is released from the desired cell is that the raw product is released from the surface of the cell! - and cannot migrate from the desired cells. As a result, the phase between the raw material and other substances used to detach the desired cells of this species! The L action occurs at the membrane 1- of the desired cell or near the desired cell region. Therefore, the f-stage to immobilize the cells and prevent product from migrating from the desired cells is not strictly frozen.

Another difference is that there is no longer a need to use anti-products to specifically target substances (eg enzymes) to the vicinity of desired cells.

Rather, the enzyme is conjugated directly to the product on the surface of the desired cells by selecting the appropriate antibody.

Taking these into account, the first specific force U that single-grains a desired cell with a characteristic molecule or product on its surface is simply a force U that acts on the product on the surface of the desired cell. It consists of adding an enzyme that specifically binds to the cell population (eg, in a stirred container or in a suspension). If the enzyme is present only in the vicinity of the desired cells (in fact, it is bound to the desired cells), the various environments described above can be used to selectively distinguish desired cells from undesired cells. can be formed by enzymes.

It will be appreciated that similar results can be obtained, if desired, by using anti-products, etc., in the -1 series for the cells that produce and release the product. Therefore, anti-products can be used to bind products on the cell surface,
Antiproduct 2, to which the enzyme is attached and which specifically binds to the raw product or product-antiproduct conjugate, can be used to target the enzyme only to the vicinity of the desired cells.

9J cells that have characteristic molecules or products on their surface? As another example of separating ti, a method similar to that shown in FIGS. 5A to 5D can be used. In this method, a carrier with an anti-product bound to its surface and containing an encapsulated agent (e.g. an enzyme or rescue substance) inside is placed on the desired cells. bound to the product. Then, using a suitable agent, only the carrier particles where the interaction between the product and the anti-product has occurred are destroyed. This destruction results in the release of enzymes or rescue substances only in the vicinity of the desired cells (assuming an arrangement that inhibits migration is employed).

Within the context of this discussion, certain methods and operations are presently considered particularly preferred.

Ensure that an environment is generated (or prevented from being generated) in the vicinity of the desired cells, either directly or indirectly, through the interaction of products produced by the desired cells with other substances. With regard to the immobilization of cells and other substances necessary to
cells in solid or hollow bead-like materials, such as pieces of acarose, Jn alginate, seratin, etc. This immobilization method is 1
- Can be used for any of the above-mentioned "111 one-ono".

The purpose of this immobilization force, V, is to completely immobilize each cell in the cell population, whether it is a desired cell producing a desired product (e.g. an antibody) or an undesired cell. The purpose of the present invention is to provide separated beads. This means that, statistically, the cell population will be suspended in the presence of one minute of bead-forming material such that each bead contains one cell. This can be done by known methods to form microbeads of uniform size. Bead formation is carried out by emulsifying the suspension in oil at a temperature or under conditions such that the bead-forming material is a liquid, and then bead formation occurs and the beads are separated from the emulsion. This can be done by gradually changing the temperature (for example, by cooling).

If the dust method employed relies on the presence of other particles around the cells, for example scavenging cells (Figure 2), carrier particles (Figures 3 and 4) or Goi J
When carrier particles containing agents (fiS51Δ) are used, the formation of beads is carried out such that these substances or particles become entrapped within the beads with the cells in the cell population.

The formed beads can contain various substances that are sensitive to the ELL methods described above, such as antibiotics to which drugs or enzymes are attached, enzymes, rescue substances, sterile substances, etc. The bound anti-AP or anti-product 2 may freely enter the beads and perform phase-LL action with the cells therein, or if binding does not occur, it may be freely washed out from the beads. Something can happen. 111) Of course, the pores of the beads can also be made so large that the cells or particles within them cannot escape.

By way of example, this immobilization method will be described with reference to the isolation method generally illustrated in FIG. The beads are formed to contain j) t- cells, whether desired or undesired cells, together with l or more [- particles carrying anti-products. In contrast to the situation shown in Figure 3, where desired cells and undesired cells exist (albeit immobilized) in a circulating medium or device, when beads are used, a single cell are physically separated from other cells.

After a suitable incubation period, the products from the desired cells bind to the anti-products on the carrier. On the other hand, in beads where the desired cells are not present, the anti-product on the carrier remains unbound.

Enzyme-conjugated anti-AP is then added to all beads. Enzyme-conjugated anti-AP diffuses into all pieces, but is immobilized only in beads containing unbound anti-product, ie, beads containing Chasho ep cells. After washing out the unbound anti-AP-enzyme, only in the presence of the enzyme, ie Chasho! The whole piece system can be subjected to a large number of possible environments that can only be measured or identified in beads containing -7I vesicles. This environment may, for example, enzymatically turn the animal colorless or colored, become fluorescent, or create lethal conditions.

As can be imagined, when the desired cells are finally selectively identified (e.g., by coloration, fluorescence, or producing light) from the cells of the cell, they are Their inclusion in pieces makes it potentially more difficult to isolate them from undesired cells that are also contained in beads. For example, an isolation method selectively keeps the desired cells alive (or does not expose them to a toxic environment)
If so, it is necessary to further isolate the beads containing live cells (ie, the desired cells). For this reason, a number of alternative methods to bead immobilization have been developed.

A first method using the above-described technique of selectively binding an enzyme to the vicinity of desired or undesired cells (i.e. within the beads) involves, as an enzyme, directly or indirectly lysing the beads after a period of time after binding. A method is employed that can selectively leave beads containing desired cells or undesired cells intact. In one embodiment of this method, alkyne M is used as the gel material constituting the piece.
Use IXM. After the enzyme has been selectively bound to or near specific cells in the beads, the beads are each surrounded by a porous, low-volume separation layer, such as a lipid material. As the enzyme, one that hydrolyzes alkyne vinegar salt is used. After hydrolysis, the calcium is removed from the entire system (eg by washing out or adding a suitable sequestering agent).

This liquefies the algine salt-resistant gel (which requires calcium to gel). As the surrounding porous layer, one having pores of a size that allows only the liquefied Kel to pass through, but allows the gel material hydrolyzed by enzymes to pass through is used. After removing the escaped material, calcium is added to the entire system, causing the alkinate to gel. Upon removal of the outer surrounding layer, the system consists of gelled beads containing only the desired cells or cells.

45 A second method of isolating certain beads involves the use of an enzyme (which selectively binds to beads containing desired or undesired cells) to replace a particular environment with another environment that makes the beads physically different. Adopt what you can change. for example,
Enzymes that release oxygen or other gases can be employed. Pieces containing gas will then float higher in the water than pieces that do not contain dregs, so they can be sorted. Therefore, when catalase is used as the enzyme and hydrogen peroxide is added to the system, beads in which hydrogen peroxide has been converted to hydrogen and oxygen will float more easily than beads in which gas has not been generated. At the same time, of course, hydrogen peroxide not converted by catalase is lethal to the cells in the beads. Therefore, in this particular embodiment, selective binding of the catalase enzyme is set to occur only on beads with the desired cells, eg, by methods similar to those shown in FIGS. 4 and 5.

A third method of isolating specific beads is to ensure that only the beads containing the desired cells remain gelled in the system through the influence of enzymes or other substances. This can be achieved through a number of operations.

In one method, when the desired cell product is an antibody, the antigen is incorporated directly into the gel material #I that makes up the beads. In addition to capturing antibodies from the desired cells, the antigen crosslinks the gel material after a suitable period of time. Therefore,
Similar to the method described above, all beads can be surrounded by a barrier layer. For example, an alkynoic acid 1n gel can be used and the gel removed from the system to liquefy the gel. The liquefied gel incorporating the antigen (no antibody bound, i.e., from beads containing cells of different qJ) can be used to properly transform the pore size of the T-layer.
It then diffuses through the barrier layer and is removed from the system. A liquefied gel in which the antigen is bound to the antibody cannot diffuse through the barrier. after that.

When Calcilla 1 is added to the gel and the barrier layer is removed, what remains are gelled beads containing the desired cells.

Another method that may be useful in this regard is
The beads containing the cells and the undesired cells contain a calcium-containing agent button and a "microcapsule" containing the calcium-containing agent. Thus, if the pieces are formed of an alkinate, the microcapsules may be liposomes containing I-memoplasm. Using a method in which the enzyme is immobilized on the root fungi only in the vicinity of the desired cells (i.e., within the beads containing the desired cells), the enzyme is then immobilized on individual pieces in the system (i.e., within the beads containing the desired cells), and then (containing cells and undesired cells) are surrounded by a barrier layer. Calcium sequestering agents (e.g. ED) can be added by increasing the temperature of the system.
TA) containing liposomes (located within all beads) are ruptured. EDTA binds with calcium ions and removes calcium from the gel, and the gel is liquefied. The barrier layer may be a liquefied gel or something that cannot be diffused.

As the second liposome in the beads containing calcium ions, one that is gradually decomposed by the enzyme used in the R1 step is used. Since the enzyme is only present in the beads containing the desired cells, calcium is released only in such beads, causing the liquefied alkinate to gel.

Once the barrier layer is removed and the system is washed, the only solid particles left are gelled beads containing the desired cells.

Another method to ensure that only the beads containing the desired cells remain intact is to cross-link the gel material so that all beads contain microcapsules containing a substance that is released from the microcapsules only in the presence of an enzyme. That's true. For example, if agarose is the material constituting the heath, the microcapsules can contain an "antibody" against agarose that binds to and crosslinks the agarose. Using a barrier layer as described above, which allows only liquefied agarose with no bound antibodies to pass through, the entire system is incubated for one year under conditions that liquefy all the agarose. When Kel is liquefied, enzymes act on the microcapsules to digest them, and the substance (crosslinking agent) inside the microcapsules is released. After gelling the agarose (+1) and removing the barrier layer, all remaining beads contain the desired cells.

By using the piece immobilization technique, it is possible to adopt a so-called "amplification" reaction in any cell center horn 4 in this invention.

In any case where the enzyme is required to act on all the beads, the amplification procedure uses liposomes present in the beads that are destroyed by the enzyme to provide substances that act on the beads. . The reason for this operation is that the total amount of enzyme that can be contained in one bead is extremely small (because the enzyme that selectively binds indirectly or directly to the vicinity of the desired cells is Since it is a product produced by
This is because the amount of enzyme present in the beads is directly proportional to the amount of product produced by the desired cells. Since the amount of product produced by the desired cells is so small, the small amount of contaminating enzyme may not be able to affect the entire bead. However, the amount of enzyme is sufficient to affect (ie, digest) microcapsules in the heath that have a relatively small surface area. When the microcapsules are digested, sufficient imitation material is released from the microcapsules to affect the entire bead. In this way, the "width" of the beads for small numbers of enzymes is greatly amplified. Furthermore, by using a large number of small microcapsules rather than a few large microcapsules, it is possible that the small microcapsules contain the desired quickly released substances (e.g., dyes, rescue substances, EDTA, calcium salts). ) can be increased using a small amount of enzyme more effectively. This is because small capsules have thinner, more easily broken surfaces. In this way, greatly amplified reactions can be achieved from only a small amount of raw material from the desired cells.

In other aspects of Enhancement 119J, enzymes are used to convert a first antigen to a second antigen.

The second antigen is then captured by an antibody bound to liposol 1 (after liposol 1 has been lysed, e.g. by complement) or by an antibody of the gel material 1 of the bead. . Thereby, the number of antibody sites on the liposome or gel that are affected can be greatly amplified. A somewhat related method that does not use enzymes is illustrated by the following specific example. For all beads,
That is, both those containing desired antibody-producing cells and those containing undesired cells that do not produce the desired antibodies are combined with anti-products and antibodies against viral phages or antibodies against habten bound to phages. anti-AP or anti-product 2. After washing, anti-AP-antiphage (or anti-hapten) or anti-product 2-antiphage (
or anti-habten) remains only in beads containing the desired cells.

A second bead then surrounds each first bead and within this surrounding bead layer binds to and cross-links the gel material of the bead or binds to the liposome in the first heath. A bacterium and a phage that produce a substance that binds to the antigen of JF8 are introduced.

The antiphage is anti-AP or anti-product 2 in the first bead.
The phages are separated from the phage and diffused into the surrounding gel, inactivating the phage. Since bacteria can only grow in the absence of live phage, they will grow and release substances only in beads containing the desired cells. The multiplicity of substances released by the bacteria diffuses into the first bead and acts on the antibodies bound to the gel or liposomes. In this way, polyacid substances are allowed to act on the beads.

In these or other methods described herein, enzymes are used to disrupt microcapsules and release specific agents (e.g., gel crosslinkers, calcium ions, dyes, etc.). It is preferable that the microcapsules can be freely moved within the beads to reach the microcapsules. This is because the enzyme is bound to anti-product 2 or anti-AP when added to the beads, and then binds to the product or product-anti-product complex in the beads, so it is fairly immobile in the beads. This is because it has been made into In some cases, the immobilized enzyme may have a strength of 1-min or be located close to 1-min of the microcapsule, or the enzyme may have a close phase 1f interaction with the microcapsule. To ensure this, it is preferable to make the enzyme "rIi-active". This can be done in a number of ways. One method uses an i'If reverse cross-linking agent, such as a disulfide, to link the enzyme to anti-product 2 or anti-AP. After the enzyme-anti-AP or enzyme-antiproduct 2 complexes are bound in the beads by interaction with the products from the desired cells,
A suitable agent is introduced into the beads that is capable of releasing bound enzymes and allowing them to move through the beads. Another method employs the anti-product 2 or anti-AP to which the enzyme is bound to have a low but sufficient affinity for the product or anti-product. After the enzyme-antiproduct 2 or enzyme-anti-AP complex binds to the product or product-antiproduct, the antibiotic lc2 or anti-AP complex has a high affinity for the product or product-antiproduct. Add AP.

This high-affinity substance replaces the previously occupied enzyme-anti-
The AP is expelled, allowing the enzyme (with bound anti-AP or anti-product 2) to move into the piece and interact with the microcapsules.

The use of enzymes to disrupt liposomal microcapsules typically slows down the process, which is preferred. Because, if this is not bitter, the enzyme-anti-AP
Alternatively, simply introducing the enzyme-antiproduct 2 complex into the beads will cause the contents of the microcapsules to be released prematurely. However, certain procedures may be adopted to ensure that no release occurs during the first phase (e.g. when the enzyme activity is very high and/or the enzymatic degradation products of the liposomes are harmful to the cells themselves). You can also do it.

In such operations, an activator (e.g. J
l: <+x Unless there is an intrinsic factor, a substance that does not act on Liposor 1 or other microcapsules is used.

The activator is not added until the enzyme-anti-AP or enzyme-anti product 2 is bound within a particular bead. Alternatively, one can select Noboso 1 and an enzyme that becomes active only at a certain temperature, and enzyme-anti-A
The temperature may not be increased until the P or enzyme-antiproduct 2 is bound within the single bead. Yet another method is to use enzyme-anti-AP or enzyme-anti-products that have low permeability through individual microcapsules and cannot forcefully pass through the enzyme-anti-AP or enzyme-antiproduct 2 to act on the liposome or group of liposomes.
For example, it is surrounded by an agarose layer or a capsule. - When the enzyme is detached from the complex using the following method, it is now large enough to pass through the acarose layer and interact with the liposomes therein. In this method, it is useful to combine the feature that the enzymatic activity on liposomes is temperature dependent. Increasing the temperature of the liposomes can be accomplished by increasing the temperature of the entire system or by induction heating incorporating small metal particles within the acarose layer containing the liposomes.

A particular embodiment utilizing a similar principle to F-duplex employs liposomes having antibodies against benicilic acid attached to their surface. Enzymes added to the system (enzyme-anti-AP
or enzyme-antiproduct 2) is penicillinase. After the enzyme conjugate is bound in the beads, penicillin bound to a carrier material is added to the system. The penicillinase enzyme converts penicillin to benicillic acid, which is captured by antibodies bound to the liposome surface. Next, antibodies against the carrier are added, followed by the addition of complement to the system, which lyses the liposomes and releases the substances contained within them.

In another specific example, urokinase is selected as the enzyme coupled to the enzyme-anti-AP or enzyme-anti product 2. The enzyme is bound in beads containing the desired molecules.
After immobilization, plasminogen, a soluble lignically inert substance, is added. Plasminogen is converted to plasmin, an active proteolytic enzyme, by immobilized urokinase. By using microcapsules made of polylysine to contain the active substance on the beads, plasmin digests these microcapsules and the substance is released. In this conjugate example, it will therefore be seen that it is not strictly required that the enzyme be truncated in order to act on the liposomes within the pieces or beads.

Any immobilized form (e.g., air-woven fiber gel, centrifuge, machine surface, beads, etc.) is suitable for all isolation techniques; ), I
'ft The desired product that produces and releases the desired product?' 11 A particularly preferred method of separating beads (described below) produces a particularly desirable product and
Contains confirmation to isolate only the desired cells.

An isolation method that uses an "antigen" having a large number of antigenic determinants, each capable of binding a different antibody, to capture "antibodies" from many clones, each grown in separate beads, the method comprising: When it is desired to expand cells or clones producing a specific antibody (e.g. fused cells producing a specific n-clonal antibody), I7X uses previously produced antibodies. One is employed that prevents the antigen from capturing other unwanted antibodies, such as. In this method, when antigen is added to the system, all types of antibodies produced by all antibody producing cells (their FAB fractions) may also be added. In this way, by adding the anti-JJrC of Chasho 9I (without the F c region, as the F,8 region) to occupy all the antigen binding sites of Chasho 9J,
This multivalent antigen captures only the desired antibodies (which include the Fc domain) produced and released by the cells of interest. The addition of the anti-Fc-conjugated enzyme then immobilizes the enzyme only in the vicinity of cells producing the desired antibody or antibodies other than those added to the system.

A similar method using antigen to selectively capture 4,+r constant antibodies can be used. For example, antigens directed against one or more antibodies produced can be added to beads containing antibody-producing cells. In a second layer around the beads, an antigen for the desired antibody can be placed. Antibodies other than the desired antibody remain within the fffJl beads because they are bound to the antigen in the first bead.

The desired antibody freely diffuses into the '52 layer where it binds to the corresponding antigen.

The most preferred method for perfusing desired cells that produce the desired product from a cell population is one in which conditions are created that affect the molding or density of the beads containing the desired or undesired cells. be. In this way, beads containing desired cells can be rapidly isolated from beads containing undesired cells.

For example, as shown in Figures 1OA to 10G, individual cells of a mixed cell population (including both desired cells that produce certain antibodies and undesired Ml+ cells) form a heath. ``1'' is placed in a permeable membrane material. A large number of red blood cells are present within each bead, and an antigen specific to the desired antibody is bound to the surface of each red blood cell. In the 1OA diagram, the antigen is indicated by a -. All beads are suspended in a medium suitable for cell growth.

A condition that exists after enough time has passed for cells to produce and release products! C1 is shown in FIG. 10B.

Red Jr. spheres only within the beads containing the desired cells.
, and a product produced by the cell that is different from this antibiotic (4.ν). In other cases, i.e., no product is produced or the desired production is not achieved. If other products are being produced, antigens will not bind to red blood cells.

Adding sleeves to the total cell population (Heathwood t for complement)
't is permeable), red blood cells with antigen-antibody complexes are lysed. Therefore, only the red blood cells contained within the beads containing the desired cells are lysed by complement. This is because only within such beads does the antigen on the red triocytes bind to the antibody. Red blood cells within all other beads remain intact.

The permeability of the heath material is chosen such that the lysed red blood cell components can pass through it to the external medium (first
0C diagram). As a result of lysate flow, the beads containing the desired cells become significantly more dense (less dense) than all other beads and can be separated from other beads by weight-based methods. I can do it.

-1-The above method is very suitable when the antigen specific for the desired antibody is bound to the red 1f sphere. However, there are many cases in which it is simply not possible to bind such antigens to red blood cells. This is because the antigen is detached from the surface of nucleated cells where the antigen resides, it is difficult to attach the antigen to red blood cells, or the different antigenicity of the desired antigen is destroyed or changed. In such cases, the source of the antigen is a nucleated cell with the antigen present on its surface.However, unlike red blood cells, nucleated cells cannot be lysed in any case. Therefore, it is extremely difficult to
Did you explain about the IT ball? It is not possible to adopt a method similar to the separation method V (for example, FIGS. 1OA to 100).

For such cases, ie, when the antigen specific to the desired antibody is carried by nuclear cells that are not easily lysed, the following method has been developed.

In one method, shown in Figures 11A to 11D,
Individual cells of the J1L cell population are placed into bead-like beads. In each bead, for example, Kan cells (the first
1A (represented by "T"). 4″: The pieces are suspended in a suitable J8 substrate.

After a suitable incubation period, the heath containing the desired cells contains the desired antibodies that specifically bind to the cancer cell-human antigen. Such a phase does not occur in beads containing cells that do not produce the desired antibody (see Figure 11B). A complex of anti-immunoglobulin (anti-product 2) and enzyme is then added to the whole cell population. This complex is able to pass through the bead material, but is immobilized only within the piece where the antigen-antibody complex resides, ie, the bead containing the desired cells (Figure 11C).

After washing out the unbound anti-immunoglobulin-enzyme complex, soluble material is added to the whole cell population, which becomes an insoluble precipitate by the action of the enzyme carried on the anti-immunoglobulin. Since the enzyme is present only within the beads containing the desired cells, insoluble precipitates are formed only within the beads containing the desired cells (Figure 11D). As a result, beads containing desired cells are much denser than beads containing undesired cells, so they can be separated by a simple method that utilizes the difference in weight.

l-H1rlj method is called an "amplification" reaction, i.e.
The weight of the beads containing the desired cells can be modified to allow more increase in rlt than due to enzyme-induced conversion and precipitation of insoluble material. This amplification can be accomplished 11 by adding to the entire system a substance that binds to the precipitate that forms within the beads containing the desired cells. This substance (due to its +1 quality, antibodies against the precipitate and t
), a large one (but able to pass through the bead material) is selected. This is where the precipitation occurs (i.e. only within the beads containing the desired cells)
The weight of such beads is much higher than that of beads for which there is no precipitate (therefore there is no "antibody" against it), i.e. beads containing undesired fAl + cells. growing. In this way, separation using the difference in claw size can be performed more easily.

This amplification method was explained earlier (1st OA to 100
It is also applicable to the method in which red blood cells carrying antigens specific for the desired antibody (Fig.) are lysed.

In this case, the semipermeable bead material is selected to retain certain components of the lysed red blood cells, in particular hemoglobin. Because lysis occurs only within the beads containing the desired cells, free hemoglobin is present only within the beads containing the desired cells. After lysis has occurred, antibodies against hemoglobin are added to the system in bulk. This antibody is then immobilized only where M# hemoglobin is present. In this way, beads containing desired cells will be much heavier than beads containing undesired cells, thus facilitating separation based on weight differences.

Another cell isolation technique in which antibodies specific for the desired product are carried by cells that cannot themselves be lysed is as follows. 1st
As shown in Figures 2A-12E, individual cells in the cell population are housed within beads of semipermeable material.

In addition to these cells, inside the beads are Kan cells carrying an antigen (-be) specific to the desired antibody, and a certain substance (-) (
(I will explain this in more detail later) is 1! -1 red blood cells (see Figure 12A).

After an appropriate incubation period, a given qJ of antibodies produced and released by the desired cells bind to the antigen (12th BIA). An enzyme-conjugated anti-product 2 (eg, anti-immunoglobulin) is then added to the entire cell population.

This is immobilized only within beads on which an antigen-antibody reaction has occurred, that is, beads containing desired cells (FIG. 12C).

Therefore, when the unbound anti-immunoglobulin-enzyme complex is washed out, the beads containing the enzyme become only those containing the desired cells.

As enzymes are identified on beads containing the desired cells, specific compounds (added to the whole cell population) are added to the dr.
A compound (- in Figure 12D) that exhibits specific antigenicity to the compound (□) that is attached to the surface of the beads, and jl
It is selected whether it can be converted into In this way, an antigen-antibody reaction occurs on the surface of red blood cells only within the beads containing the desired cells (FIG. 12D). In this state, for example, complement can be added to the system to lyse red blood cells only within the beads containing the desired cells. The components of the lysed red blood cells pass through the bead layer and flow out (Figure 12E), so that the beads containing the desired cells are replaced by the beads containing the undesired cells (which still have intact red blood cells). This makes it possible to separate beads using the difference in weight.

In all of the above examples where Kang cells are used as "carriers" for specific antigens, Kang cells proliferate and divide while various reactions occur or reagents or compounds are added. is imagined. ” - The single-legged method mentioned is
Because it takes advantage of the relative density difference between beads containing desired cells and beads containing undesired cells, the growth rate of Kan cells in each dose is uniform, which is often the case. If not, an error will occur.

For example, if isolation method 2 forms an insoluble precipitate within the beads containing the desired cells, thereby making them heavier than the beads containing the undesired cells (see Section 11A (see Figure 11D), l or 2 or more
It is possible that the antigen-bearing cancer cells in the second bead containing the undesired cells divide faster than the cancer cells in the one or more beads containing the desired cells. If the rate of division of cancer cells in such beads (containing undesired cells) is greater than 1 minute, they are considered to contain the desired cells, although they do not contain insoluble precipitates. It could be as far east as that. In this way they are mixed up with beads containing the desired cells.

In order to prevent this, two methods are generally adopted. In one method, various interactions are involved in including (or not including) a substance that is present (or is present) in the bead containing the desired cells, but not present (or present) in the bead containing the undesired cells. It is made to occur an extremely large number of times so that the difference in severity is much larger than the difference in weight due to the difference in the growth rate of cancer cells. Alternatively, the KAN cells can be irradiated before mixing with other cells and housing them within the beads, allowing them to perform their function (providing antigens to capture antibodies produced by the desired cells). ) can also be made to die at some point after completing the process. Additionally or alternatively, the beads can be cooled to significantly slow cell metabolism and proliferation, thereby preventing changes in bead weight due to cell proliferation.

In all the methods described above, the desired cells themselves can be "protected" from the action of any agents that cause differences in the beads (color, weight, fluorescence, etc.). For example, individual cells of a cell population can first be housed within a beaded semipermeable membrane material. These beads are then housed within another semipermeable membrane material. Substances such as red blood cells and cancer cells are then housed within this [outer layer J]. As a semipermeable membrane material that houses the cells themselves, they allow products produced by the cells to pass through and interact with agents (e.g., antigen-bearing red blood cells or cancer cells); The bead is selected so that it does not allow the agent to pass into the internal bead.

In this way, the differentiation between beads containing desired cells and beads containing undesired cells takes place within the outer layer. Once the beads containing the desired cells are isolated, the outer layer can be removed to leave only the beads containing 9J of cells. The desired cells are then isolated using the various It can be recovered from the beads without interaction with the substances and without the presence of these substances.

Cells that separate beads based on differences in assembly ('/FH degree) - Another important factor for FM technology -
is the uniformity of weight and density of the beads before starting the operation. As will be explained in more detail later, the formation of hinizu from cell populations? The procedure is such that cells of li- and, if applicable, other M11 cells (erythrocytes, cancer cells) of 1 or 2 or more strains are systematically contained within individual beads. However, it has been found that some cells may contain more or fewer cells than others, and furthermore, these may differ in size, cell size, and density. If such variations exist when carrying out the weight- or density-dependent isolation methods described above, they may result in a gap between beads containing desired cells and beads containing undesired cells (e.g. due to lysis). or by precipitation formation);
The difference in quantity or density may be comparable to the difference in weight or density that the beads themselves have from the beginning. In this case, many beads will be mistakenly separated because they are initially heavier or lighter than other beads.

This problem can be solved by making the intentionally created bulk or density difference much larger than the inherent weight or density difference present in the initial beads. This can be done using the amplification techniques described above. However, a more preferred solution is to subject the formed beads to such means that beads of very similar weight and density come together before being used in the isolation procedure. For example, if all beads are 4+
In addition to a vertical column containing a liquid with a constant viscosity and density, beads of the same or similar weight and density can be collected at different depths in the liquid. can. Only beads that are present at the same depth are employed for use in the isolation method. Thereby, Φ#,
It is ensured that the differences in amount and density observed above are due to intentionally induced striations and not to variations that were originally present in the starting beads.

1- As mentioned, many methods can be utilized to form beads containing individual cells in a population of cells and other materials (eg, antigen-bearing red blood cells, cancer cells, carriers, etc.). Generally, in these methods, cell populations (and other materials) are suspended in a suitable gel-forming material such as alkyne a JlX or agarose. Stir the suspended liquid continuously to ensure uniformity, take a small amount of the suspended p7:J liquid using a T1-shaped device, for example, and add it to the medium for forming the droplets into beads. I'll put it on. For example, if alginic acid fj,4 is the gel-forming material, the drop can be placed on a calcium ion-containing solution (eg, calcium chloride) to gel the alkinate. If the gel-forming material is agarose, the drops can be placed in an oil solution kept at a sufficiently low temperature to cause the agarose to gel. A mixture of alginate and acarose can also be employed as the gel-forming material, in which case the drops are
It is placed on a calcium-containing solution kept at a low temperature.

Cells (and other materials) and gel-forming material are fed through different feed ports, and the drops of mixed cells and medium are then added to a gelling solution (e.g. calcium-containing 1
It is also possible to use a drop forming and placing device that places the drops in a solution (or cold oil). Beads are also formed by emulsifying the cells and a gel-forming material to form small discrete droplets containing the cells and gel medium and exposing the droplets to an environment that causes them to gel into discrete spherical beads. You can also do that.

Another method of forming beads containing individual cells in a cell population uses a syringe with holes to contain a suspension of cells and gel-forming material. The cylinder is placed in a gelling medium and rotated to shake out small discrete droplets of gel-forming material and cells from the holes. These drops immediately come into contact with a medium that causes the gel-forming material to gel (eg cold oil, calcium-containing solution) and beads are formed.

In individual bead formation mechanisms, the concentration of cells, the concentration of gel-forming material, and the size of the droplets formed are such that each droplet (from which all beads are ultimately created) is one of the cells in the population. It is set to contain a certain number of cells and, if appropriate, other things such as antigen-bearing red blood cells, cell caps.

1- The gel-like beads or microparticles formed by any of the methods mentioned above can be used as beads per se in the various cell isolation methods mentioned above. However, -+a, beads containing the components present in the gel-like particles can be formed by surrounding each gel-like particle with a semipermeable material and then dissolving the gel-like material of the particle. is preferred. For example, alginate microparticles are surrounded by a layer of polylysine (by electrostatic attraction) and the beads thus formed are placed in a medium that removes calcium from the microparticles (e.g. a calcium sequestering agent-containing solution or a calcium-free solution). It can be applied to This liquefies the microparticles, thereby resulting in beads composed of polylysine containing the cells and other components that were present in the initially formed microparticles.

A similar method can also be used when the gel-like microparticles consist of agarose or an agarose-alginate mixture. In this case, the agarose microparticles are surrounded by a semi-permeable membrane material such as polylysine, and then the agarose gel is dissolved into polylysino beads, eg, added to a hot sleeve or other liquid. (Polylysine has little or no static attraction to acarose, so
In forming the agarose microparticles, it is desirable to use sufficient small scales of the alkinate to electrostatically attract the polylysine and surround the microparticles. In this case, the alginate moieties in the resulting beads can be dissolved as described above.

How to adopt a second piece layer, i.e. an external bead layer
In No. 1, i.e., various bead sorting agents (and their reactions) are confined within an outer layer separated from the cell-containing beads, the cell-containing beads are separated from, for example, alginate, agarose, polylysine, etc. Various bead forming techniques can be used to surround the external bead material.

The "isolation" method of this publication has a wide range of applicability in finding and isolating any desired cells from a cell population in which desired cells and so-called undesired cells coexist. To reiterate the points that have been made in considerable detail in the first part of this specification, the h.
Horn: is useful, for example, when it is desired to find all cells in a cell population that produce a specific product (eg, insulin or a specific antibody). In this case, undesired cells are cells that are not producing the desired product. At the same time, however, this coarse separation of desired cells from undesired cells results in the production of the desired product in the greatest quantity (i.e., the fastest production of the desired product). ' cells can be located and isolated. It is highly desirable to locate and 'I'# these high producing cells.

This is because such cells, when cultured, produce the highest amount of the desired product per unit of cost, thereby facilitating the production of products such as antibodies and insulin. This is because production) is significantly optimized.

To further illustrate, the method shown in FIGS. 11A-1D for isolating cells producing antibodies to a particular antigen, for example, involves determining the weight and density of beads containing such desired cells. It is based on increasing the number of beads selectively over other beads in the mix. This weight gain is achieved by enzymatically converting added soluble compounds into insoluble precipitates in the beads containing the desired cells. This conversion takes place only in the beads containing the desired cells since the necessary enzymes are only present in the beads containing the desired cells. The enzyme is only present in these beads because the I2 form of the enzyme, eg anti-immunoglobulin, is immobilized only within the beads containing the desired cells.

This is because only within such beads are antigen-antibody complexes to which anti-immunoglobulin can bind.

The reason that antigen-antibody complexes are present only in beads containing the desired cells is that antibodies specific for the antigen (carried in nuclear cells) are produced only in such beads. It is from. It is therefore clear that the absolute amount of enzyme present in a bead containing the desired cells is directly proportional to the amount of antibody produced by the cells (the enzyme cell is immobilized within the bead). is proportional to the amount of anti-immunoglobulin, the latter is proportional to the number of antigen-antibody complexes in the beads,
Furthermore, it is proportional to the absolute amount of antibody produced by the cells in the beads).

Therefore, in this method, all beads containing the desired cells (i.e. cells producing the specific antibody sought) will be heavier than all other beads in the population, and therefore they will be affected by gravity. They can be separated by virtue of their ability to sink deeper than all other beads in a suitable liquid medium. Among these heavy beads containing the desired cells, those that sink faster or deeper than other beads containing the desired cells are the desired beads. Because more insoluble precipitate is formed therein. Since the ψ of the precipitate produced is directly proportional to the amount of antibody produced by the cells in the beads,
These beads necessarily contain the desired cells that are producing the largest amount of antibody at the fastest rate. Thus, from among the first isolated cells, those cells that are fastest producing the most destructive antibodies of all desired cells based on their sedimentation rate and/or sedimentation depth in the medium. The value of r can be further optimized by simply adding beads containing . By culturing these cells and repeating the same operations as described above, it is possible to further isolate the most highly productive cells from among the highly productive cells. This culture-separation procedure can be repeated as many times as desired to find cells that produce the highest number of products per intermediate time. These cells are then inoculated with the desired antibody. Used as a source for production and recovery (i.e., in a suitable medium group).Since cells with a high antibody Jr. The culture medium, culture room, etc.) have been optimized.

According to the isolation method of the present invention, the cells producing the most desired product can therefore be separated from the cell population per medium time. Why do certain cells produce more of Mars' desired raw Jr product than others? It doesn't matter as long as n# is done. specific m
l! The reasons why the production of desired biological products is increased due to +incubation include, for example, the inherent genetic constitution of the cells;
The genetic mechanism of cells is artificially altered: #I
Cells are known for spontaneous gene amplification, which means that when a cell divides, a K4 cell has twice as many genes (coding for the production of a particular product) as the parent cell's genes. I can list things I have experienced.

The above discussion also applies to isolation methods in which desired cells are distinguished from undesired cells, such as by the beads containing the desired cells being lighter than others or by being rescued from a toxic environment. The same applies. For example, the isolation method described earlier may be effective against lethal methotrex
・Includes selective protection of only desired cells from the environment. In this method, individual cells in a cell population are
Encapsulated in beads with nucleated cells having anti-products (eg, antigens) specific for the desired products (eg, antibodies) produced by the desired cells.

The heath is suspended in a vehicle containing methotrex-1. and dihydrofolic acid. Methotrexe i. binds irreversibly to cellular folate reductase, preventing the conversion of dihydrofolate to the vital folinin enzyme. Next, 1.
Anti-products (e.g. anti-immunoglobulins) combined with specific immunoglobulins (e.g. derived from mammalian cells), which are effective in stimulating the system, act on the system.
II can be obtained. Anti-immunoglobulins (and enzymes) are immobilized only within beads on which antigen-antibody reactions have occurred, ie, beads containing cells producing the desired antibodies. The presence of enzymes in these beads converts dihydrofolate or folinic acid, allowing these cells to survive in the mel-)lexade environment despite their own folate reductase being inactivated. In this way, the only beads containing live cells will be those containing the desired cells, and these can be separated from other beads.

This invention allows for a large number of extraordinary and simple methods to be used when separating "desired" cells from "undesired" cells. 1t method can be performed. For example, if the desired product produced by a desired cell is necessary for the maintenance of life in other cells or organisms, what is necessary is to It is only necessary to house the desired product together with the required cell or cells.

If a desired product is produced by a cell within a bead, the cells that need that product to live will
can grow within this bead.

With all other beads, cells that require the product to live die. Thus, a bead containing the desired cells that produce the desired product becomes heavier and denser than all other beads over time due to proliferation of the cells within it. One example of this method is to find desired cells that are producing T cell growth factors necessary for T cell survival. Beads are the desired T
T,III follicles can grow within them if they produce cell growth factors.

A similar example may include the isolation of an organism producing a desired biological product, such as an antibiotic. Individual organisms in the population are housed in beads with organisms whose growth is inhibited by the antibiotic ("reactive organisms").

If the organisms from the population are those that produce antibiotics, the growth of the reactive organisms within the beads is inhibited and -force,
On other beads, the reacting organisms grow. Therefore, beads containing the desired organism, ie, the organism producing the desired antibiotic, will be lighter than all other beads.

Another practical embodiment of the method of this invention is the isolation of cells that do not produce a particular product. for example,
In procedures for producing fused cells that produce monoclonal antibodies by cell fusion, the immortal parent cells (eg, myeloma cells) of the fused cells must not themselves produce antibodies. This is because, otherwise, the production of these antibodies would be inherited by the fused cells, and therefore,
The fusion side 1 antibody is not a monoclonal antibody, but an anti-(14J
This is because a Jij compound is produced. According to the present invention, for example, a procedure for easily finding myeloma cells (for use in cell fusion) that do not produce antibodies by themselves is provided. Individual myeloma cells in a myeloma cell population are housed within beads with appropriately immobilized anti-immunoglobulins or staphylococci (antigens specific for the particular antibodies produced by the myeloma are not required since (This is because we are not trying to detect myeloma producing a particular antibody, but rather all antibody production by myeloma cells.) From the bead population, all beads containing any antibody-producing cells are then separated by any of the various lysis or enzyme-dependent methods described above. In another practical application of the isolation method of the present invention, the desired myeloma cells, that is, myeloma cells that do not produce antibodies by themselves, are contained in the beads in which no reaction occurred.
? 'ft K cells may be monitored to see if they are producing products (either at all or to a certain extent). In an operation in which cells producing a specific product (e.g. antibody) are cultured in an appropriate medium and the antibodies produced by the cells are collected continuously or periodically, the cultured cells are The ability to produce a product of many hours is excellent in economy (i.e., the cost of culture operations, equipment, etc. required to produce antibodies of about 1 hour).Culture operation for producing large quantities of antibodies, etc. in 1 hour However, if the cells being cultured produce only a small amount of antibodies or no longer produce antibodies, the economy becomes less economical. Even if the method is economically efficient, it is necessary to periodically test whether the cells continue to produce a large amount of antibodies (or whether they have completely stopped producing antibodies). Preferably, in this way, undesired low-producing cells (specific producing cells) can be removed from the culture system, saving on medium and product recovery.

According to the invention - the above-mentioned test is carried out periodically (
or periodically), and the cells thus taken are supported - on other materials necessary for any of the above-mentioned isolation techniques, e.g. on a suitable carrier (e.g. red blood cells, nucleated cells), etc. The bead contains antigens or anti-products, etc., which may be used or form part of the bead material itself. Generally, the cells that are cultured are fused cells that produce clonal products (antibodies). Thus, the anti-product need not be specific for the product produced by the cell, but may simply be an anti-immunoglobulin or staphylococcal. This is because the fused cells produce 1
This is because it is a type of product.

The beads are then subjected to any of the isolation methods described above in order to isolate beads containing cells that are producing the most desired product per unit time. After discarding all other beads and lysing the bead material, these desired high-producing cells are returned to the culture system for continued production and recovery of product.

This IJJ mid-life process can be carried out in batch, continuous or semi-continuous mode for all or individual steps during -. From an economic point of view, of course, continuous type 1 is preferable. A sequential means of separating desired cells from unwanted cells by an isolation method based on selective killing of unwanted cells is illustrated in FIG.

In Figure 13, the size is greatly exaggerated.
Hollow fibers 14 made of plastic material are reduced in size. The hollow fiber 14 has a circle f;'
The rim-shaped housing 18 is divided into four regions (L II, III, and IV) at its outer periphery. a population of cells (some of which are producing live Ir products), an oil/water medium, and an anti-product against the desired product (e.g., if the desired product is an antibody). , whereas the antigen of $-￥ +p) is
A series of tJ
12. An oil-in-ice droplet 16 containing cells from the cell population and an antigen specific for the desired antibody is released from the needle 12 into a hollow fiber, t14.

Within the hollow fiber 14, a drop containing cells is subjected to -a fixed amount of methotrexate, thereby inactivating all folate reductase in the cells themselves and inhibiting the cells from converting dihydrofolate to folinic acid. inhibit. Unless the cells are rescued from this condition, the lack of folinic acid will cause them to die by the time they reach the end of the hollow fiber 14.

Cell rescue is limited to the desired cells as follows.

f of the hollow fiber 14: In the fS1 region (I), cell-containing J
The drops are contacted with a complex of anti-immunoglobulin and bacterial folate reductase, which is applied through the permeable fiber surface. The anti-immunoglobulin-enzyme complex remains only in the droplets where the antigen has reacted with the specific (desired) antibody, ie, in the bay containing the desired cells. Excess unbound anti-immunoglobulin-enzyme complex is present in the hollow fibers 14 in areas 1 and 11, in which the drops are appropriately washed.

In the next region 11I along the hollow fiber 14.

Dihydrofolic acid is added through the permeable fiber surface.

Dihydrofolate is used only in drops where folate reductase resistant to methotrexate is present, i.e.
It is only converted to folinic acid in the bay containing the cells producing it. As a result, these desired microbubbles live, but all other cells lacking holinin resistance die.

In the next region (IV) of the hollow fiber M#14, substances necessary for producing living ft111 cells, such as Jr'', earth, oxygen-containing waste, etc., can be added.

All cell-containing droplets are then passed through tube 22 (which is a wide extension of the hollow HhMll 4) to separate droplets containing live (desired) cells from those containing dead cells. reach the area of The latter is discarded via 'Lj'0 and the former continues through tube 22 to the means of breaking up the drop and releasing the living cells themselves.

If it is desired to isolate the most prolific antibody-producing cells, the separated live cells can be passed through the hollow fiber 14 to form three droplets (containing the antigen). The fibers are sent again to the container 10 where they are stirred to remove the fibers, and the hollow fibers 14 are passed through once again. However, this time,
Increase the dose of methotrexate. By repeating this cycle with increasing amounts of methotrexate, the end result will be the cells that produce the most amount of the desired antibody per unit time.

With the forces i', I; and the device shown in FIG. 13, the hollow fiber 14 has a permeability of -1
It can be made very different. By doing so, substances can be added freely in some areas, while substances in the fibers (eg folic acid produced in area III) can be prevented from escaping in other areas.

In this invention, a desired cell (a cell that produces and releases a certain product or a cell that has a specific product as part of its surface) is placed only in the vicinity of the cell for which the product is desired. cells are separated from unwanted cells based on their ability to create an environment that can be used to separate cells from unwanted cells. 1- As is clear from the above description, a number of such methods can be used to achieve this result. The methods described, and the materials used in these methods, are illustrative rather than limiting, and in fact, various modifications may be made without departing from the scope, spirit and essential characteristics of this invention. It would be obvious to Nagisa skilled in the art that the modification etc. ? In many of the methods described here by way of example, the essential step is to capture the desired product (produced by the desired cell) with a substance (anti-product) that exhibits specificity for it; It is immobile. This anti-product may be antigenic if the desired product is an antibody, or it may be an antibody if the desired 11-product is antigenic. Furthermore, the anti-product may be one that exhibits electrostatic affinity for the product or may be a synthetic chemical that exhibits specific binding affinity for the product.

It is also possible to permanently alter the desired cell itself so that it produces the desired product modified in a form that facilitates the capture of the desired product by the anti-product. be. For example, if it is desired to isolate insulin-producing cells from a cell population, the cell population may be subjected to one of the two isolation methods using an antibody against insulin as the capture anti-product. can be applied to find desired cells producing insulin. When it is desired to isolate only cells that produce extremely large amounts of insulin, this isolation method can be repeated on insulin-producing cells, as described above. When such isolation operations are performed repeatedly, insulin can be more easily captured by anti-products by linking, for example, a gene encoding +f polylysine to the insulin-encoding gene of these cells. The insulin that is subsequently produced within the cell has polylysine and can therefore be captured by substances that exhibit a static attraction to the highly positively charged polylysine moieties of insulin. . This and a force like 17+1 can be used to find cells that have undergone gene amplification. This is because when the Insu 1 non-pickled Buddha child is amplified, the PO 1 no 1 lysine gene that has been converted in succession is also amplified, and the Insu 1 non-t that has been converted by such M111 blood, this number. They are careful to add polylysine to the 1-

[Brief explanation of the drawing]

1A and 18, 2A and 2B, 3A to 30, 4A to 40, and 5A to 5B,
Diagrams each schematically illustrating methods for isolating desired cells producing and releasing specific products;
Figures 6A and 6B are diagrams showing the gel (based on jδ) for immobilizing cells and products; Figure 7 is the interior of a centrifuge for immobilizing cells and products. Figure 8 is a side view of the stippled surface for immobilizing cells and products; Figures 9A and 9B are
10A to 10C, 118 to 11D, and 12A to 12E are
A diagram schematically showing a method for producing a desired product and isolating cells; FIG. 13 is an apparatus for isolating cells producing a desired product. FIG. D...desired cells, U...cells of Chasho l-1l),
C...Carrier, T...Cancer cell, E...Enzyme, RB
C... Red blood cell applicant's agent Suzu 7 [i″r + invitation of Takehiko drawing (no change in content) JPO Chief Minoru Kazuo Wakasugi M An 29 Name of invention Method for isolating cells 3, Submit supplementary IE Relation to the case of ``Bio-Response Incorporated'' February 28, 1980 6 and City JF, subject 7 Contents of the amendment Engraving of the drawing as shown in the attached sheet (no change in content)

Claims (1)

Claims: (1) A method for isolating desired living cells secreting cellular products with specific binding affinity from a mixed cell population, comprising the steps of: (b) obtaining a cell cluster containing desired and undesired cells; (b) subjecting said cell population to a predetermined environment for a sufficient period of time for it to produce and release said product; keep within. The environment separates the cells such that the product accumulates and is concentrated only in the immediate vicinity of the cell from which it is released, and the product is concentrated in the immediate vicinity of the cell from which it is released. (c) Before or after maintaining the cell population in the environment, the cell population is prevented from migrating from the vicinity where the products are produced and released. Afterwards, the product is combined with a first substance that specifically binds to the product, and the first substance and the product are combined to form a complex only in the vicinity of desired cells, and the complex is formed. to form a distinguishable environment in the vicinity of desired cells; and (d) interact with said distinguishable environment to cause physical, chemical, or at least one second effecting a survival change;
The desired cells are selected by applying the substance to the cell population. (2) The complex or combination formed by the first substance is
a barrier environment is formed in the vicinity of the desired cells, and the second substance responds to the barrier environment to cause a different effect on all desired cells than on all undesired cells. 2. The method according to claim 1, wherein a phase IL effect is caused and ITf is enabled to select desired cells by a phase W effect of the second substance. (3) The substance f'+rr'52 is bound to the substance i3, which is a drug that can kill living cells when it enters them, and the complex is placed in the vicinity of the desired cells. , forming a barrier environment that prevents the second substance from contacting and killing desired cells, thereby selecting desired cells and killing undesired cells. . (4) The first substance is treated so that it dies after carrying out normal metabolism, and specifically binds to the product to form the complex and forms a circular part. A scavenging cells with an enhanced ability to take in molecules from the outside world when combined as a complex: the predetermined environment shunts the positions of the cells of the cell population and the scavenging cells such that the scavenging cells surround the individual cells of the cell population, and the predetermined environment Preventing the movement of the cellular products from the cells from which they were secreted, so that the scavenging cells encounter the cellular products only in the vicinity of the desired cells. :
The 1Afi follicular product is produced and secreted by the desired cells and left for a sufficient period of time for it to bind to the scavenging cells in the vicinity of the desired cells; and the second substance is produced and secreted by the desired cells; is lethal to the population and the scavenging cells, and the extent to which the complex of the product and the scavenging cells prevents the scavenging cells in the vicinity of the desired cells from contacting the desired cells and the second substance; second to
Selecting the desired cells by incorporating a substance therein, thereby preventing the killing of the desired cells; killing the unwanted cells by a lethal environment; killing the scavenging cells by pretreatment thereof;・Method of precepts. (5) the first substance is supported on carrier particles, and the carrier particles are brought together with a population of microbubbles before being placed in the predetermined environment; An enzyme is bound to a substance having a foot temperature of 2, which is surrounded by 1jθ carrier particles in a predetermined environment; or the second substance is capable of specifically binding the free first substance to be bound and directs said enzyme preferentially to the vicinity of the undesired cells;
removing from the mixed cell population all unbound second substances that are bound to the enzyme and bound to the cellular product or to the complex of carrier particles and cellular product; When the second substance combines with the first substance of 'M',
lethal, colored, colorless, that selectively acts on unwanted cells;
A third substance, which is converted by the enzyme into a fluorescent or insoluble compound, is added to a cell population containing the second substance, selecting the desired cells, and the second cell binds the product to the complex. In some cases, a lethal third substance, which is detoxified by an enzyme, is added to the cell population containing the second substance, killing the unwanted cells and killing the desired cells with the lethal third substance. 2. The method of claim 1, wherein desired cells are selected by rescuing them from a cell. (6) the first substance is supported on carrier particles, the carrier particles are combined with a cell population before being placed in the predetermined environment, and the carrier particles are loaded with enzymes; wherein the cell population is surrounded by the carrier particles in the predetermined environment, and only carrier particles present in the vicinity of the desired cells encounter cellular products. selectively bind a second substance only to the complex of the carrier particles and the product, dissolve the carrier particles in the complex, and release the enzyme from it; release the second substance; lethal, soluble,
or add a heavy third substance, which is detoxified by enzymes if it is lethal;
If it is soluble, it is converted by enzymes into an insoluble precipitate; if it is heavy, it is converted by enzymes into a lighter compound; If the third substance is +Tl m by being rescued from a lethal environment
2. The method of claim 1, wherein the third substance is selected by precipitation if it is of a different nature or by flotation if it is different. (7) said first substance is supported on carrier particles, said carrier particles are brought together with a cell population before being placed in said predetermined environment, and said carrier particles are loaded with a rescue substance; 1) The cell population described above is surrounded by the carrier particles in the pre-filled environment; only the carrier particles present in the immediate vicinity of the desired cells are Encountering and binding to the cellular product: selectively binding the second substance only to the carrier particle-product complex;
The carrier particles in the complex are dissolved, and the rescue substance is then released only in the immediate vicinity of the desired cells, forming a barrier around the desired cells; a lethal third substance; 2. The method of claim 1, wherein the released rescue substance is added to the cell population, thereby saving desired cells by the rescue substance barrier and killing unwanted cells by a third substance. (8) The defined environment is a semi-permeable I-membrane which retains the cells in the cell population inside but allows the first and second substances to diffuse through its walls. They are hollow beads; the second substance has an enzyme and binds only to the complex or only to the first substance of JIk; the unbound second substance is transferred to cells. a second substance bound to the complex and interacting with the bead material of the bead containing the desired cells; alternatively, the second substance bound only to the first substance; Pairing the beads containing the undesired cells with the bead material: adding a third substance, which is a piece material liquefying substance, to form a discernible environment by liquefying the bead material or maintaining its gel-like properties; 2. The method according to claim 1, wherein the desired cells are selected. (9) The first substance is supported by carrier particles, and the carrier particles are attached to the cell collection [
and the carrier particles are red blood cells or liposomes that surround the cells of said cell population within a predetermined environment: said predetermined environment. is a membrane-permeable bead that retains and retains the cells of the cell population; only carrier particles within the bead containing the desired cells are exposed to and bound to all the biological products; bead wall material; is permeable to the second substance: allows the second substance to bind only to the complex of carrier particles and cellular products, and dissolves only the carrier particles bound as a complex. or the second substance is a complement-fixed enzyme; the unbound second substance is removed from the beads; and if the second substance causes a lytic effect, the carrier particles - The lysate released from the bead is translucent 11. escape through the J wall, so that the beads containing the desired cells are on top of the other beads in the liquid: or if the second substance is a complement-fixed enzyme. , adding a third substance to the agent which dissolves the carrier particles and releases a lysate which escapes from this to the outside through the walls of the beads, thereby containing the desired cells in the liquid. 2. A method according to claim 1, wherein the beads are placed in a smaller area than other beads. (1(1) The cellular product is a first antibody, and the first substance contains an antigen specific for the antibody and a second substance that has affinity for the antigen. the first substance and the cell population are housed in a semi-permeable enveloping material in the predetermined environment, and the first substance and the cell population are contained in a semi-permeable enveloping material in the predetermined environment; The first secreted by the desired cells in
for any F48 antibody with a weaker affinity than this.
The substance with a foot temperature of 2 after being left for a sufficient period of time to replace the Fc region is a substance that binds to the Fc region of the replaced first antibody, and these capsules are placed in the capsule containing the first antibody. 2. A method according to claim 1, which creates an environment that is distinguishable from all other capsules. (11) The method according to any one of claims 1 to H'SlO'J-1, wherein the predetermined environment (iif) is a hollow gel bead or a hollow fiber.