WO1984000815A1

WO1984000815A1 - Cell differentiation based upon particle uptake

Info

Publication number: WO1984000815A1
Application number: PCT/US1983/001164
Authority: WO
Inventors: Elizabeth A Komives; William G Thilly
Original assignee: Massachusetts Inst Technology
Priority date: 1982-08-05
Filing date: 1983-08-01
Publication date: 1984-03-01
Also published as: IT8322467A0; IT8322467A1; IL69413A0; IT1164396B; CA1199598A; IL69413A; EP0126085A1

Abstract

Method of enumerating and isolating mutant cells and other variant cells in a cell population. A cell population is treated with a selective agent which allows variant cells to remain phagocytic while rendering non-variant cells non-viable or non-phagocytic. A suspension of discriminator particles, such as fluorescent or magnetic particles, is mixed and incubated with the cells. The variant cells take up the particles, while the non-variant cells do not take up the particles. The cell population is then analyzed to determine which cells have taken up discriminator particles.

Description

CELL DIFFERENTIATION BASED UPON PARTICLE UPTAKE

Description

Government Support

The invention described herein was supported in whole or in part by a grant from the U. S. Department of Energy.

Technical Field

This invention is in the fields of cell biology, chemistry, and mutation analysis.

Background Art

In numerous types of biological work, it is useful to analyze a population of cells to identify, enumerate, and possibly isolate variant cells. Two important uses for such procedures are mutation assays and carcinogenicity analyses, which determine whether various substances cause damage to or alteration of the genetic makeup of various types of cells. As used herein, the terms "variant cell" and "variant" are used interchangeably to refer to a cell which has one or more characteristics that differ from the char¬ acteristics of other cells within a cell population that includes the variant cell. For example, mutant cells within a population of non-mutant cells are variants. As another example, a phagocytic cell in a population of non-phagocytic cells is a variant. Simi¬ larly, a viable cell in a population of non-viable cells is a variant.

Numerous types of assays depend upon the ability of cells to form colonies as an indicator of viability or variant characteristics. For example, a culture of cells may be exposed to a mutagen, and then to a selec¬ tive agent. Only those cells which have mutated in a

OMP specific way (for example, by inactivation of a partic¬ ular gene) are capable of surviving in the selective agent. The exposed cells are then cultured and the number of colonies formed provides an indication of the number of cells which survived exposure to the selective agent. However, such assays suffer from several draw¬ backs. First, they require a relatively long period of time to perform, e.g., more than a week. Second, numerous types of cells, including many types of human cells, do not form colonies. This renders them unsuit¬ able for mutation assays which involve colony growth. Third, mutation rates tend to be relatively low. For example, uninduced mutation rates tend to range from

-8 -4 about 1 x 10 to about 1 x 10 , depending upon the type of cell and the type of mutation involved. There¬ fore, in order to allow for accurate statistical anal¬ yses, a very large number of cells must be placed in suitable growing conditions in order to accurately determine the number that are viable. Various other analytical techniques exist which do not require colony growth. Such techniques usually provide a rough approximation of the general condition of a cell culture. For example, a dye such as Trypan Blue or Eosin B may be added to a culture of cells. These dyes will permeate into cells which have damaged membranes, while being excluded from cells which have intact membranes. ,A cell culture is viewed under a microscope after it has been contacted with dye. If a large number of cells appear to be stained by the dye, this indicates that numerous cells within the culture are dead. However, such dyes may be excluded by the membrane of a cell which is dead yet still intact. Even if dyes were sufficiently specific, it would be very difficult to locate and isolate a small' number of unstained mutants in a large population of stained cells. Therefore, staining techniques are not highly accurate, and they cannot be used to enumerate or isolate rare variants within a cell culture.

Flow Cytometry Flow cytometry is an established procedure which is known to those skilled in the art. The following is a simplified description of how a flow cytometer operates. A suspension of cells is placed in a vial. By means of a vacuum, cells and fluid are drawn from the vial into a plastic tube, and are passed through a "flow cell" or "cuvette". The cuvette has an inside diameter which is sufficiently small (about 40 u ) so that only one cell at a time passes through it. A laser beam with a known wavelength shines upon all fluid and cells that pass through the cuvette. If a cell or a particle is fluorescent it will emit light with a wavelength that differs from the wavelength of the laser beam. A photodetector is directed toward the cuvette, usually at right angles to the laser beam. This photodetector typically has a controllable band width, so that only light which has a wavelength within a predetermined range will be detected. The photo¬ detector is exci.ted by the fluorescent wavelength, but not by the wavelength of the laser beam. When the photodetector detects emitted light with a fluorescent wavelength, it creates an electric impulse. This may be amplified or otherwise processed by electronic circuitry within the flow cytometer. For example, the number of impulses may be counted to provide an indication of the number of fluorescent cells that pass through the cuvette. In addition, the intensity of the fluorescent signal from each cell may be determined, allowing various types of histograms to be plotted. Several flow cytometers are commercially avail- able, e.g. , the Cytofluorograf system FC 4800A (Ortho Instruments, Westwood, MA) , which can be equipped with an oscilloscope or other analyzer, e.g., the Tektronix 2102 Distribution Analyzer (Ortho Instr.).

In a slightly more complex and sophisticated arrangement, a flow cytometer may be coupled with a cell sorting device, which may be briefly described as follows. A suspension of cells flows through a cuvette and then through a set of electrical charging plates. The cells move at a known, controlled speed so that a known amount of time elapses while each cell is travelling from the cuvette to the charging plates. Whenever a fluorescent cell is detected by the photo¬ detector, an electronic charge is imposed on the charging plates as the fluorescent cell passes through the plates. This imparts a charge to each fluorescent cell. All cells which pass through the cuvette and the charging plates subsequently pass through an electromagnetic field. Non-fluorescent cells (which have not been electrically charged by the plates) travel in a rela- tively straight line and are collected in a first receptacle. Fluorescent cells (which have received an electric charge from the charging plates) are deflected by the electromagnetic field into a different flow path, and are collected in a separate receptacle. Several instruments are commercially available which perform this function, such as the Cytofluorograf system 50H (Ortho Instr.).

There have been numerous uses of fluorescent cell sorting. Such uses can be divided into two general categories, depending upon whether the fluorescent substances are located outside or inside the cell.

The first category usually involves contacting a culture of cells with fluorescent antibodies, particles, or other substances which bind or cling to the surface of one or more types of cells within a cell culture. See, e.g., U. S. Patent 3,853,987 (Dreyer, 1974);

OMPI A«. Rembaum et al, Science 208: 364 (1980) . Such methods can analyze the presence of antigens or other substances on the surfaces of cells. However, such methods suffer from at least three limitations which impede their use in analyzing rare variant cells; (1) They can be used to analyze variations in the surface characteristics of cells. However, numerous types of mutations that are of interest do not cause changes on the surfaces of cells, and therefore are not detectable by such procedures. (2) The surface molecules which differentiate such cells tend to remain on the cells for substantial periods of time after the cells have died. Therefore, such techniques cannot be used reliably to distinguish or isolate viable cells from non-viable cells. (3) Such techniques have not been sufficiently refined to provide deter¬ minations that are sufficiently sensitive for accurate mutation analysis. For example, in one published experiment, fluorescent cell sorting was used to concentrate the variant population by a factor of

3000 times; see J. M. Jovin et al, "Cell Separation," TIBS p. 214 at 217 (August, 1980) . However, the con¬ centrated culture required subsequent analysis by human observation through a light microscope to deter-, mine the variant fraction.

The second category of fluorescent cell sorting involves fluorescent substances that are ingested by or permeate into cells. Such techniques often utilize substances which are referred to as "fluorogenic. " Such substances are not fluorescent, but they can be metabolized into fluorescent metabolites. For example, a substance commonly used for this purpose comprises fluorescein diacetate, which is a fluorescein molecule which has been modified by the addition of two acetate moieties.

OMPI_ ~ Fluorescein diacetate is not fluorescent. However, after it enters a cell, the acetate moieties may be removed by enzymes referred to as intracellular esterases, which exist in most types of cells. The resulting fluorescein molecule is fluorescent. Since intracellular esterases are active in virtually all living cells, this procedure provides a useful ap¬ proximation of the number of viable cells within a cell culture. However, the cleavage of fluorescein diacetate can occur whenever fluorescein diacetate contacts a functional esterase, regardless of whether the cell which produced the esterase is viable or non-viable. Esterases often remain functional for a substantial period of time after a cell has died. If fluorescein diacetate permeates into a dead cell and then contacts an esterase, it will be cleaved and become fluorescent; if it remains in the cell, it may cause a false-positive reading. This interferes with the accuracy and interpretation of such assays, and renders them insufficiently accurate for most analyses of rare variants.

Particle Uptake

Various studies have been performed which involve the uptake of particles by cells. For example, F. Schroeder, Biochimica et Biophysica Acta 649: 162

(1981) utilized nonfluorescent polystyrene microspheres to analyze the effects of choline analogs on phago¬ cytosis. This article indicates that the following parameters are important: 1. the ratio of microspheres to cells. Schroeder utilized 800 to 2000 microspheres per cell during the incubation. 2. the size of the microspheres. Schroeder utilized microspheres with an average diameter of 0.76 μ , and observed very little uptake of 0.497 urn beads.

•?^*. 3. the duration of incubation, Schroeder incubated fibroblast cells with microspheres for 5 to 10 minutes.

A general review of particle uptake is contained in R. I. Kavet et al, J. of Reticuloendothelial Society 27: 201 (1980).

A team of researchers has recently announced the use of flow cytometry to analyze phagocytosis. See J. A. Steinka p et al, "Phagocytosis: Flow Cytometric Quantitation with Fluorescent Microspheres," Science 215: 64-66 (1 January 1982) . In this work, rats were anes¬ thetized, and a saline solution containing fluorescent latex spheres, 1.83 urn in diameter, was instilled into their lungs. The rats were sacrificed and the lung cells were analyzed to determine whether the spheres had been taken into the cells by phagocytosis. The authors of that paper demonstrated that sodium azide inhibits phagocytosis. They also determined the per¬ centage of phagocytic cells, which is important.in assessing the ability of an animal to resist airborne disease and particulate pollutants. However, this method of analysis is not sufficiently sensitive and accurate to utilize in mutation analysis. For example, Steinkamp et al indicated that comparison of flow cyto- metric analyses with fluorescent microscope analyses revealed discrepancies of several percentage points. Such degrees of accuracy were confirmed by the Applicants in independent research. This level of accuracy was sufficient to accomplish the objectives of Steinkamp et al. However, discrepancies of a single percentage point would render analyses of rare variants grossly inaccurate. For example, one percent is 10,000 times higher than a mutation rate of 1 x 10

Magnetic Detection and Isolation Techniques Several researchers have developed methods for cell sorting that utilize magnetic substances. See,

*- UEAcT

CMPI e.g., T. M. Jovin et al, cited above, at 218; S. Margel et al, Journal of I munological Methods 28: p. 341 (1979); R. S. Molday et al, Nature 268: 437 (1977). Such work typically involves a solution of small, mag- netic particles which are affixed to ligands. The magnetic ligands are mixed and incubated with a suspen¬ sion of cells. The ligands form complexes with certain types of molecules on the surface of certain types of cells. After the reaction has been allowed to proceed for a substantial period of time, the cell suspension is then passed through a magnetic field. Those cells which have become affixed to magnetic particles by means of the ligands are attracted to the poles of the magnets. The remaining cells are washed away. The magnetic particles are then released from the magnet by eliminating the magnetic field. This allows for the cells to be removed and collected.

Such techniques can be used only to analyze variations in the surface characteristics of cells. However, as mentioned above, numerous types of mutations that are of interest do not cause changes on the surfaces of cells. Such mutations cannot be detected by the magnetic techniques described above.

In addition, such techniques are not sufficiently sensitive or accurate for mutation analysis. Although P. L. Kronick et al, Science 200: 1074-1076 (1978) reported accuracies of better than 98% and 99% for negative and positive fractions, these do not approach the sensitivity levels that are required for analysis of rare variants.

Disclosure of the Invention

This invention comprises a method of enumerating and isolating mutant cells and other variant cells in a cell population. This method comprises the follow- ing steps. A cell population is treated in a manner such that only variant cells are capable of taking up discriminator particles. For example, this may be accomplished by treating the cells with a selective agent which allows variant cells to remain viable while rendering non- variant cells non-viable. A suspension of particles, such as microscopic spheres, is mixed with the cells. The particles comprise one or more substances which can be readily taken up by the variant cells, and which contain one or more compounds which exhibit, or which can be metabolized into compounds which exhibit, some type of discriminator signal such as fluorescence or mag¬ netism. The cells are incubated with the suspension of particles for a sufficient period of time to allow viable cells to take up particles. The cell population is then processed by using a device, such as a flow cytometer or a magnet, which can be activated by the discriminator signal being used. The cells which have taken up the discriminator particles may be identified, enumerated, isolated if desired, or otherwise analyzed. This invention requires careful control of several parameters and steps, including the size and the composition of the particles, the methods and substances used to render non-variant cells incapable of taking up particles, the methods and substances used to allow or induce variant cells to take up particles, and the methods used to analyze the cells after particle uptake.

Brief Description of the Drawings Figure 1 is a flow chart indicating a sequence of processing steps.

Figure 2 indicates the effect of duration of 6-TG contact on particle uptake.

Figure 3 is a histogram indicating the number of ingested particles per cell.

?I Figu e 4 indicates the kinetics of particle uptake. Figure 5 indicates the effects of particle/cell ratio on particle uptake.

Figure 6 compares the results of particle uptake assays with conventional plating assays.

Figure 7 indicates the results of particle uptake assays of cultures with known variant fractions.

Best Mode of Carrying Out the Invention

In one preferred embodiment of this invention, microspheres comprising fluorescent carboxylated polystyrene (FCP) were utilized. These microspheres were purchased from a commercial supplier. A sub¬ stantial amount of variability was encountered, which required preliminary testing to determine which lots were suitable for use for the purposes of this inven¬ tion. Three important parameters were:

1. Size. Low degrees of phagocytosis were observed utilizing spheres that were smaller than 0.5 um or larger than 1.8 μm in diameter. Optimal size is believed to be in a range of about 1.0 to about 1.5 um.

2. The tendency of the beads to aggregate. Aggregation of beads tended to reduce the rate of phagocytosis by cells. 3- Leaching of the dye. Fluorescent dye leached out of at least one lot of microspheres and caused non-viable cells to become fluorescent. The cells used in the work described herein were human diploid lymphoblast cells, designated the TK6 _cell line, grown in suspension culture. This invention may be utilized with any type of cell which takes up particles; however, if the particle uptake of a cer¬ tain type of cell is low, the accuracy of the analysis may be impeded. The selective agent utilized in mutation experiments was 6-thioguanine (6-TG) . 6-TG kills cells which con¬ tain functional hypoxanthine - guanine - phosphoribosyl- transferase (HGPRT) , an enzyme which creates purines by a salvage pathway. If 6-TG is present in the culture medium, cells with functional HGPRT genes (referred to as HGPRT cells) will be killed, while mutant cells with non-functional HGPRT genes (referred to as HGPRT- cells) will survive. In order to reduce the number of spontaneous

HGPRT mutants in a culture, the cells may be contacted with CHAT, which contains cytidine, hypoxanthine, aminopterine, and thymidine. CHAT kills HGPRT- cells. This reduces the number of spontaneous mutants in a culture, thereby increasing the sensitivity of a muta¬ tion assay.

Microspheres and cells were prepared and incubated in the manner summarized in the flow chart in Figure 1. In the first step, microspheres were suspended in culture medium RPMI-1640. This medium should not contain horse serum, which tends to cause aggregation and adherence of the microspheres. A sufficient number of microspheres were added to the medium to provide for a final con¬ centration of about 50 to about 200 microspheres per cell. The rate of phagocytosis can be increased by increasing the number of microspheres per cell; one published article (F. Schroeder, 1981, cited above) indicates that particle uptake increases as the par¬ ticle/cell ratio increases to about 2000 particles/ cell. However, concentrations of greater than 200 microspheres per cell tended to cause severe problems of adherence of uningested microspheres to the cells. The suspension of microspheres was filtered through a filter with 3 u diameter pores to remove aggregates of microspheres.

TK6 cells were centrifuged to remove serum-containing medium and resuspended in the serum-free suspension of microspheres. The cell and microsphere suspension was placed in a spinner flask with a diameter of about 2.3 inches, and incubated at 37°C for 4 hours at a stirring speed of 60 to 120 rpm. Lower spinner rates were found to allow the cells to settle to the bottom of the flask, while higher rates were found to decrease the phagocytic efficiency of the cells. Optimal stirring rates for flasks of any size may be determined through routine experimention. After incubation, the suspension of cells and microspheres was centrifuged through a centrifuge gradient such as Percoll Gradient (Pharmacia Fine Chemicals, Piscataway, NJ) diluted 1:10 in phosphate buffered saline (PBS) . This allowed for about 90% of the uningested microspheres to be removed from these cells. It was extremely difficult to separate the remaining uningested microspheres from the cells. To overcome this problem, the following technique was developed. Polystyrene petri dishes were coated with a film of concanavalin-A (con-A) . TK6 cells have a sub¬ stantially greater affinity for this substance than do carboxylated polystyrene microspheres. Although con-A was a suitable adherent for TK6 cells, other compounds may be preferable for other types of cells. A suspension of cells and microspheres was placed on the con-A plate, and incubated. The cells formed a monolayer on these plates. Depending on the volume of suspension and the diameter of the plate, a sus- pension of cells takes about 1 hour to settle and adhere to a con-A coated plate. Uningested micro¬ spheres were subsequently washed off the plate by using PBS. After the PBS wash, if a substantial number of uningested microspheres remain attached to the plate, it is possible to incubate the cells with a solution containing a proteolytic enzyme such as trypsin for a brief period and rinse the plate with PBS to remove microspheres which were detached by the trypsin. Treatment with 0.005% trypsin for 5 minutes caused less than 5% of the cells on a plate to become detached.

Due to difficulties encountered in removing the cells from the con-A plate, it was decided to analyze the cells while still on the plate, uti- lizing a fluorescent light microscope. A layer of oil was placed over the cells to keep them from drying out during examination. Mutation rates were determined and compared with expected muta¬ tion rates. The results are described in Example 4 and displayed in Figure 7.

As used herein, the term "discriminator charac¬ teristic" includes any property or effect which can be utilized to determine the presence, absence, con¬ centration, or quantity of one or more particles. For example, fluorescence, magnetism, radioactivity, electron opacity, color, or phar alogical activity may be utilized as discriminator characteristics.

The term "particle" is used herein to mean any solid composition of matter which is too large to pass through an intact cell membrane by diffusion or permeation. The term "discriminator particle" refers to a particle with one or more discriminator characteristics. This invention may be used to isolate variant cells. As used herein, the term- "isolate" refers to the removal of the large majority of undesired cells from a population of desired cells. It is not essential to this invention that such isola- tion be complete; a population of isolated variant cells might contain one or more non-variant cells.

This invention also may be utilized to con¬ centrate a population of variant cells rendering the population more suitable for subsequent anal- ysis by other means such as a light microscope.

As used herein, the terms "particle uptake," "uptake," and "taking up" refer to any process by which a particle enters a cell. Such processes are often referred to by a variety of terms, including phagocytosis, endocytosis, and ingestion.

Several types ^'of analysis are available using the method of this invention. Variant cells may be enumerated by determining the number of cells which have taken up discriminator particles. A variant fraction may be determined by dividing the number of variant cells by the number of cells in the cell population being analyzed.

In order to increase the accuracy of such a determination, a variant fraction should be ad- justed by one or more of the following correction factors:

1. A doubling factor. This factor reflects the reproduction of variant cells during the procedure. This factor is necessary to take into account the fact that viable variant cells normally will continue to reproduce while they are being con¬ tacted with a selective agent, and while they are being incubated with the parti- cles. Unless determined otherwise, this factor i •s presumed to be equal to 2X _f where x is the amount of time that elapses while the invention is being performed divided by the doubling time of the variant cells under the conditions of the invention. 2. A particle uptake efficiency factor. This factor reflects the fact that, in a large ^• control population which is incubated with particles, only a certain fraction of the cells take up particles. The exact fraction depends upon several factors, including the length of the incubation period and the number of particles per cell. For accurate analyses of small variant fractions, the phagocytic efficiency should be close to

1.0, e.g., greater than about 0.9. If the phagocytic efficiency is substantially less than 0.9, a large cell population should be analyzed to accurately determine a very small variant fraction such as a mutant fraction. 3. A processing recovery factor. This factor reflects the fact that in various preferred embodiments of this invention, the cells must be processed in certain ways. For

OMPI example, it may be desired to centrifuge a cell population after it has been con- ted with a selective agent in order to remove the selective agent from the medium. It may also be desired to centri¬ fuge a mixture of cells and particles after incubation to remove uningested micro¬ spheres from the cell population. In any type of processing step such as centri- fugation, it normally is impossible to recover all of the cells that were in the starting population. To account for the loss of cells during processing, a recovery factor may be determined for each step, or for all of the processing steps. The recovery factor is equal to the number of cells present in an aliquot after a pro¬ cessing step or series of steps is completed, divided by the number of cells that were in the aliquot before the processing step. Recovery factors may be approximated by using control populations which have not been contacted with a selective agent. The number of cells in an aliquot may be determined at any stage of processing by thoroughly mixing a suspension of cells by means such as shaking or vortexing, determining the concentration of cells per volume by analyzing the suspension on a cell counter, and multiplying the cell concentration by. the volume of the suspension. The overall equation for determining the variant fraction may be expressed as

variant _ # of fluorescinq cells fraction f ^~ of cells ^ /processing^ /particle \ /doubling^"") (factor /

There are numerous methods and compounds which may be utilized to render non-variant cells incapable of taking up particles. For example, selective agents such as nucleotide analogs may be used to kill cells with specific types of enzymes. A variety of nucleo¬ tide analogs are known to those skilled in the art, such as 6-thioguanine, 8-azaguanine, and trifluoro- deoxythymidine.

Various other compounds with different types of selective effects, such as chemotherapeutic agents (for example, methotrexate and daunomycin) may also be utilized to divide a cell population into categories which may be regarded as variant and non-variant. This invention may be utilized to study the effects of such compounds, or for various other purposes. This invention is not limited to use with cells which are capable of growing in suspension. Anchorage- dependent cells may be grown in a monolayer on appropriate types of solid surfaces. A suspension of particles may be incubated with such a monolayer, and phagocytosis normally

OMPI will occur. The particles that have not been taken up may be subsequently removed by washing, and, if necessary, treatment with a substance such as dilute trypsin. The cells may subsequently be removed from the solid surface by various means known to those skilled in the art, such as extended treatment with trypsin. Such cells may subsequently be analyzed by flow cyto etric methods. Alternately, such cells may be analyzed while remaining attached to the solid surface, by the methods described above. A variety of techniques may be utilized to isolate or concentrate variant cells using the methods of this invention. For example, cells which have been incubated with fluorescent parti- cles may be placed on a microscope slide or petri dish and observed using a fluorescent microscope. Cells which have ingested particles may be identi¬ fied by the fluorescence of the particles, and removed from the plate or slide by using mechani- cal means such as a micropipette. Alternately, cells which have been incubated with fluorescent particles may be processed by a flow cytometer equipped with a cell sorter. Cells which have been incubated with magnetic particles may be isolated or concentrated by using an electromagnet or other magnetic device.

EXAMPLES

Example 1: Materials and Equipment Used

The cells used in the work described herein were human diploid lymphoblast cells, designated the TK6 cell line. This cell line, described by T. Skopek et al, .Bioche Biophys. Res. Com un. 84: 411 (1978) is heterozygous for thymidine kinase. The starter culture was provided by Dr. A. Bloom, College of Physicians and Surgeons, Columbia University, New York City, NY. The cells were grown in suspension culture using

RPMI 1640 culture medium (Flow Laboratories, McLean, VA or Grand Island Biological Company, Grand Island, NY) , supplemented with 10% horse serum (Flow or GIBCO) . Stock cultures were grown in spinner cultures and passaged daily to 3 x 10 cells per ml . Cell counts were made using a Coulter cell counter (Coulter Electronics, Hialeah, FL) . Doubling times are 15 to 17 hours in spinner culture, and 17 to _20 hours in stationary culture. In all mutant fraction experiments, the selective agent 6-thioguanine (6-TG, Sigma Chemical Co., St. Louis, MO) was used. In order to reduce the number of spontaneous HGPRT mutants in a culture, the cells were grown in CHAT medium, comprising RPMI 1640 medium with 10% horse serum containing 10 M cytidine,

2 x 10 -4M hypoxanthine, 2 x 10-7 M aminopterin, and

1.75 x 10 -5 M thymidine.

The microspheres utilized in the experiments were carboxylated polystyrene (Polysciences Co., Warrington, PA) . The beads chosen exhibit green fluoresence at a peak of 492 nm at an excitation of 450 nm. - _n-

Example 2: Uptake of Particles by Cells

The incubation procedure described below is summarized in the flow chart in Figure 1. A suspension

9 of microspheres was prepared by adding 2 x 10 micro- spheres per ml to serum-free RPMI 1640 medium. The suspension was filtered through a filter with 3 i diameter pores to remove aggregates of beads.

Separately, a suspension of TK6 cells containing

1 - 2 x 10 cells were centrifuged into a pellet. The cells were resuspended in the microsphere suspension. The cell and microsphere suspension was placed in a spinner flask with a diameter of about 2.25 inches, and incubated at 37° C for 4 hours at a stirring speed of 60 to 120 rpm. After incubation, the suspension was centrifuged through Percoll gradient (Pharmacia Fine Chemicals, Piscataway, NJ) diluted 1:10 in phosphate buffered saline (PBS) . This allowed for about 90% of the uningested microspheres to be removed from the cells. A control culture which was not exposed to 6-TG was processed simultaneously. This culture was utilized to determine the doubling time of the cells, the reco^¬ very factor, and the particle uptake efficiency factor as shown on Figure 1.

OMPI Example 3: Analysis of Cells After Particle Uptake

TK6 cells were analyzed after being incubated with fluorescent microspheres by either of two methods. In one method, a small quantity of the cellular sus- pension was placed on a microscope slide. The cells were covered with a cover slip, which was sealed under the slide by means of clear nail polish. By using a cover slip, the cells were constrained more or less within a single focal plane. This made it somewhat simpler to distinguish fluorescent microspheres which had been ingested by the cells by the cells from micro- spheres which merely adhered to cells. However, this method was not entirely satisfactory, so a second method was developed. Polystyrene petri dishes 3 cm in diameter were contacted with an aqueous solution of concanavalin-A (con-A) . The plates were incubated for one hour at room temperature, and excess aqueous solution was poured off. TK6 cells have a substantially greater affinity for con-A than do carboxylated- polystyrene

7 microspheres. Approximately 1-2 x 10 cells, which had been incubated with microspheres as described in Example 2, were suspended in 2.5 ml of PBS. The suspension was placed on a con-A coated plate and allowed to incubate for one hour. The plate was then washed five times with PBS to remove uningested microspheres.

If a substantial number of microspheres remained on the plate, the plate was incubated for 5 minutes in a solution of 0.005 percent trypsin, then washed five times with PBS. The trypsin caused the majority of uningested microspheres to become detached from the plate while causing less than 5% of the cells to become detached.

Analysis was performed on Zeiss Model III R S microscope. Excitation was by a mercury arc lanp equipped with suitable filters.

By using the foregoing techniques, a variety of results were obtained which are displayed in Figures 2 through 5.

Figure 2 indicates the effect of duration of 6-TG contact on particle intake. On the horizontal axis is plotted the number of hours that TK 6 cells were incubated in 5 pg/ml 6-TG before they were centrifuged to remove the 6-TG and resuspended with microspheres. The variant fraction is plotted on the vertical axis. This figure indicated that a contact time in excess of about 10 hours is suffi¬ cient to render non-variant HGPRT cells non- phagocytic. Figure 3 is a histogram indicating the number of cells which ingested from 1 to 25 microspheres per cell.

Figure 4 indicates uptake of microspheres by TK6 cells. As shown in that figure, the amount of uptake tends to level off after about 4 hours. These results were taken from a relatively early experiment when only 50 microspheres per cell were incubated with the cells; therefore, the percentage of cells containing microspheres is substantially lower than percentages that can be obtained utilizing 200 micro¬ spheres per cell.

OMPI Figure 5 indicates the effects of the particle to cell ratio on the uptake of cells.

In each of the figures, vertical bars around- a data point indicate 95% confidence limits.

Example 4: Evaluation of Particle Uptake Method

The accuracy and sensitivity of the particle uptake method of this invention was assessed by comparing the results obtained by that method against the results obtained by conventional plating assays. In this experiment, TK6 cells were contacted with varying concentrations of methyl nitrosourea (MNU) . Plating assays were conducted by the methods described in E.E.Furth et al, Anal. Biochem. 108: 1-8(1980).

Particle uptake was performed using the incubation methods described in Example 1 and Figure 1; analysis under a light microscope was performed utilizing microscope slides with sealed coverslips as described in Example 3. The results of this comparison are shown in Figure 6.

In a separate experiment, a population of cells was created by mixing a population of mutant HGPRT TK6 cells with a population of wild-type HGPRT TK6 cells. Controlled quantities of mutant cells were mixed with the wild-type cells, so that a known mutant fraction, ranging from 10 -6 to 10-4, could be tested. The results are shown in Figure 7. The data points indicated by open circles reflect the number of fluorescent cells after exposure to 6-TG

OMPI - .~- 2>> A4-

and evaluation as described in Example 2 and Figure 1. However, those values must be adjusted to account for spontaneous mutation rates which are known to exist in TK6 cells. The data points indicated by solid triangles were obtained by subtracting the spontaneous mutation rates, often referred to as background, from the uncorrected observations.

Industrial Applicability

This invention has industrial applicability in reducing the time and effort required to assess certain mutagenic effects and other effects that industrial chemicals and processes have upon cells.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equi¬ valents are considered to be within the scope of this invention, and are covered by the following claims.

Claims

1. A method of identifying variant cells, comprising: a. subjecting a population of cells to first conditions which cause non-variant cells to become incapable of taking up particles having at least one discriminator characteristic; b. contacting said population of cells with particles having at least one discriminator characteristic under second conditions which allow variant cells within said population of cells to take up said particles; and c. analyzing said population of cells to determine which cells have taken up one or more of said particles.

2. A method of isolating variant cells, comprising: a. subjecting a population of cells to first conditions which cause non-variant cells to become incapable of taking up particles having at least one discriminator characteristic; b. contacting said population of cells with particles having at least one discriminator characteristic under second conditions which allow variant cells within said population of cells to take up said particles; and c. separating said population of cells into two or more subpopulations, at least one of which consists essentially of cells which have not taken up said particles and at least one of which consists essentially of cells which have taken up said particles.

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3. A method of enumerating variant cells, comprising: a. subjecting a population of cells to first conditions which cause non-variant cells to become incapable of taking up particles having at least one discriminator characteristic; b. contacting said population of cells with particles having at least one discriminator characteristic under second conditions which allow variant cells within said population of cells to take up said particles; and c. determining the number of cells which have taken up said particles.

4. A method of determining the variant fraction of a cell population, comprising: a. subjecting a population of cells to first conditions which cause non-variant cells to become incapable of taking up particles having at least one discriminator characteristic; b. contacting said population of cells with particles having at least one discriminator characteristic under second conditions which allow variant cells within said population of cells to take up said particles; c.. determining the number of cells which have taken up said particles; and d. dividing the number of cells which have taken up said particles by the number of cells in said cell population.

5. A method of Claims 1 or 2 wherein said variant cells are mutant cells in a population of non- mutant cells.

6. A method of Claims 1 or 2 wherein less than 0.1 percent of the cells in said population of cells are variant cells.

7. A method of Claims 1 or 2 wherein said first conditions comprise contacting said population of cells with a selective agent.

8. A method of Claim 7 wherein said selective agent comprises a nucleotide analog.

9. A method of Claim 8 wherein said nucleotide analog is selected from the following group: 6-thioguanine, 8-azaguanine, and trifluorodeoxy- thymidine.

10. A method of Claims 1 or 2 wherein said particles comprise microspheres with an average diameter of about 0.6 to about 1.5 microns.

11. A method of Claims 1 or 2 wherein said cells are contacted with about 100 to about 300 particles per cell.

12. A method of Claims 1 or 2 wherein said second conditions comprise incubation in stirred cul¬ ture medium.

13. A method of Claims 1 or 2 wherein said cells are centrifuged after being contacted with said particles to remove particles that have not been taken up by cells.

14. A method of Claims 1 or 2 wherein said discrimi¬ nator characteristic comprises fluorescence.

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15. A method of Claim 14 wherein said cells are analyzed or separated using a flow cytometer.

16. A method of Claims 1 or 2 wherein said dis¬ criminator characteristic is selected from the following group: magnetism, radioactivity, electron apacity, color, or pharmalogical activity.

17. A method of Claim 4 wherein said variant fraction fraction is corrected by one or more of the following factors: a. a doubling factor; b. a particle uptake efficiency factor; and c. a processing recovery factor.

18. A method of Claim 17 wherein said doubling factor is equal to 2x, wherein x is equal to the amount of time that elapses while the method is being performed divided by the doubling time of the variant cells under said first and second conditions.

19. A method of Claim 17 wherein said particle uptake efficiency factor is determined by con¬ tacting a control population of cells which has not been subjected to said first .conditions with said particles under said second conditions, determining the number of cells in said popu¬ lation which take up said particles, and dividing that number by the number of cells in said population.

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